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Cancers, Volume 11, Issue 8 (August 2019) – 175 articles

Cover Story (view full-size image): Cancer cells have a fundamentally different metabolism than noncancerous cells, and this difference is manifested in the endogenous metabolites they produce. Metabolomics aims to study metabolic differences in biological systems and has recently been applied to the discovery of tumor biomarkers in solid tumors. Previous studies reported the utility of the enzyme SSAT-1 as a cancer detection tool. SSAT-1 is a key protein involved in the synthesis of the polyamines spermine and spermidine that are involved in physiological cellular processes. This study led by Singhal and Rolfo et al. evaluated a panel of 14 metabolites associated in the SSAT-1/polyamine pathway that correctly discriminated between lung cancer patients from healthy controls, demonstrating the utility of metabolomics for lung cancer detection and adding further evidence on the role of liquid biopsy in cancer interception. View this paper
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18 pages, 1918 KiB  
Review
Fluorescence-Guided Surgery for Hepatoblastoma with Indocyanine Green
by Yohei Yamada, Michinobu Ohno, Akihiro Fujino, Yutaka Kanamori, Rie Irie, Takako Yoshioka, Osamu Miyazaki, Hajime Uchida, Akinari Fukuda, Seisuke Sakamoto, Mureo Kasahara, Kimikazu Matsumoto, Yasushi Fuchimoto, Ken Hoshino, Tatsuo Kuroda and Tomoro Hishiki
Cancers 2019, 11(8), 1215; https://doi.org/10.3390/cancers11081215 - 20 Aug 2019
Cited by 66 | Viewed by 6298
Abstract
Fluorescence-guided surgery with indocyanine green (ICG) for malignant hepatic tumors has been gaining more attention with technical advancements. Since hepatoblastomas (HBs) possess similar features to hepatocellular carcinoma, fluorescence-guided surgery can be used for HBs, as aggressive surgical resection, even for distant metastases of [...] Read more.
Fluorescence-guided surgery with indocyanine green (ICG) for malignant hepatic tumors has been gaining more attention with technical advancements. Since hepatoblastomas (HBs) possess similar features to hepatocellular carcinoma, fluorescence-guided surgery can be used for HBs, as aggressive surgical resection, even for distant metastases of HBs, often contributes positively to R0 (complete) resection and subsequent patient survival. Despite a few caveats, fluorescence-guided surgery allows for the more sensitive identification of lesions that may go undetected by conventional imaging or be invisible macroscopically. This leads to precise resection of distant metastatic tumors as well as primary liver tumors. Full article
(This article belongs to the Special Issue Hepatoblastoma and Pediatric Liver Tumors)
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Figure 1
<p><b>a</b>,<b>b</b>: Multiple fluorescent spots are observed under near-infrared (NIR) in the liver. Of note, some nodules show a rim-type fluorescence pattern, indicated by an arrowhead (combined fetal and embryonal subtype, post-chemotherapy).</p>
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<p><b>a</b>,<b>b</b>: The hilum of the liver in Patient 12. A giant tumor with uneven fluorescence in the left lobe along with intrahepatic metastasis (diffuse pattern) is indicated by an arrowhead. Of note, the common bile duct is also visualized, presumably due to residual fluorescence, indicated by an arrow (mixed epithelial and mesenchymal, post-chemotherapy).</p>
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<p><b>a</b>–<b>g</b>: Mixed epithelial and mesenchymal type with teratoid features, post-chemotherapy. The formalin-fixed cross section of the liver is shown (<b>a</b>), and the tumor is encircled with a yellow-dotted line. The tumor is heterogeneous macroscopically, and fluorescence can be observed only in the small area marked with a white-dotted line (<b>b</b>: white-light mode, <b>c</b>: NIR mode, <b>d</b>: mapping mode). A histological analysis showed that while the fluorescent area corresponded to well-differentiated HB (<b>e</b>), the non-fluorescent area consisted of poorly differentiated HB (<b>f</b>) and an osteoid lesion (<b>g</b>). Scale bar: 100 µm.</p>
Full article ">Figure 3 Cont.
<p><b>a</b>–<b>g</b>: Mixed epithelial and mesenchymal type with teratoid features, post-chemotherapy. The formalin-fixed cross section of the liver is shown (<b>a</b>), and the tumor is encircled with a yellow-dotted line. The tumor is heterogeneous macroscopically, and fluorescence can be observed only in the small area marked with a white-dotted line (<b>b</b>: white-light mode, <b>c</b>: NIR mode, <b>d</b>: mapping mode). A histological analysis showed that while the fluorescent area corresponded to well-differentiated HB (<b>e</b>), the non-fluorescent area consisted of poorly differentiated HB (<b>f</b>) and an osteoid lesion (<b>g</b>). Scale bar: 100 µm.</p>
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<p><b>a</b>–<b>d</b>: Multiple metastatic HBs in the transplanted liver in vivo (<b>a</b>,<b>b</b>) and ex vivo (<b>c</b>,<b>d</b>). This patient underwent the second living donor liver transplantation [<a href="#B44-cancers-11-01215" class="html-bibr">44</a>].</p>
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<p><b>a</b>,<b>b</b>: Several pulmonary metastases are visualized in NIR mode (patient 19; transitional liver cell tumor).</p>
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<p>Peritoneal metastases in the patient 8, which were successfully removed with the help of NIR mode. Normal white light mode viewing the abdominal cavity (<b>a</b>,<b>c</b>) and NIR mode (<b>b</b>,<b>d</b>).</p>
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<p>Pleural metastasis visualized with the Pinpoint system. Normal white-light mode (<b>a</b>), NIR mode (<b>b</b>), and overlay mode (<b>c</b>) are shown. The tumor is visualized as a green color overlaid on the white-light mode view.</p>
Full article ">Figure 8
<p>ICG is distributed to the whole body and accumulates in the liver (within a few minutes after the intravenous injection). ICG is then excreted into the biliary system and persists for up to 20–24 h. While liver tumors display non-fluorescent spots in NIR mode at a very early stage after the injection of ICG, such studies have not been performed in lungs with metastatic HBs. The selective retention of ICG can be observed in HBs in both the liver and lungs around 24 h, but excreted fluorescence remains in the bowel loops. By 72–96 h after the injection, bowel-retained ICG is excreted with feces. HB tissues retain ICG for up to two weeks. The pattern of fluorescence may vary depending on the dose of ICG, detecting device, liver function, and pathology.</p>
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<p><b>a</b>,<b>b</b> Non-specific fluorescence in the pancreas and bowel loops. The arrowhead indicates the intense fluorescence in the pancreas, but a thorough inspection denied the presence of metastases. Non-specific fluorescence in the bowl loops was also observed (indicated by an arrow).</p>
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20 pages, 3361 KiB  
Article
Determination of the Potential Tumor-Suppressive Effects of Gsdme in a Chemically Induced and in a Genetically Modified Intestinal Cancer Mouse Model
by Lieselot Croes, Erik Fransen, Marieke Hylebos, Kimberly Buys, Christophe Hermans, Glenn Broeckx, Marc Peeters, Patrick Pauwels, Ken Op de Beeck and Guy Van Camp
Cancers 2019, 11(8), 1214; https://doi.org/10.3390/cancers11081214 - 20 Aug 2019
Cited by 30 | Viewed by 4394
Abstract
Gasdermin E (GSDME), also known as deafness autosomal dominant 5 (DFNA5) and previously identified to be an inducer of regulated cell death, is frequently epigenetically inactivated in different cancer types, suggesting that GSDME is a tumor suppressor gene. In [...] Read more.
Gasdermin E (GSDME), also known as deafness autosomal dominant 5 (DFNA5) and previously identified to be an inducer of regulated cell death, is frequently epigenetically inactivated in different cancer types, suggesting that GSDME is a tumor suppressor gene. In this study, we aimed to evaluate the tumor-suppressive effects of GSDME in two intestinal cancer mouse models. To mimic the silencing of GSDME by methylation as observed in human cancers, a Gsdme knockout (KO) mouse was developed. The effect of GSDME on tumorigenesis was studied both in a chemically induced and in a genetic intestinal cancer mouse model, as strong evidence shows that GSDME plays a role in human colorectal cancer and representative mouse models for intestinal cancer are available. Azoxymethane (AOM) was used to induce colorectal tumors in the chemically induced intestinal cancer model (n = 100). For the genetic intestinal cancer model, Apc1638N/+ mice were used (n = 37). In both experiments, the number of mice bearing microscopic proliferative lesions, the number and type of lesions per mouse and the histopathological features of the adenocarcinomas were compared between Gsdme KO and wild type (WT) mice. Unfortunately, we found no major differences between Gsdme KO and WT mice, neither for the number of affected mice nor for the multiplicity of proliferative lesions in the mice. However, recent breakthroughs on gasdermin function indicate that GSDME is an executioner of necrotic cell death. Therefore, it is possible that GSDME may be important for creating an inflammatory microenvironment around the tumor. This is in line with the trend towards more severe inflammation in WT compared to Gsdme KO mice, that we observed in our study. We conclude that the effect of GSDME in tumor biology is probably more subtle than previously thought. Full article
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<p>qRT-PCR analyses on <span class="html-italic">Gsdme</span> KO and WT mice. qRT-PCR analyses for <span class="html-italic">Gsdme</span> mRNA expression on <span class="html-italic">Gsdme</span> KO (<span class="html-italic">n</span> = 7) and WT (<span class="html-italic">n</span> = 9) mice, both on brain (<span class="html-italic">n</span> = 15) and colorectal (<span class="html-italic">n</span> = 16) tissues were performed. The Calibrated Normalized Relative Quantity (CNRQ) ± standard error (se) is represented for every sample. The expression patterns in colon and in brain tissues were similar. There was only very low to no measurable <span class="html-italic">Gsdme</span> expression in <span class="html-italic">Gsdme</span> KO mice, in contrast to WT mice where <span class="html-italic">Gsdme</span> expression was higher and more variable.</p>
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<p>Representative example of a Western Blot on samples of <span class="html-italic">Gsdme</span> KO and WT mice. Western Blotting was performed with a primary rabbit anti-DFNA5/GSDME-N-Terminal antibody (ab215191) and a secondary Goat Anti-Rabbit IgG (H+L)-HRP Conjugate (#1706515). Clear GSDME protein bands (<math display="inline"><semantics> <mo>~</mo> </semantics></math>57 kDa) were seen, both in the brain and colorectal tissue of WT mice. GSDME could not be detected in <span class="html-italic">Gsdme</span> KO brain or colorectal tissues. In all samples, clear bands for β-actin could be seen (<math display="inline"><semantics> <mo>~</mo> </semantics></math>42 kDa). WT = wild type mice; KO = <span class="html-italic">Gsdme</span> knockout mice; B = brain tissue; C = colorectal tissue.</p>
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<p>Large intestine of a representative mouse. The large intestine was opened longitudinally and divided into four parts: proximal, mid 1, mid 2, and distal.</p>
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<p>Microscopic images of proliferative lesions in AOM-treated mice. (<b>A</b>) Typical hyperplasia, indicated by the arrow, in the large intestine. (<b>B</b>) Atypical hyperplasia, indicated by the arrow, in the large intestine. (<b>C</b>) Adenoma in the large intestine. (<b>D</b>) Adenoma in the small intestine. (<b>E</b>) Adenocarcinoma in the large intestine. (<b>F</b>) Magnification of the adenocarcinoma in the large intestine (E). The arrow indicates infiltration of the adenocarcinoma in the submucosa. Scale bars are indicated on the images.</p>
Full article ">Figure 5
<p>Distribution of the number of adenocarcinomas per mouse in <span class="html-italic">Gsdme</span> KO and WT mice. There were 20 out of 46 (43.5%) <span class="html-italic">Gsdme</span> KO and 25 out of 54 (46.3%) WT mice with one or more colon adenocarcinomas, with a median of one adenocarcinoma/mouse (range: 1–6) for the <span class="html-italic">Gsdme</span> KO mice and a median of one adenocarcinoma/mouse (range: 1–7) for the WT mice.</p>
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<p>Percentage of adenocarcinomas with different grades of mononuclear cell infiltration in WT and <span class="html-italic">Gsdme</span> KO mice. Slight inflammation was more often associated with adenocarcinomas in WT mice compared to <span class="html-italic">Gsdme</span> KO mice, while in the latter group more often no inflammation was present.</p>
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<p>Distribution of the number of adenocarcinomas per mouse in <span class="html-italic">Apc<sup>1638N/+</sup> Gsdme</span> KO and <span class="html-italic">Apc<sup>1638N/+</sup> Gsdme</span> WT mice. There were 10 out of 13 (76.9%) <span class="html-italic">Apc<sup>1638N/+</sup> Gsdme</span> KO and 16 out of 24 (66.7%) <span class="html-italic">Apc<sup>1638N/+</sup> Gsdme</span> WT mice with one or more adenocarcinomas in the small intestine, with a median of two adenocarcinomas/mouse (range: 1–3) for the <span class="html-italic">Gsdme</span> KO mice, and a median of two adenocarcinomas/mouse (range: 1–9) for the WT mice.</p>
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16 pages, 913 KiB  
Review
LAG3: The Biological Processes That Motivate Targeting This Immune Checkpoint Molecule in Human Cancer
by Cinzia Solinas, Edoardo Migliori, Pushpamali De Silva and Karen Willard-Gallo
Cancers 2019, 11(8), 1213; https://doi.org/10.3390/cancers11081213 - 20 Aug 2019
Cited by 82 | Viewed by 12234
Abstract
The programmed cell death 1 (PD-1) pathway is an important regulator of immune responses in peripheral tissues, including abnormal situations such as the tumor microenvironment. This pathway is currently the principal target for immunotherapeutic compounds designed to block immune checkpoint pathways, with these [...] Read more.
The programmed cell death 1 (PD-1) pathway is an important regulator of immune responses in peripheral tissues, including abnormal situations such as the tumor microenvironment. This pathway is currently the principal target for immunotherapeutic compounds designed to block immune checkpoint pathways, with these drugs improving clinical outcomes in a number of solid and hematological tumors. Medical oncology is experiencing an immune revolution that has scientists and clinicians looking at alternative, non-redundant inhibitory pathways also involved in regulating immune responses in cancer. A variety of targets have emerged for combinatorial approaches in immune checkpoint blockade. The main purpose of this narrative review is to summarize the biological role of lymphocyte activation gene 3 (LAG3), an emerging targetable inhibitory immune checkpoint molecule. We briefly discuss its role in infection, autoimmune disease and cancer, with a more detailed analysis of current data on LAG3 expression in breast cancer. Current clinical trials testing soluble LAG3 immunoglobulin and LAG3 antagonists are also presented in this work. Full article
(This article belongs to the Special Issue Signaling Pathways and Immune Checkpoint Regulation in Cancer)
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<p>LAG3 biology on immune cells (<b>A</b>) and in the tumor microenvironment (TME) (<b>B</b>). (<b>A</b>). LAG3 is expressed on CD4<sup>+</sup> (Th) and CD8<sup>+</sup> (CTL) T cells, plasmacytoid dendritic cells (pDC) and NK cells in the TME (<b>B</b>). Its principle ligands include: MHC-II expressed on antigen presenting cells (APC) and tumor cells, LSECtin expressed on melanoma cells and galectin-3 expressed on some T cells and stromal cells in the TME (<b>B</b>). In its soluble form, LAG3 (sLAG3) impairs monocyte differentiation to dendritic cells (DC) and macrophages. In the TME, interactions mediated by LAG3 and its ligands are inhibitory (<b>B</b>).</p>
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<p>Molecular mechanisms of LAG3 function. LAG3 can be expressed on CD4<sup>+</sup> (<b>A</b>), CD8<sup>+</sup> T and NK cells (<b>B</b>). Its interaction with its ligands (stable pMHC-II complexes; LSECtin; FGL-1; galectin-3) expressed on different cells (immune, stromal, liver and tumor cells), generates an inhibition of CD4<sup>+</sup>, CD8<sup>+</sup> and NK T cell proliferation, cytokine production and cytolitic function. These effects are mediated by the cytoplasmic motif of the LAG3 receptor, which is named KIEELE.</p>
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<p>Targeting effector and regulatory T cells with LAG3 antagonistic antibodies (<b>A</b>) and activating antigen presenting cells with soluble LAG3 Immunoglobulin (Ig) (<b>B</b>). (<b>A</b>) Binding of LAG3 antagonistic antibodies (Abs) to LAG3 prevents its interaction with the respective ligands (LSECtin, galectin-3 and FGL-1 for CD8<sup>+</sup> T cells; antigen presenting cells (APC) for CD4<sup>+</sup> T cells). This contributes to releasing the break to the activation of tumor-infiltrating lymphocytes (TIL) and is achieved with the use of a combined blockade with anti-PD-1/PD-L1 Abs. (<b>B</b>) Binding of a soluble LAG3 Ig to APC engages MHC-II molecules on the cell surface. This generates an upregulation of co-stimulatory molecules, such as CD80, CD86 and CD40, and leads to the secretion of pro-inflammatory cytokines and chemokines by APC. APC activation through soluble LAG3 agonistic Ab may induce a rapid release of pro-inflammatory cytokines by a subpopulation of NK cells and activation of memory CD8<sup>+</sup> T cells in an antigen independent manner. APC: Antigen presenting cell; CCL: chemokine (C-C motif) ligand; CXCL: chemokine (C-X-C motif) ligand; DC: dendritic cell; FGL-1: fibrinoigen like protein - 1; IFN: interferon; Ig: immunoglobulin; IL: interleukin; LSECtin: liver and lymph node sinusoidal endothelial cell C-type lectin; NK: natural killer; PD-1: programmed cell death -1; Teff: effector T cells; TNF: tumor necrosis factor; Tregs: regulatory T cells.</p>
Full article ">
14 pages, 1307 KiB  
Article
Circulating Tumor Cell Enumeration and Characterization in Metastatic Castration-Resistant Prostate Cancer Patients Treated with Cabazitaxel
by Ingeborg E. de Kruijff, Anieta M. Sieuwerts, Wendy Onstenk, Jaco Kraan, Marcel Smid, Mai N. Van, Michelle van der Vlugt-Daane, Esther Oomen-de Hoop, Ron H.J. Mathijssen, Martijn P. Lolkema, Ronald de Wit, Paul Hamberg, Hielke J. Meulenbeld, Aart Beeker, Geert-Jan Creemers, John W.M. Martens and Stefan Sleijfer
Cancers 2019, 11(8), 1212; https://doi.org/10.3390/cancers11081212 - 20 Aug 2019
Cited by 23 | Viewed by 4234
Abstract
(1) Background: Markers identifying which patients with metastatic, castration-resistant prostate cancer (mCRPC) will benefit from cabazitaxel therapy are currently lacking. Therefore, the aim of this study was to identify markers associated with outcome to cabazitaxel therapy based on counts and gene expression profiles [...] Read more.
(1) Background: Markers identifying which patients with metastatic, castration-resistant prostate cancer (mCRPC) will benefit from cabazitaxel therapy are currently lacking. Therefore, the aim of this study was to identify markers associated with outcome to cabazitaxel therapy based on counts and gene expression profiles of circulating tumor cells (CTCs). (2) Methods: From 120 mCRPC patients, CellSearch enriched CTCs were obtained at baseline and after 6 weeks of cabazitaxel therapy. Furthermore, 91 genes associated with prostate cancer were measured in mRNA of these CTCs. (3) Results: In 114 mCRPC patients with an evaluable CTC count, the CTC count was independently associated with poor progression-free survival (PFS) and overall survival (OS) in multivariable analysis with other commonly used variables associated with outcome in mCRPC (age, prostate specific antigen (PSA), alkaline phosphatase, lactate dehydrogenase (LDH), albumin, hemoglobin), together with alkaline phosphatase and hemoglobin. A five-gene expression profile was generated to predict for outcome to cabazitaxel therapy. However, even though this signature was associated with OS in univariate analysis, this was not the case in the multivariate analysis for OS nor for PFS. (4) Conclusion: The established five-gene expression profile in CTCs was not independently associated with PFS nor OS. However, along with alkaline phosphatase and hemoglobin, CTC-count is independently associated with PFS and OS in mCRPC patients who are treated with cabazitaxel. Full article
(This article belongs to the Collection Cancer Biomarkers)
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<p>PFS and OS in relation to dichotomized CTC count at baseline. Kaplan Meier curves of (<b>A</b>) progression-free survival (PFS) and (<b>B</b>) overall survival (OS) in relation to circulating tumor cell (CTC) count at baseline. CTC counts are divided into two categories of &lt; 5 CTCs and ≥ 5 CTCs.</p>
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<p>PFS and OS in relation to CTC groups. Kaplan Meier curves of (<b>A</b>) progression-free survival (PFS) and (<b>B</b>) overall survival (OS) in relation to circulating tumor cell (CTC) count. CTC counts are divided into four categories: (1) &lt;5 CTCs at baseline and &lt;5 CTCs during treatment, (2) ≥5 CTCs at baseline and &lt;5 CTCs during treatment, (3) &lt;5 CTCs at baseline and ≥5 CTCs during treatment and (4) ≥5 CTCs at baseline and ≥5 CTCs during treatment.</p>
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<p>Patient inclusion. Flowchart of patient inclusion. In blue all patients who are included in the CABARESC study and the side CTC enumeration study. In green the patients included for this manuscript with available CTC enumeration. In Orange the patients included for this manuscript with available gene expression data.</p>
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12 pages, 1921 KiB  
Article
RAC1B Suppresses TGF-β-Dependent Chemokinesis and Growth Inhibition through an Autoregulatory Feed-Forward Loop Involving PAR2 and ALK5
by Hannah Otterbein, Koichiro Mihara, Morley D. Hollenberg, Hendrik Lehnert, David Witte and Hendrik Ungefroren
Cancers 2019, 11(8), 1211; https://doi.org/10.3390/cancers11081211 - 20 Aug 2019
Cited by 7 | Viewed by 3805
Abstract
The small GTPase RAC1B functions as a powerful inhibitor of transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition, cell motility, and growth arrest in pancreatic epithelial cells. Previous work has shown that RAC1B downregulates the TGF-β type I receptor ALK5, but the molecular details of [...] Read more.
The small GTPase RAC1B functions as a powerful inhibitor of transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition, cell motility, and growth arrest in pancreatic epithelial cells. Previous work has shown that RAC1B downregulates the TGF-β type I receptor ALK5, but the molecular details of this process have remained unclear. Here, we hypothesized that RAC1B-mediated suppression of activin receptor-like kinase 5 (ALK5) involves proteinase-activated receptor 2 (PAR2), a G protein-coupled receptor encoded by F2RL1 that is crucial for sustaining ALK5 expression. We found in pancreatic carcinoma Panc1 cells that PAR2 is upregulated by TGF-β1 in an ALK5-dependent manner and that siRNA-mediated knockdown of RAC1B increased both basal and TGF-β1-induced expression of PAR2. Further, the simultaneous knockdown of PAR2 and RAC1B rescued Panc1 cells from a RAC1B knockdown-induced increase in ALK5 abundance and the ALK5-mediated increase in TGF-β1-induced migratory activity. Conversely, Panc1 cells with stable ectopic expression of RAC1B displayed reduced ALK5 expression, an impaired upregulation of PAR2, and a reduced migratory responsiveness to TGF-β1 stimulation. However, these effects could be reversed by ectopic overexpression of PAR2. Moreover, the knockdown of PAR2 alone in Panc1 cells and HaCaT keratinocytes phenocopied RAC1B’s ability to suppress ALK5 abundance and TGF-β1-induced chemokinesis and growth inhibition. Lastly, we found that the RAC1B knockdown-induced increase in TGF-β1-induced PAR2 mRNA expression was sensitive to pharmacological inhibition of MEK-ERK signaling. Our data show that in pancreatic and skin epithelial cells, downregulation of ALK5 activity by RAC1B is secondary to suppression of F2RL1/PAR2 expression. Since F2RL1 itself is a TGF-β target gene and its upregulation by TGF-β1 is mediated by ALK5 and MEK-ERK signaling, we suggest the existence of a feed-forward signaling loop involving ALK5 and PAR2 that is efficiently suppressed by RAC1B to restrict TGF-β-driven cell motility and growth inhibition. Full article
(This article belongs to the Special Issue Rho Family of GTPases in Cancer)
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<p>Effect of RAC1B knockdown or ectopic overexpression on TGF-β1-induced gene expression of PAR2. (<b>A</b>) Panc1 cells were transfected twice with 50 nM each of either control (Co) siRNA or RAC1B siRNA. Forty-eight hours after the second round of transfection, cells were treated with TGF-β1 for 24 h and subsequently subjected to qPCR analysis for PAR2. (<b>B</b>) As in (<b>A</b>), except that Panc1 cells were cotransfected with 25 nM each of Co siRNAs specific for RAC1B and ALK5, or 25 nM each of RAC1B siRNA + ALK5-specific Co siRNA, or 25 ng each of RAC1B siRNA + ALK5 siRNA prior to TGF-β1 treatment and qPCR analysis of PAR2. Data in (<b>A</b>) and (<b>B</b>) represent the normalized mean ± SD from three independent experiments and are displayed relative to control siRNA set arbitrarily at 1. (<b>C</b>) Three individual clones of Panc1 cells with stable ectopic expression of HA-tagged RAC1B (HA-RAC1B) were subjected to immunoblot analysis of ALK5. The graph below the immunoblot shows results from densitometry-based quantitative analysis. (<b>D</b>) As in (<b>C</b>) except that cells were treated, or not, with TGF-β1 for 48 h and subjected to qPCR analysis of PAR2. Data in (<b>C</b>) and (<b>D</b>) represent the normalized mean ± SD of three parallel wells from one representative experiment out of three experiments performed with very similar results. The asterisks indicate significance.</p>
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<p>Effect of PAR2 knockdown on RAC1B knockdown-induced ALK5 expression in Panc1 cells. (<b>A</b>) Panc1 cells were transfected twice with 25 nM each of Co siRNAs specific for RAC1B and PAR2, 25 nM each of RAC1B siRNA + Co siRNA specific for PAR2, or 25 nM each of RAC1B siRNA + PAR2 siRNA. Forty-eight hours after the second transfection, these Panc1-RAC1B-KD cells were subjected to RNA isolation and qPCR analysis for ALK5. Data represent the normalized mean ± SD from three independent experiments and are displayed relative to control siRNA-transfected cells set arbitrarily at 1.0. (<b>B</b>) As in (<b>A</b>) except that RAC1B-knockdown cells were analyzed by immunoblotting for ALK5, RAC1B, and HSP90. The graph below the immunoblot shows results from densitometry-based quantitative analysis of ALK5. Data represent the mean ± SD from three experiments. (<b>C</b>) As in (<b>A</b>), except that the indicated transfectants were treated, or not, with TGF-β1 for 24 h prior to qPCR analysis of ALK5. (<b>D</b>) As in (<b>A</b>), except that cells were cotransfected with ALK5 siRNA instead of PAR2 siRNA and subjected to qPCR analysis of PAR2. (<b>E</b>) Panc1-RAC1B-KO cells (KO), and vector control cells (V) were transfected twice with 50 nM each of either Co siRNA or PAR2 siRNA. Forty-eight hours after the second transfection, the cells were subjected to immunoblot analysis of ALK5 and GAPDH. The asterisks in (<b>A</b>–<b>C</b>,<b>E</b>) indicate significance. The thin vertical lines in the immunoblot images of panels B and E indicate that irrelevant lanes have been removed.</p>
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<p>Effect of PAR2 depletion or ectopic overexpression on TGF-β-regulated chemokinesis in Panc1 cells with RAC1B knockdown or stable overexpression, respectively. (<b>A</b>) Panc1 cells were transfected twice with 25 nM each of Co siRNAs for RAC1B and PAR2, or 25 nM each of RAC1B siRNA and PAR2-specific Co siRNA, or 25 nM RAC1B siRNA + 25 nM PAR2 siRNA. Forty-eight hours after the second round of transfection, cells were assayed for migratory activity on an xCELLigence platform in the presence of TGF-β1. The graph shows a representative experiment. Data are the mean ± SD from 3–4 wells per condition. Differences between Panc1 + RAC1B siRNA + PAR2 siRNA + TGF-β1 (black curve, tracing C) and Panc1 + RAC1B siRNA + TGF-β1 (magenta curve, tracing B) are significant at 01:00 and all later time points. Successful inhibition of RAC1B and PAR2 was verified by immunoblotting and qPCR analysis, respectively. (<b>B</b>) Panc1 cells with ectopic expression of HA-RAC1B (clone 4) were transiently transfected with Myc-DKK-tagged PAR2, or empty vector, and 48 h later subjected to real-time cell migration assay in the presence of TGF-β1. Panc1 cells with stable expression of empty pCGN vector (Panc1-vector) rather than HA-RAC1B were used as control for the migration-inhibitory effect of HA-RAC1B. Shown is a representative experiment (mean ± SD from 3–4 wells per condition). Differences between Panc1-HA-RAC1B + PAR2-Myc-DKK + TGF-β1 (black curve, tracing C) and Panc1-HA-RAC1B + empty vector + TGF-β1 (magenta curve, tracing B) are significant at 04:00 and all later time points. Ectopic expression of HA-RAC1B and PAR2-Myc-DKK were verified in immunoblots using anti-HA and anti-Myc antibodies, respectively (inset).</p>
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<p>Effect of PAR2 knockdown on TGF-β1-induced growth inhibition in Panc1 cells. (<b>A</b>) Panc1 cells were transfected twice with 50 nM of either PAR2 siRNA or Co siRNA. Forty-eight hours after the second round of transfection cells were treated, or not, with TGF-β1 for 24 h, then detached and counted. Data are the mean ± SD of three experiments and are displayed as % reduction in cell numbers of TGF-β1-treated cells relative to numbers of untreated control cells. The asterisk indicates a significant difference. (<b>B</b>) As in (<b>A</b>) except that cells were lysed after TGF-β1 treatment and processed for immunoblotting of ALK5, and β-actin as a loading control.</p>
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<p>Effect of inhibition of MKK-p38 or MEK-ERK signaling on TGF-β1-induced regulation of PAR2 mRNA under conditions of a RAC1B knockdown. Panc1 cells were transfected twice with 50 nM each of either control (Co) siRNA or RAC1B (R1B) siRNA. Twenty-four hours after the second transfection, cells were serum-starved overnight and treated with vehicle (dimethylsulfoxide, 0.1%), SB203580 (10 μM), UO126 (10 μM), or SB431542 (5 μM). Thirty minutes after the addition of inhibitors, cells received TGF-β1 and were incubated for another 48 h in medium with 0.5% fetal bovine serum followed by qPCR analysis of PAR2. Data represent the normalized mean ± SD from three independent experiments and are displayed relative to Co siRNA-transfected, vehicle-treated cells set arbitrarily at 1. The asterisks indicate significant differences.</p>
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<p>Schematic diagram illustrating RAC1B regulation of ALK5 via PAR2. TOP: RAC1B suppresses the abundance of basal and TGF-β1/ALK5-dependent mRNA (right, solid red lines) and protein (right, stippled red line) of PAR2. Reduction in the protein abundance of PAR2 leads to downregulation of ALK5 mRNA and, hence, protein abundance and/or membrane localization (center) and, thereby, of TGF-β1-induced Smad and non-Smad signaling (top left), culminating in reduction in TGF-β-induced PAR2 gene expression (bottom right) as well as cell migration and growth inhibition (bottom left). In addition, RAC1B suppresses autoinduction of <span class="html-italic">TGFBR1</span> by TGF-β1 (top). Stimulatory interactions are indicated by green arrows and inhibitory interactions by red lines. Stippled lines indicate the possibility that RAC1B targets PAR2 not directly but through intermediary proteins of as yet unknown identity, while the dotted arrow indicates that ALK5 autostimulation only occurs under conditions of RAC1B inhibition. The question marks indicate possible effects on protein stability independent of transcriptional events. P, phosphate residue. BOTTOM: Summary of effect: RAC1B attenuates (red lines) upregulation of PAR2 by activated ALK5 (upward green arrow), which in turn affects the upregulation of ALK5 (green line/arrow). The RAC1B inhibitory effect on basal PAR2 expression, which is ALK5-independent (see <a href="#cancers-11-01211-f002" class="html-fig">Figure 2</a>D), has been omitted here for reasons of clarity.</p>
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28 pages, 499 KiB  
Review
The Importance of Gender-Related Anticancer Research on Mitochondrial Regulator Sodium Dichloroacetate in Preclinical Studies In Vivo
by Donatas Stakišaitis, Milda Juknevičienė, Eligija Damanskienė, Angelija Valančiūtė, Ingrida Balnytė and Marta Maria Alonso
Cancers 2019, 11(8), 1210; https://doi.org/10.3390/cancers11081210 - 20 Aug 2019
Cited by 13 | Viewed by 4990
Abstract
Sodium dichloroacetate (DCA) is an investigational medicinal product which has a potential anticancer preparation as a metabolic regulator in cancer cells’ mitochondria. Inhibition of pyruvate dehydrogenase kinases by DCA keeps the pyruvate dehydrogenase complex in the active form, resulting in decreased lactic acid [...] Read more.
Sodium dichloroacetate (DCA) is an investigational medicinal product which has a potential anticancer preparation as a metabolic regulator in cancer cells’ mitochondria. Inhibition of pyruvate dehydrogenase kinases by DCA keeps the pyruvate dehydrogenase complex in the active form, resulting in decreased lactic acid in the tumor microenvironment. This literature review displays the preclinical research data on DCA’s effects on the cell pyruvate dehydrogenase deficiency, pyruvate mitochondrial oxidative phosphorylation, reactive oxygen species generation, and the Na+–K+–2Cl cotransporter expression regulation in relation to gender. It presents DCA pharmacokinetics and the hepatocarcinogenic effect, and the safety data covers the DCA monotherapy efficacy for various human cancer xenografts in vivo in male and female animals. Preclinical cancer researchers report the synergistic effects of DCA combined with different drugs on cancer by reversing resistance to chemotherapy and promoting cell apoptosis. Researchers note that female and male animals differ in the mechanisms of cancerogenesis but often ignore studying DCA’s effects in relation to gender. Preclinical gender-related differences in DCA pharmacology, pharmacological mechanisms, and the elucidation of treatment efficacy in gonad hormone dependency could be relevant for individualized therapy approaches so that gender-related differences in treatment response and safety can be proposed. Full article
(This article belongs to the Special Issue Mitochondria and Cancer)
18 pages, 1014 KiB  
Review
Involvement of Actin in Autophagy and Autophagy-Dependent Multidrug Resistance in Cancer
by Magdalena Izdebska, Wioletta Zielińska, Marta Hałas-Wiśniewska and Alina Grzanka
Cancers 2019, 11(8), 1209; https://doi.org/10.3390/cancers11081209 - 20 Aug 2019
Cited by 17 | Viewed by 5747
Abstract
Currently, autophagy in the context of cancer progression arouses a lot of controversy. It is connected with the possibility of switching the nature of this process from cytotoxic to cytoprotective and vice versa depending on the treatment. At the same time, autophagy of [...] Read more.
Currently, autophagy in the context of cancer progression arouses a lot of controversy. It is connected with the possibility of switching the nature of this process from cytotoxic to cytoprotective and vice versa depending on the treatment. At the same time, autophagy of cytoprotective character may be one of the factors determining multidrug resistance, as intensification of the process is observed in patients with poorer prognosis. The exact mechanism of this relationship is not yet fully understood; however, it is suggested that one of the elements of the puzzle may be a cytoskeleton. In the latest literature reports, more and more attention is paid to the involvement of actin in the autophagy. The role of this protein is linked to the formation of autophagosomes, which are necessary element of the process. However, based on the proven effectiveness of manipulation of the actin pool, it seems to be an attractive alternative in breaking autophagy-dependent multidrug resistance in cancer. Full article
(This article belongs to the Special Issue The Role of Autophagy in Cancer Progression and Drug Resistance)
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<p>The course of the autophagy along with the main proteins involved in the process. The description of the figure can be found in the text above.</p>
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<p>The participation of actin in autophagy process. The description of the figure can be found in the text above.</p>
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16 pages, 1048 KiB  
Review
Antioxidant Defenses: A Context-Specific Vulnerability of Cancer Cells
by Jordan A. Cockfield and Zachary T. Schafer
Cancers 2019, 11(8), 1208; https://doi.org/10.3390/cancers11081208 - 20 Aug 2019
Cited by 38 | Viewed by 7644
Abstract
Reactive oxygen species (ROS) are well known for their capacity to cause DNA damage, augment mutagenesis, and thereby promote oncogenic transformation. Similarly, agents that reduce ROS levels (antioxidants) are frequently thought to have anti-cancer properties given their propensity to minimize DNA damage and [...] Read more.
Reactive oxygen species (ROS) are well known for their capacity to cause DNA damage, augment mutagenesis, and thereby promote oncogenic transformation. Similarly, agents that reduce ROS levels (antioxidants) are frequently thought to have anti-cancer properties given their propensity to minimize DNA damage and mutagenesis. However, numerous clinical studies focused on antioxidants suggest that this is a facile premise and that antioxidant capacity can be important for cancer cells in a similar fashion to normal cells. As a consequence of this realization, numerous laboratories have been motivated to investigate the biological underpinnings explaining how and when antioxidant activity can potentially be beneficial to cancer cells. Relatedly, it has become clear that the reliance of cancer cells on antioxidant activity in certain contexts represents a potential vulnerability that could be exploited for therapeutic gain. Here, we review some of the recent, exciting findings documenting how cancer cells utilized antioxidant activity and under what circumstances this activity could represent an opportunity for selective elimination of cancer cells. Full article
(This article belongs to the Special Issue Metabolic Reprogramming and Vulnerabilities in Cancer)
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<p>The impacts on tumor progression by oxidative stress regulation by nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and exogenous antioxidants. Under normal conditions, Nrf2 is bound in a complex with kelch-like ECH associated protein 1 (Keap1) (red curves) and cullin-3 (Cul-3) containing E3 ubiquitin ligase (green oval) where it is targeted for proteasomal degradation. Nonetheless, upon increases in stimuli that promote Nrf2 stabilization (e.g., oxidative stress, oncogenic signaling, mutations in Nrf2 or Keap1 prohibiting their binding, or interference of the Nrf2–Keap1 interaction by iASPP), Nrf2 accumulates in the cytosol and translocates to the nucleus wherein it binds to the antioxidant response element (ARE) and initiates an antioxidant program. Among the genes transcribed by Nrf2 is the cystine/glutamate antiporter <span class="html-italic">SCL7A11</span>. With its upregulation due to Nrf2, extracellular cystine uptake increases, and when combined with glycine (orange sphere) and glutamate (red sphere), the antioxidant glutathione is synthesized and contributes to decreasing reactive oxygen species (ROS) leading to tumor progression and drug resistance. Exogenous antioxidants likewise promote tumor progression by decreasing intracellular ROS levels. When oxidative stress is reduced, Nrf2 promotes the transcription of heme oxygenase which catabolizes free heme and stabilizes basic leucine zipper transcription factor 1 (BACH1). Thereafter, BACH1 induces glycolysis-dependent metastasis via the transcription of <span class="html-italic">Hexokinase 2</span> and <span class="html-italic">Gapdh</span>. Lastly, in the case of the antioxidant vitamin C, which enters cells via the glucose transporter GLUT1, diminishing glutathione levels were observed to occur leading to subsequent increase in energetic crisis and cell death.</p>
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<p>Mitigation and tolerance of ROS within extracellular matrix (ECM)-detached cancer cells Detachment from the extracellular matrix is a known stimulus for oxidative stress accumulation. Since continued ROS accumulation is understood to induce apoptosis in cells, cancer cells must employ mechanisms to address this obstacle and proceed in growth. Among these mechanisms lies two modes of addressing ROS during ECM-detachment: mitigation or tolerance. Through investigations of ECM-detachment-induced cell death, receptor-interacting protein kinase 1 (RIPK1) was surprisingly found to regulate mitophagy by promoting PINK1 stabilization and resulting in an increase in mitochondrial ROS (mitoROS). Upon inhibition or absence of RIPK1, PINK1 is susceptible to cleavage and mitoROS decreases through the reducing effects of IDH2 to generate NADPH. Additionally, increases in the Ca<sup>2+</sup> transporter TRPA1 have been observed in a variety of cancers. During ECM-detachment, TRPA1 was observed to promote the increase in Ca<sup>2+</sup> influx into cells which lead to increases in the levels of antiapoptotic Mcl1 and ultimately resulted in evasion of apoptosis despite the presence of high ROS levels. Thus, tumor cells are enabled to be tolerant of oxidative stress during ECM-detachment by the aid of TRPA1.</p>
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<p>Glucose metabolism is highly crucial for cancer cells. This six-carbon monosaccharide can not only be used to fuel the energetic needs of cancer cells, but also to address the oxidative stress that accumulates as a byproduct of proliferation, metabolism, and growth. One mechanism employed to address oxidative stress is the use of the serine/threonine-protein phosphatase 2A (PP2A). During glycolysis, 6-phosphofructo-2-kinase (PFK-2) drives the commitment of glucose flux through the glycolytic pathway by influencing the phosphorylation of fructose-6-phosphate (F6P) and converting it into fructose-1,6,-bisphosphate (FBP). PP2A can reverse the flux of glucose to flow into the pentose phosphate pathway (PPP) by de-phosphorylating PFK-2. When this occurs, an increase of NADPH is generated and subsequent decreases in ROS is observed. The mechanism in this pathway is denoted by solid, black arrows, while the consequences of de-phosphorylated PFK-2 are marked by dashed, black arrows. Along the same note of rewired metabolism to address ROS accumulation, constitutive activation of casein kinase 2 (CK2) has been observed in many cancers, yet its mechanism was poorly understood. Recently, it was found that CK2 activity is dependent upon glucose metabolism, and from glycolysis, CK2 signaling relies on the metabolic enzyme lactase dehydrogenase (LDHA) to promote cancer cell migration and invasion. The CK2/LDHA signaling pathway is marked by solid, red arrows and the results of this signaling is marked by dashed, red arrows. Intriguingly, although LDHA promotes the conversion of pyruvate into lactate at the end of glycolysis in the cytosol, oxidative stress induces its translocation to the nucleus wherein it promotes the conversion of α-ketobutyrate (α-KB) into α-hydroxybutyrate (α-HB) which promotes a complex formation with LDHA and disruptor of telomeric silencing 1-like (DOTL1) to drive a Nrf2-dependent transcription of antioxidant genes as well as Wnt target genes. The consequence of these upregulated genes is diminished ROS levels and promotion of tumor growth. The ROS-induced LDHA translocation pathway is marked by solid, green arrows, while the consequences are indicated with dashed, green arrows. Other Abbreviations: glucose-6-phosphate (G6P); hexokinase (HK); glucose-6-phosphate dehydrogenase (G6PDH); 6-phosphogluconolactone (6PG); 6-phosphogluconate (6P-Gluconate); ribose-5-phosphate (R5P).</p>
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23 pages, 2521 KiB  
Review
Tumor Cell Dormancy: Threat or Opportunity in the Fight against Cancer
by Rana Jahanban-Esfahlan, Khaled Seidi, Masoud H. Manjili, Ali Jahanban-Esfahlan, Tahereh Javaheri and Peyman Zare
Cancers 2019, 11(8), 1207; https://doi.org/10.3390/cancers11081207 - 19 Aug 2019
Cited by 81 | Viewed by 8308
Abstract
Tumor dormancy, a clinically undetectable state of cancer, makes a major contribution to the development of multidrug resistance (MDR), minimum residual disease (MRD), tumor outgrowth, cancer relapse, and metastasis. Despite its high incidence, the whole picture of dormancy-regulated molecular programs is far from [...] Read more.
Tumor dormancy, a clinically undetectable state of cancer, makes a major contribution to the development of multidrug resistance (MDR), minimum residual disease (MRD), tumor outgrowth, cancer relapse, and metastasis. Despite its high incidence, the whole picture of dormancy-regulated molecular programs is far from clear. That is, it is unknown when and which dormant cells will resume proliferation causing late relapse, and which will remain asymptomatic and harmless to their hosts. Thus, identification of dormancy-related culprits and understanding their roles can help predict cancer prognosis and may increase the probability of timely therapeutic intervention for the desired outcome. Here, we provide a comprehensive review of the dormancy-dictated molecular mechanisms, including angiogenic switch, immune escape, cancer stem cells, extracellular matrix (ECM) remodeling, metabolic reprogramming, miRNAs, epigenetic modifications, and stress-induced p38 signaling pathways. Further, we analyze the possibility of leveraging these dormancy-related molecular cues to outmaneuver cancer and discuss the implications of such approaches in cancer treatment. Full article
(This article belongs to the Special Issue Circulating Tumor Cells (CTCs))
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<p>The implication of the immune system in tumor cell dormancy.</p>
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<p>The implication of ECM and p38 signaling in tumor dormancy.</p>
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<p>Tumor dormancy as a therapeutic opportunity to fight cancer back.</p>
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11 pages, 2514 KiB  
Article
Rapid Depletions of Subcutaneous Fat Mass and Skeletal Muscle Mass Predict Worse Survival in Patients with Hepatocellular Carcinoma Treated with Sorafenib
by Kenji Imai, Koji Takai, Takao Miwa, Daisuke Taguchi, Tatsunori Hanai, Atsushi Suetsugu, Makoto Shiraki and Masahito Shimizu
Cancers 2019, 11(8), 1206; https://doi.org/10.3390/cancers11081206 - 19 Aug 2019
Cited by 45 | Viewed by 3884
Abstract
The aim of this study was to investigate whether rapid depletions of fat mass and skeletal muscle mass predict mortality in hepatocellular carcinoma (HCC) patients treated with sorafenib. This retrospective study evaluated 61 HCC patients. The cross-sectional areas of visceral and subcutaneous fat [...] Read more.
The aim of this study was to investigate whether rapid depletions of fat mass and skeletal muscle mass predict mortality in hepatocellular carcinoma (HCC) patients treated with sorafenib. This retrospective study evaluated 61 HCC patients. The cross-sectional areas of visceral and subcutaneous fat mass and skeletal muscle mass were measured by computed tomography, from which the visceral fat mass index (VFMI), subcutaneous fat mass index (SFMI), and skeletal muscle index (L3SMI) were obtained. The relative changes in these indices per 120 days (ΔVFMI, ΔSFMI, and ΔL3SMI) before and after sorafenib treatment were calculated in each patient. Patients within the 20th percentile cutoffs for these indices were classified into the rapid depletion (RD) group. Kaplan–Meier analysis revealed that with respect to ΔL3SMI (p = 0.0101) and ΔSFMI (p = 0.0027), the RD group had a significantly poorer survival. Multivariate analysis using the Cox proportional-hazards model also demonstrated that ΔL3SMI (≤−5.73 vs. >−5.73; hazard ratio [HR]: 4.010, 95% confidence interval [CI]: 1.799–8.938, p = < 0.001) and ΔSFMI (≤−5.33 vs. >−5.33; HR: 4.109, 95% CI: 1.967–8.584, p = < 0.001) were independent predictors. Rapid depletions of subcutaneous fat mass and skeletal muscle mass after the introduction of sorafenib indicate a poor prognosis. Full article
(This article belongs to the Special Issue Cancer Cachexia)
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<p>Kaplan–Meier curves for overall survival time divided into the presence or absence of sarcopenia (<b>a</b>) and rapid depletion (RD) or non-rapid depletion (non-RD) groups in ΔL3SMI (≤−5.73 and &gt;−5.73 cm<sup>2</sup>/m<sup>2</sup>/120 days) (<b>b</b>), ΔSFMI (≤−5.33 and &gt;−5.33 cm<sup>2</sup>/m<sup>2</sup>/120days) (<b>c</b>), and ΔVFMI (≤−3.95 and &gt;−3.95 cm<sup>2</sup>/m<sup>2</sup>/120 days) (<b>d</b>).</p>
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<p>Kaplan–Meier curves for overall survival time divided into four groups according to the cutoffs for ΔL3SMI (−5.73 cm<sup>2</sup>/m<sup>2</sup>/120 days) and ΔSFMI (−5.33 cm<sup>2</sup>/m<sup>2</sup>/120 days).</p>
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<p>Patient flow in this study.</p>
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<p>Outline and formula for ΔL3SMI, ΔSFMI, and ΔVFMI. L3 skeletal muscle index (L3SMI) was the cross-sectional area of the muscle (cm<sup>2</sup>) at the L3 level of the computed tomography (CT) image normalized by the square of the height (m<sup>2</sup>). Subcutaneous fat mass index (SFMI) and visceral fat mass index (VFMI) were the cross-sectional areas of the subcutaneous and visceral fat (cm<sup>2</sup>), respectively, at the umbilical point normalized by the square of the height (m<sup>2</sup>).</p>
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21 pages, 1318 KiB  
Article
The Complex Interaction between the Tumor Micro-Environment and Immune Checkpoints in Breast Cancer
by Vanessa Barriga, Nyanbol Kuol, Kulmira Nurgali and Vasso Apostolopoulos
Cancers 2019, 11(8), 1205; https://doi.org/10.3390/cancers11081205 - 19 Aug 2019
Cited by 64 | Viewed by 14207
Abstract
The progression of breast cancer and its association with clinical outcome and treatment remain largely unexplored. Accumulating data has highlighted the interaction between cells of the immune system and the tumor microenvironment in cancer progression, and although studies have identified multiple facets of [...] Read more.
The progression of breast cancer and its association with clinical outcome and treatment remain largely unexplored. Accumulating data has highlighted the interaction between cells of the immune system and the tumor microenvironment in cancer progression, and although studies have identified multiple facets of cancer progression within the development of the tumor microenvironment (TME) and its constituents, there is lack of research into the associations between breast cancer subtype and staging. Current literature has provided insight into the cells and pathways associated with breast cancer progression through expression analysis. However, there is lack of co-expression studies between immune pathways and cells of the TME that form pro-tumorigenic relationships contributing to immune-evasion. We focus on the immune checkpoint and TME elements that influence cancer progression, particularly studies in molecular subtypes of breast cancer. Full article
(This article belongs to the Special Issue Cancer Vaccines: Research and Applications)
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<p>Tumor-associated immune cells in the tumor microenvironment (TME) of breast cancer models. Within the TME there is an array of resident cells contributing to the progression and metastasis of breast cancer cells. The different residents and their associated secretory elements including stimulatory growth factors, chemokines and cytokines are shown. The expression of these residents within the TME of breast cancer patients may aid in discovering new markers associated with specific subtype leading to earlier diagnosis and better clinical outcome. [ARG1. Arginase 1; CAF. Cancer-associated Fibroblast; CD163. Macrophage scavenger receptor; CCL2; Chemokine Ligand 2; CXCL8-CXCR1/2. Chemokine Ligand 8-Chemokine Receptor 1 &amp; 2; CXCL12. Chemokine Ligand 12; COX-2. cyclooxygenase-2; EMT. Epithelial-Mesenchymal Transition; ER− Estrogen Receptor Negative; FGF. Fibroblast Growth Factor; GM-CSF. Granulocyte-Macrophage Colony-Stimulating Factor; HGF. Hepatocyte Growth Factor; IDO. Idoleamine-2, 3-Dioxygenase; IL. Interleukin; iDC. immature Dendritic Cells; mDC. mature Dendritic Cells; MDSCs. Myeloid-derived suppressor cells; M2 TAMS. M2 subtype Tumor-associated Macrophage; NK CELL. Natural Killer; N2 TAN. N2 Subtype Tumor-associated Neutrophil; PDGF-R. Platelet-Derived Growth Factor Receptor; PD-1. Programmed cell death protein 1; PGE<sub>2</sub>. Prostaglandin E2; ROS. Reactive Oxygen Species; SDF-1. Stromal cell-derived factor-1; TGF-β. Transforming Growth Factor-beta; Th Cells. T-helper cells; TILs. Tumor-Infiltrating Lymphocytes; TREG. T-regulatory cells; VEGF. Vascular endothelial growth factor].</p>
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<p>Immune checkpoints of immunosuppressive actions associated with breast cancer. Immune checkpoints of inhibitory pathways are fundamental in the immune system to maintain self-tolerance and modulate immune responses. In breast cancer, some of these immune checkpoints and immunosuppressive factors have been associated with subtype specificity through their expressions on breast cancer cells. The different immune cells and their ligand-receptor interactions and secreted stimulatory growth factors, chemokines and cytokines are shown. The expression of these immune markers in the TME in breast cancer may or not be subtype-specific but are important in circumventing immune recognition or to immobilize effector T cells. Thus, the expression of these ligands and receptors may be associated with breast cancer stage and clinical outcome. (AKT. serine/threonine kinase or protein kinase B; ARG1. Arginase 1; Bcl-xL. B-cell lymphoma-xtra large; CAFs. Cancer-associated fibroblasts; CTLA4. cytotoxic T-lymphocyte-associated protein 4; ER− Estrogen Receptor Negative; GM-CSF. Granulocyte-Macrophage Colony-Stimulating Factor; HER2+. Human Epidermal Growth Factor Receptor 2; IDO. Indoleamine-2,3-dioxygenase; IFN-γ. interferon gamma; IFN-γR. interferon gamma receptor; IL. Interleukin; JAK. Janus kinase; iNOS. Inducible nitric oxide synthase; MDSC. Myeloid-derived suppressor cells; MHC. major histocompatibility complex; MMP. Matrix Metalloproteinases; mTOR. Mammalian target of rapamycin; MUC. Mucins; NK. natural killer; NF-κB. nuclear factor-κB; NLR. Neutrophil-lymphocyte ratio; NO. Nitric Oxide; PI3K. PI3K.phosphoinositide 3-kinase; PD-1. programmed death-1; PD-L1. Programmed death-ligand1; PGE<sub>2</sub>. Prostaglandin E2; PR−. Progesterone Receptor Negative; ROS. Reactive Oxygen Species; SHP. Src homology protein-tyrosine phosphatase; Siglec 9. Sialic acid-binding lectins 9; STAT. Signal transducer and activator of transcription; TAMs. Tumor-associated macrophages; TANs. Tumor-associated neutrophils; TGF-β. Transforming Growth Factor-beta; TIL. Tumor-Infiltrating Lymphocytes; TNBC. Triple Negative Breast Cancer; Treg. regulatory T cell; TCR. T cell receptor; VEGF. Vascular endothelial growth factor).</p>
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20 pages, 6994 KiB  
Article
Maintenance BEZ235 Treatment Prolongs the Therapeutic Effect of the Combination of BEZ235 and Radiotherapy for Colorectal Cancer
by Yu-Hsuan Chen, Chun-Wei Wang, Ming-Feng Wei, Yi-Shin Tzeng, Keng-Hsueh Lan, Ann-Lii Cheng and Sung-Hsin Kuo
Cancers 2019, 11(8), 1204; https://doi.org/10.3390/cancers11081204 - 19 Aug 2019
Cited by 11 | Viewed by 3707
Abstract
Our previous study demonstrated that administration of NVP-BEZ235 (BEZ235), a dual PI3K/mTOR inhibitor, before radiotherapy (RT) enhanced the radiotherapeutic effect in colorectal cancer (CRC) cells both in vitro and in vivo. Here, we evaluated whether maintenance BEZ235 treatment, after combinatorial BEZ235 + RT [...] Read more.
Our previous study demonstrated that administration of NVP-BEZ235 (BEZ235), a dual PI3K/mTOR inhibitor, before radiotherapy (RT) enhanced the radiotherapeutic effect in colorectal cancer (CRC) cells both in vitro and in vivo. Here, we evaluated whether maintenance BEZ235 treatment, after combinatorial BEZ235 + RT therapy, prolonged the antitumor effect in CRC. K-RAS mutant CRC cells (HCT116 and SW480), wild-type CRC cells (HT29), and HCT116 xenograft tumors were separated into the following six study groups: (1) untreated (control); (2) RT alone; (3) BEZ235 alone; (4) RT + BEZ235; (5) maintenance BEZ235 following RT + BEZ235 (RT + BEZ235 + mBEZ235); and (6) maintenance BEZ235 following BEZ235 (BEZ235 + mBEZ235). RT + BEZ235 + mBEZ235 treatment significantly inhibited cell viability and increased apoptosis in three CRC cell lines compared to the other five treatments in vitro. In the HCT116 xenograft tumor model, RT + BEZ235 + mBEZ235 treatment significantly reduced the tumor size when compared to the other five treatments. Furthermore, the expression of mTOR signaling molecules (p-rpS6 and p-eIF4E), DNA double-strand break (DSB) repair-related molecules (p-ATM and p-DNA-PKcs), and angiogenesis-related molecules (VEGF-A and HIF-1α) was significantly downregulated after RT + BEZ235 + mBEZ235 treatment both in vitro and in vivo when compared to the RT + BEZ235, RT, BEZ235, BEZ235 + mBEZ235, and control treatments. Cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), 53BP1, and γ-H2AX expression in the HCT116 xenograft tissue and three CRC cell lines were significantly upregulated after RT + BEZ235 + mBEZ235 treatment. Maintenance BEZ235 treatment in CRC cells prolonged the inhibition of cell viability, enhancement of apoptosis, attenuation of mTOR signaling, impairment of the DNA-DSB repair mechanism, and downregulation of angiogenesis that occurred due to concurrent BEZ235 and RT treatment. Full article
(This article belongs to the Special Issue Colorectal Cancers)
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<p>Schematic diagram of the therapeutic protocol. (<b>A</b>) In vitro experiments. CRC cells were separated into six groups: control group (untreated), NVP-BEZ235 alone group (BEZ alone), radiotherapy (RT) alone group (RT alone), RT + BEZ235 group (RT + BEZ), RT + BEZ235 + maintenance BEZ235 group (RT + BEZ + mBEZ), and BEZ235 + maintenance BEZ235 (BEZ + mBEZ). (<b>B</b>) In vivo experiments. Immunocompromised mice were subcutaneously injected with 1 × 10<sup>6</sup> HCT116 cancer cells and then divided into six groups.</p>
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<p>BEZ235 maintenance treatment following RT + BEZ235 treatment resulted in significantly less cell survival and more apoptosis markers in CRC cells. (<b>A</b>) In three CRC cell lines (HCT116, HT29, and SW480), a colony formation study (1000 cells per well) showed that the rate of cell survival significantly decreased after RT + BEZ235 + mBEZ235 treatment. In addition, RT + BEZ235 + mBEZ235 treatment exhibited a RT dose-dependent reduction in clonogenic survival fraction. The results are expressed as the mean ± standard error (SE) of three experiments. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. (<b>B</b>) Quantification of apoptosis via annexin V-propidium iodide staining of the HCT116, HT29, and SW480 cells after various treatments. Completely apoptotic cells were more prominently found after RT + BEZ235 + mBEZ235 treatment. (<b>C</b>) Western blotting showed that BEZ235 maintenance treatment substantially enhanced the level of apoptosis (cleaved caspase-3 [CASP 3] and cleaved PARP) induced by radiation. PARP, CASP 3, and β-actin served as the controls. The band intensities were analyzed by ImageJ software. The relative ratios of the cleaved protein to non-cleaved protein amounts were quantified and indicated underneath each gel. The relative ratio of the control group is arbitrarily presented as 1.</p>
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<p>BEZ235 maintenance treatment following RT + BEZ235 treatment sensitized CRC cells to radiation by attenuating mTOR signaling- and angiogenesis-associated molecules. (<b>A</b>) BEZ235 maintenance treatment caused a marked decrease in radiation-induced phosphorylation levels of rpS6 and eIF4E in the CRC cells. The relative amounts of phosphorylated and non-phosphorylated proteins were quantified. The relative ratio of the control group was defined as 1. (<b>B</b>) VEGF-A concentrations were analyzed via enzyme-linked immunosorbent assay (ELISA). The results are expressed as the mean ± standard error (SE) of three experiments. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. (<b>C</b>) BEZ235 maintenance treatment significantly inhibited HIF-1α expression when compared with RT and RT + BEZ235 treatment in three CRC cells. The relative amounts of phosphorylated and non-phosphorylated proteins were quantified. The relative ratio of the control group was defined as 1.</p>
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<p>BEZ235 maintenance treatment following RT + BEZ235 treatment increased the susceptibility of CRC cells to radiation by inhibiting DNA repair mechanisms and increasing the effects of DNA damage. (<b>A</b>) BEZ235 maintenance treatment following RT + BEZ235 treatment significantly inhibited p-ATM and p-DNA-PKcs in three CRC cell lines compared to the BEZ235, RT, RT + BEZ235, and control treatments. The relative amounts of phosphorylated and non-phosphorylated proteins were quantified. The relative ratio of the control group was defined as 1. (<b>B</b>) RT + BEZ235 treatment followed by BEZ235 maintenance treatment significantly increased γ-H2AX and 53BP1 expression in three CRC cell lines compared to the BEZ235, RT, RT + BEZ235, and control treatments. (<b>C</b>) Cells were treated as described in <a href="#cancers-11-01204-f001" class="html-fig">Figure 1</a>A and labeled with anti-53BP1 primary antibody and DyLight 488-conjugated secondary antibody on days 1, 4, and 7 of the treatment period. The 53BP1 foci were observed using immunofluorescence with a fluorescence microscope. Nuclei were counterstained with DAPI. Immunofluorescence analysis (left panel) accompanied by quantification analysis (right panel) showed that BEZ235 maintenance treatment showed prolonged DNA damage (more 53BP1 foci) in three CRC cell lines until day 7 when compared to the other treatment groups. The number of 53BP1 foci was counted using 20 cells for each treatment. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. Scale bar: 10 μm. (<b>D</b>) Cells were treated as described in <a href="#cancers-11-01204-f001" class="html-fig">Figure 1</a>A and labeled with the anti-γ-H2AX primary antibody and DyLight 488-conjugated secondary antibody. The amount of γ-H2AX was maintained at a consistent level after BEZ235 maintenance treatment until day 7 when compared to the RT + BEZ235, RT, BEZ235, BEZ235 + mBEZ235, and control treatments in all three CRC cells. The amount of γ-H2AX was counted using 20 cells for each condition. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. Scale bar: 10 μm.</p>
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<p>BEZ235 maintenance treatment following RT + BEZ235 treatment significantly inhibited CRC xenograft tumor growth. (<b>A</b>) Maintenance BEZ235 following RT + BEZ235 treatment significantly suppressed xenograft tumor growth when compared with other treatment groups, especially RT + BEZ235 treatment without BEZ235 maintenance. The results of the tumor volume are expressed for <span class="html-italic">n</span> = 6 in each treatment group. *** <span class="html-italic">p</span> &lt; 0.001 compared with the control group; *** <span class="html-italic">p</span> &lt; 0.001 compared with the RT + BEZ235 group. (<b>B</b>) The body weight of mice in the five groups did not differ, and no marked changes were observed during the treatment period. (<b>C</b>) Representative pictures of each treatment group. Mice were photographed during the seventh week after treatment. Scale bar: 10 mm.</p>
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<p>Expression of the apoptosis-, mTOR signaling pathway-, DNA-DSB repair-, DNA damage-, and angiogenesis-related molecules in CRC xenograft tissue following each treatment by immunohistochemistry. (<b>A</b>) BEZ235 maintenance treatment following RT + BEZ235 treatment (RT + BEZ235 + mBEZ235) significantly increased cleaved caspase-3 (CASP 3) expression when compared with other four treatments on day 43. (<b>B</b>) p-rpS6 and p-eIF4E expression after RT + BEZ235 + mBEZ235 treatment was downregulated when compared with those after RT + BEZ235 or control treatment on day 43. (<b>C</b>) RT + BEZ235 + mBEZ235 treatment significantly downregulated p-ATM and p-DNA-PKcs expression when compared with RT + BEZ235 and control treatment on day 43, whereas 53BP1 expression was significantly increased after RT + BEZ235 + mBEZ235 treatment. (<b>D</b>) RT + BEZ235 + mBEZ235 treatment significantly downregulated VEGF-A and HIF-1α when compared with other treatments on day 43. Scale bar = 100 μm.</p>
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<p>Maintenance BEZ235 treatment after BEZ235 combined with radiation treatment reduced cell viability and inhibited tumor growth in a colorectal cancer cell model. (<b>A</b>) Although radiotherapy (RT) inhibited tumor growth of colorectal cancer (CRC) cells, RT also upregulated the expression of AKT/mTOR signaling molecules (p-AKT, p-eIF4E, and p-rpS6) and DNA double-strand break (DSB) repair-related molecules (p-ATM and p-DNA-PKcs). (<b>B</b>) Maintenance BEZ235 treatment prolonged the therapeutic effect of concurrent BEZ235 + RT treatment by attenuating mTOR signaling activation, impairing the DNA-DSB repair mechanism, inhibiting angiogenesis (VEGF-A and HIF-1α), and enhancing apoptosis via cleaved caspase-3, cleaved PARP, and radiation-induced DNA damage (γ-H2AX and 53BP1).</p>
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16 pages, 2022 KiB  
Article
Baseline Genomic Features in BRAFV600-Mutated Metastatic Melanoma Patients Treated with BRAF Inhibitor + MEK Inhibitor in Routine Care
by Baptiste Louveau, Fanelie Jouenne, Coralie Reger de Moura, Aurelie Sadoux, Barouyr Baroudjian, Julie Delyon, Florian Herms, Adele De Masson, Laetitia Da Meda, Maxime Battistella, Nicolas Dumaz, Celeste Lebbe and Samia Mourah
Cancers 2019, 11(8), 1203; https://doi.org/10.3390/cancers11081203 - 18 Aug 2019
Cited by 9 | Viewed by 3636
Abstract
In BRAFV600mut metastatic melanoma, the combination of BRAF and MEK inhibitors (BRAFi, MEKi) has undergone multiple resistance mechanisms, limiting its clinical benefit and resulting in the need for response predicting biomarkers. Based on phase III clinical trial data, several studies have previously [...] Read more.
In BRAFV600mut metastatic melanoma, the combination of BRAF and MEK inhibitors (BRAFi, MEKi) has undergone multiple resistance mechanisms, limiting its clinical benefit and resulting in the need for response predicting biomarkers. Based on phase III clinical trial data, several studies have previously explored baseline genomic features associated with response to BRAFi + MEKi. Using a targeted approach that combines the examination of mRNA expression and DNA alterations in a subset of genes, we performed an analysis of baseline genomic alterations involved in MAPK inhibitors’ resistance in a real-life cohort of BRAFV600mut metastatic melanoma patients. Twenty-seven patients were included in this retrospective study, and tumor samples were analyzed when the BRAFi + MEKi therapy was initiated. The clinical characteristics of our cohort were consistent with previously published studies. The BRAFi + MEKi treatment was initiated in seven patients as a following-line treatment, and had a specific transcriptomic profile exhibiting 14 genes with lower mRNA expression. However, DNA alterations in CCND1, RB1, and MET were only observed in patients who received BRAFi + MEKi as the first-line treatment. Furthermore, KIT mRNA expression was significantly higher in patients showing clinical benefit from the combined therapy, emphasizing the tumor-suppressor role of KIT already described within the context of BRAF-mutant melanoma. Full article
(This article belongs to the Special Issue Oncogenic Forms of BRAF as Cancer Driver Genes)
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Figure 1

Figure 1
<p>Clinical course of the 27 included patients. Events occurring during the BRAFi + MEKi treatment are shown. White bars indicate the absence of BRAFi + MEKi therapy. Patients were censored at the last available date of follow-up if death did not occur.</p>
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<p>Kaplan–Meier survival plot for progression-free survival of the 27 included patients.</p>
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<p>Landscape of baseline DNA alterations (mutations and copy number variations) for the 24 patients with available data. Amplifications and deletions were defined as CNV &gt; 5 and CNV &lt; 0.5, respectively. Patients are ranked according to the total number of alterations detected.</p>
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<p>Principal component analysis of mRNA expression for the 25 patients with available data (∆Ct, normalization on <span class="html-italic">B2M</span>). Patients from Cluster 1 were more likely to have been treated with BRAFi + MEKi as a first-line treatment.</p>
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<p>Differential gene expression analysis according to clinical course. (<b>a</b>) Differential gene expression in patients with no progression free survival (PFS) event vs. patients with PFS event (<span class="html-italic">n</span> = 25). (<b>b</b>) Differential gene expression in patients with no PFS event vs. PFS event (only patients with no brain metastases, <span class="html-italic">n</span> = 20). Differences in gene expression are expressed as fold change (2^-∆∆Ct), and genes are ranked by their level of significance. ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p><b>Differential gene expression analysis according to line of treatment</b> (<b>a</b>) Differential gene expression in patients with BRAFi + MEKi as the first-line vs. patients treated with BRAFi + MEKi as the following-line of treatment. Differences in gene expression are expressed as fold change (2^-∆∆Ct), and genes are ranked by their level of significance. ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05. (<b>b</b>) Heatmap of mRNA expression. Colors represent the relative expression of a gene in each sample centered on the mean and scaled to the standard deviation (red: high and blue: low).</p>
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11 pages, 455 KiB  
Article
Soluble HLA in the Aqueous Humour of Uveal Melanoma Is Associated with Unfavourable Tumour Characteristics
by Annemijn P. A. Wierenga, Gülçin Gezgin, Els van Beelen, Michael Eikmans, Marijke Spruyt-Gerritse, Niels J. Brouwer, Mieke Versluis, Robert M. Verdijk, Sjoerd G. van Duinen, Marina Marinkovic, Gregorius P. M. Luyten and Martine J. Jager
Cancers 2019, 11(8), 1202; https://doi.org/10.3390/cancers11081202 - 18 Aug 2019
Cited by 19 | Viewed by 3735
Abstract
A high HLA expression in uveal melanoma (UM) is part of the prognostically unfavorable inflammatory phenotype. We wondered whether the presence of soluble HLA (sHLA) in the aqueous humour is associated with clinical, histopathological or genetic tumour characteristics, and represents tumour HLA expression [...] Read more.
A high HLA expression in uveal melanoma (UM) is part of the prognostically unfavorable inflammatory phenotype. We wondered whether the presence of soluble HLA (sHLA) in the aqueous humour is associated with clinical, histopathological or genetic tumour characteristics, and represents tumour HLA expression and intratumoural inflammation. Aqueous humour from 108 UM patients was analysed for the presence of sHLA, using a Luminex assay specific for HLA Class I. Clinical and genetic parameters were compared between sHLA-positive and negative eyes. A qPCR analysis was performed on tumour tissue using a Fluidigm assay. In 19/108 UM-containing eyes, the sHLA level in the aqueous was above the detection limit. Tumours in sHLA-positive eyes were significantly larger, more frequently involved the ciliary body, and more often showed monosomy 3, gain of chromosome 8q and loss of BAP1 staining. Melanoma-related survival was worse in patients with sHLA-positive aqueous humour. sHLA in the aqueous did not represent the tumour’s HLA expression and did not relate to immune cell infiltration in the tumour. We conclude that UM-containing eyes may contain sHLA in the aqueous humour, where it is a prognostically-unfavourable sign and may influence local immune responses. Full article
(This article belongs to the Special Issue Uveal Melanoma)
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<p>Kaplan–Meier survival curve showing melanoma-related survival, since enucleation, based on the sHLA expression in the aqueous humour of 108 UM patients. Curves represent the negative and positive sHLA groups. Both groups differ significantly in survival (Log Rank test <span class="html-italic">p</span> = 0.025).</p>
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20 pages, 4216 KiB  
Article
Progesterone Receptor B signaling Reduces Breast Cancer Cell Aggressiveness: Role of Cyclin-D1/Cdk4 Mediating Paxillin Phosphorylation
by Francesca Ida Montalto, Francesca Giordano, Chiara Chiodo, Stefania Marsico, Loredana Mauro, Diego Sisci, Saveria Aquila, Marilena Lanzino, Maria Luisa Panno, Sebastiano Andò and Francesca De Amicis
Cancers 2019, 11(8), 1201; https://doi.org/10.3390/cancers11081201 - 17 Aug 2019
Cited by 19 | Viewed by 4907
Abstract
Progesterone-Receptor (PR) positivity is related with an enhanced response to breast cancer therapy, conversely cyclin D1 (CD1) is a retained marker of poor outcome. Herein, we demonstrate that hydroxyprogesterone (OHPg) through progesterone receptor B (PR-B) reduces breast cancer cell aggressiveness, by targeting the [...] Read more.
Progesterone-Receptor (PR) positivity is related with an enhanced response to breast cancer therapy, conversely cyclin D1 (CD1) is a retained marker of poor outcome. Herein, we demonstrate that hydroxyprogesterone (OHPg) through progesterone receptor B (PR-B) reduces breast cancer cell aggressiveness, by targeting the cytoplasmic CD1. Specifically, OHPg diminishes CD1 expression by a transcriptional regulation due to the recruitment of PR-B at a canonical half-PRE site of the CD1 promoter, together with HDAC1, determining a chromatin conformation less prone for gene transcription. CD1, together with its kinase partner Cdk4, regulates cell migration and metastasis, through the association with key components of focal adhesion, such as Paxillin (Pxn). Kaplan-Meier analysis shows that low Pxn expression was associated with increased distant metastasis-free survival in luminal A PR+ breast carcinomas. Interestingly, OHPg treatment reduced Pxn content in T47-D and MCF-7 cells; besides, the interaction between endogenous cytoplasmic CD1/Cdk4 with Pxn was reduced. This was consistent with the reduction of p-Ser83Pxn levels, crucially causing the delay in cell migration and a concomitant inhibition of Rac1 activity and p-PAK. Collectively, these findings support the role of PR-B in breast epithelial cell integrity and reinforce the importance in targeting PR-B as a potential strategy to restrict breast tumor cell invasion and metastasis. Full article
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<p>Hydroxyprogesterone (OHPg)-treated breast cancer cells show low motility, migration and invasion. (<b>A</b>) T47-D phalloidin staining of F-actin (stress fibers, red). 4′,6-diamidino-2-phenylindole (DAPI), nuclear staining. (<b>B</b>,<b>C</b>) Wound-healing assay (insets: time 0). T47-D and MCF-7 cells were transfected with non specific (NS) or targeted against Progesterone-Receptor (PR)-B siRNA. MDA-MB-231 were transfected with vector control (VC) or progesterone receptor B (PR-B) expression vector. (<b>D</b>) Transmigration assay, (<b>E</b>) Invasion assay. Columns are the mean of three independent experiments each in triplicate; bars, SD; * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle treated cells. ** <span class="html-italic">p</span> ≤ 0.05 vs. OHPg-treated cells.</p>
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<p>Hydroxyprogesterone (OHPg)-treated breast cancer cells show low motility, migration and invasion. (<b>A</b>) T47-D phalloidin staining of F-actin (stress fibers, red). 4′,6-diamidino-2-phenylindole (DAPI), nuclear staining. (<b>B</b>,<b>C</b>) Wound-healing assay (insets: time 0). T47-D and MCF-7 cells were transfected with non specific (NS) or targeted against Progesterone-Receptor (PR)-B siRNA. MDA-MB-231 were transfected with vector control (VC) or progesterone receptor B (PR-B) expression vector. (<b>D</b>) Transmigration assay, (<b>E</b>) Invasion assay. Columns are the mean of three independent experiments each in triplicate; bars, SD; * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle treated cells. ** <span class="html-italic">p</span> ≤ 0.05 vs. OHPg-treated cells.</p>
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<p>OHPg effects on N-cadherin (N-cadh), E-cadherin (E-cadh) and Vimentin expression in breast cancer cells. (<b>A</b>) Immunoblot analyses for PR-B and N-cadh expression. MDA-MB-231 cells transfected with vector control or PR-B expression vector were treated for 24 h, as indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), control for loading. Columns refer to three independent experiments, as the mean of the band optical density expressed as fold over vehicle, which was assumed to be 1; bars, SD. * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle-treated cells. ** <span class="html-italic">p</span> ≤ 0.05 vs. OHPg-treated cells. (<b>B</b>) Immunoblot analyses for Vimentin and E-cadh expression in T47-D and MCF-7 cells, as indicated. GAPDH and β-Actin, control for loading * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle-treated cells.</p>
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<p>OHPg-treated breast cancer cells show a reduction of the cytoplasmic cyclin D1 (CD1) amount. (<b>A</b>) Immunoblot analyses for PR-B, progesterone receptor A (PR-A), CD1 expression in indicated cells and (<b>B</b>) in T47-D and MCF-7 cells transfected as indicated. Columns are the mean of three independent experiments in which CD1 band intensities were evaluated in terms of optical density arbitrary units, and expressed as fold over vehicle-treated NS siRNA cells, which was assumed to be 1; bars, SD. * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle-treated NS siRNA cells. ** <span class="html-italic">p</span> ≤ 0.05 vs. OHPg-treated NS siRNA cells. (<b>C</b>) Immunoblot analyses for CD1 expression in MCF-7 cells treated at different times (h) as indicated by numbers. * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle-treated cells. (<b>D</b>) Real-time polymerase chain reaction (PCR) assay of CD1 mRNA expression in T47-D (upper panel) and MCF-7 cells (lower panel), transfected and treated at different times as indicated. 18S rRNA was determined as the control. * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle treated NS siRNA cells. ** <span class="html-italic">p</span> ≤ 0.05 vs. 24 h OHPg-treated NS siRNA cells. (<b>E</b>) Immunoblot analyses for CD1 expression.MCF-7 cells were pretreated with MG132 for 2 h and then co-treated with OHPg at different times (h) as indicated by numbers. * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle-treated cells. ** <span class="html-italic">p</span> ≤ 0.05 vs. OHPg-treated cells.</p>
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<p>Effects of OHPg/PR-B on CD1 transcriptional activity. (<b>A</b>) Left panel: Diagram of the different CD1 gene promoter deletion constructs. Right panel: MCF-7 cells were transiently transfected, treated for 24 h with vehicle, 10 nM OHPg and 1µM RU 486, as indicated. Columns refer to three independent experiments expressed as fold change over vehicle, which was assumed to be 1; bars SD; * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle. ** <span class="html-italic">p</span> ≤ 0.05 vs. OHPg. (<b>B</b>) MDA-MB-231 cells were co-transfected with vector control, D1Δ-2960 and PR-B or PR-A or DNA binding domain (mDBD) expression vectors, then treated as indicated; bars, SD; * <span class="html-italic">p</span> ≤ 0.05 vs. vector. ** <span class="html-italic">p</span> ≤ 0.05 vs. vehicle PR-B. (<b>C</b>) Chromatin Immunoprecipitation (ChIP)-qPCR. T47-D and MCF-7 cells treated with vehicle or OHPg for 6 h. Protein-DNA complexes were immune-precipitated with antibodies indicated. Columns are the mean of three independent experiments. Bars, SD; * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle.</p>
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<p>OHPg effects on CD1/Cdk4/Paxillin (Pxn) interaction and Pxn phosphorylation. (<b>A</b>) Transmigration assay, (<b>B</b>) Invasion assay. Cells were co-transfected with vector control, CD1 or phosphorylation site mutant of CD1 (CD1 T286A) expression plasmids. Columns are the mean of three independent experiments each in triplicate; bars, SD; * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle treated vector control cells. ** <span class="html-italic">p</span> ≤ 0.05 vs. OHPg-treated vector control cells (<b>C</b>) Kaplan–Meier distant metastasis-free survival analysis in luminal A PR+ breast carcinoma patients (<span class="html-italic">n</span> = 122) with high and low Pxn expression analyzed as described in the Materials and Methods. Kaplan-Meier survival graph, and hazard ratio (HR) with 95% confidence intervals and logrank <span class="html-italic">p</span> value (<b>D</b>) Co-immunoprecipitation analysis (right panel). Cytoplasmic extracts were immunoprecipitated with anti-Pxn or anti-CD1 antibodies, as indicated, and immunoblotted with anti-Pxn, anti-CD-1 and anti-Cdk4. Input (left panel), samples without immunoprecipitation. βActin, loading control. IgG was used as the negative control. (<b>E</b>) Immunoblotting for pSer83 Pxn and Pxn expression in T47-D and MCF-7 cells, as indicated. GAPDH, loading control. Columns indicate the mean of relative ratio pSer83 vs. total Pxn. * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle-treated cells.</p>
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<p>Phosphomimetic Pxn reverses the low invasive potential OHPg-treated cells. (<b>A</b>) Transmigration assay and (<b>B</b>) Invasion assay. Cells were co-transfected with a single phosphomimetic mutant of Pxn (Pxn S83E) or a vector control. Columns are the mean of three independent experiments each in triplicate; bars, SD; * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle-treated cells. ** <span class="html-italic">p</span> ≤ 0.05 vs. OHPg-treated cells (<b>C</b>) Immunoblot analyses for Rho, Rac in T47-D, MCF-7 and in MDA-MB-231 cells transfected as indicated. β-Actin, loading control. Images show the results of one representative experiment out of three. (<b>D</b>) Immunoblot analyses for pPAK in MDA-MB-231 cells and in (<b>E</b>) T47-D, MCF-7 cells transfected as indicated. Columns are the mean of three independent experiments each in triplicate; bars, SD; * <span class="html-italic">p</span> ≤ 0.05 vs. vehicle-treated cells. ** <span class="html-italic">p</span> ≤ 0.05 vs. MDA-MB-231 VC vehicle-treated cells.</p>
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<p>Proposed model for OHPg/PR-B-induced mesenchymal-epithelial transition in breast cancer cells. See text for details.</p>
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8 pages, 785 KiB  
Communication
SRSF2 Mutations in Uveal Melanoma: A Preference for In-Frame Deletions?
by Natasha M. van Poppelen, Wojtek Drabarek, Kyra N. Smit, Jolanda Vaarwater, Tom Brands, Dion Paridaens, Emine Kiliç and Annelies de Klein
Cancers 2019, 11(8), 1200; https://doi.org/10.3390/cancers11081200 - 17 Aug 2019
Cited by 18 | Viewed by 4198
Abstract
Background: Uveal melanoma (UM) is the most common primary ocular malignancy in adults in the Western world. UM with a mutation in SF3B1, a spliceosome gene, is characterized by three or more structural changes of chromosome 1, 6, 8, 9, or 11. [...] Read more.
Background: Uveal melanoma (UM) is the most common primary ocular malignancy in adults in the Western world. UM with a mutation in SF3B1, a spliceosome gene, is characterized by three or more structural changes of chromosome 1, 6, 8, 9, or 11. Also UM without a mutation in SF3B1 harbors similar chromosomal aberrations. Since, in addition to SF3B1, mutations in U2AF1 and SRSF2 have also been observed in hematological malignancies, UM without a SF3B1 mutation—but with the characteristic chromosomal pattern—might harbor mutations in one of these genes. Methods: 42 UMs were selected based on their chromosomal profile and wildtype SF3B1 status. Sanger sequencing covering the U2AF1 (exon 2 and 7) hotspots and SRSF2 (exon 1 and 2) was performed on DNA extracted from tumor tissue. Data of three UM with an SRSF2 mutation was extracted from the The Cancer Genome Atlas (TCGA). Results: Heterozygous in-frame SRSF2 deletions affecting amino acids 92–100 were detected in two UMs (5%) of 42 selected tumors and in three TGCA UM specimens. Both the UM with an SRSF2 mutation from our cohort and the UM samples from the TCGA showed more than four structural chromosomal aberrations including (partial) gain of chromosome 6 and 8, although in two TCGA UMs monosomy 3 was observed. Conclusions: Whereas in myelodysplastic syndrome predominantly missense SRSF2 mutations are described, the observed SRSF2 mutations in UM are all in-frame deletions of 8–9 amino acids. This suggests that the R625 missense SF3B1 mutations and SRSF2 mutations in UM are different compared to the spliceosome gene mutations in hematological cancers, and probably target a different, as yet unknown, set of genes involved in uveal melanoma etiology. Full article
(This article belongs to the Special Issue Uveal Melanoma)
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Figure 1
<p>Single nucleotide polymorphism (SNP) array profile with the B-allele frequency from two uveal melanoma samples with an <span class="html-italic">SRSF2</span> mutation. On the x-axes the chromosomes are displayed. (<b>a</b>) Uveal melanoma 1 (UM1). (<b>b</b>) Uveal melanoma 2 (UM2).</p>
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<p>Mutations in the <span class="html-italic">SRSF2</span> gene from two out of the 42 analyzed uveal melanoma patients. (<b>a</b>) and (<b>c</b>) Uveal melanoma sample (UM1) with a p.Y92_H99del displayed in SeqPilot V4.3.0 (JSI medical systems, Ettenheim, Germany) (<b>a</b>) and in SeqScape V3.0 (Thermo Fisher Scientific, Waltham, MA, USA) (<b>c</b>). (<b>b</b>,<b>d</b>) Uveal melanoma (UM2) with a p.G93_H100del displayed in SeqPilot V4.3.0 (<b>b</b>) and in SeqScape V3.0 (<b>d</b>).</p>
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20 pages, 6651 KiB  
Article
Single-Cell Analysis of Multiple Steps of Dynamic NF-κB Regulation in Interleukin-1α-Triggered Tumor Cells Using Proximity Ligation Assays
by Christin Mayr-Buro, Eva Schlereth, Knut Beuerlein, Ulas Tenekeci, Johanna Meier-Soelch, M. Lienhard Schmitz and Michael Kracht
Cancers 2019, 11(8), 1199; https://doi.org/10.3390/cancers11081199 - 16 Aug 2019
Cited by 7 | Viewed by 4280
Abstract
The frequently occurring heterogeneity of cancer cells and their functional interaction with immune cells in the tumor microenvironment raises the need to study signaling pathways at the single cell level with high precision, sensitivity, and spatial resolution. As aberrant NF-κB activity has been [...] Read more.
The frequently occurring heterogeneity of cancer cells and their functional interaction with immune cells in the tumor microenvironment raises the need to study signaling pathways at the single cell level with high precision, sensitivity, and spatial resolution. As aberrant NF-κB activity has been implicated in almost all steps of cancer development, we analyzed the dynamic regulation and activation status of the canonical NF-κB pathway in control and IL-1α-stimulated individual cells using proximity ligation assays (PLAs). These systematic experiments allowed the visualization of the dynamic dissociation and re-formation of endogenous p65/IκBα complexes and the nuclear translocation of NF-κB p50/p65 dimers. PLA combined with immunostaining for p65 or with NFKBIA single molecule mRNA-FISH facilitated the analysis of (i) further levels of the NF-κB pathway, (i) its functionality for downstream gene expression, and (iii) the heterogeneity of the NF-κB response in individual cells. PLA also revealed the interaction between NF-κB p65 and the P-body component DCP1a, a new p65 interactor that contributes to efficient p65 NF-κB nuclear translocation. In summary, these data show that PLA technology faithfully mirrored all aspects of dynamic NF-κB regulation, thus allowing molecular diagnostics of this key pathway at the single cell level which will be required for future precision medicine. Full article
(This article belongs to the Special Issue NF-kappaB signalling pathway)
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<p>Specificity of proximity ligation assay- (PLA) based detection of p65/IκBα complexes as revealed by the analysis of IL-1α-triggered wild type or p65-deficient mouse embryonic fibroblasts (MEFs). (<b>A</b>) Scheme of the PLAs used to determine p65/IκBα heterodimers including three types of negative controls that were used throughout this study to assess background signals. At the end of the PLA procedure, protein complexes were visualized as fluorescent spots by microscopic imaging and signals per cell were counted for quantification. (<b>B</b>) Wild type (p65 +/+) or p65-deficient MEFs (p65 −/−) were left untreated or were stimulated with IL-1α (10 ng/mL) for 60 min. Cells were fixed and analyzed by PLA by adding antibodies recognizing p65 (F-6) and IκBα (E130). After the PLA reaction, the nuclei were stained with Hoechst 33342. Representative merged images are displayed. (<b>C</b>) PLA signals from individual stained cells were quantified using the Duolink<sup>®</sup>ImageTool software. Box plots visualize the distribution of p65/IκBα complexes (PLA signals) per cell. Data from two independent experiments were pooled. (<b>D</b>) The table summarizes the numbers of analyzed cells, PLA signals per cell, relative changes and the significance of changes as obtained by the Mann–Whitney Rank Sum Test (<span class="html-italic">p</span> ≤ 0.001).</p>
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<p>Sensitivity of PLA-based detection of p65/IκBα heterodimers as revealed by the analysis of IL-1α-triggered kinetic changes in complex formation. HeLa cells were left untreated or treated with IL-1α (10 ng/mL) for different time points as indicated. Subsequently cells were fixed and analyzed by PLA using the anti-p65 (F-6) and anti-IκBα (E130) antibodies. As an internal control, antibodies were omitted or used individually. Nuclear DNA was stained with Hoechst 33342. (<b>A</b>) Representative merged images are displayed. (<b>B</b>,<b>C</b>) Data from three independent experiments were pooled. Evaluation and statistical analyses were performed as described for <a href="#cancers-11-01199-f001" class="html-fig">Figure 1</a>. Distribution of PLA signals is shown in (<b>B</b>) and the summary and statistics of all relevant data are depicted in (<b>C</b>).</p>
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<p>Global functional analysis of p65/IκBα complex formation by co-immunoprecipitation compared to PLA analysis specifically in cells with nuclear translocation of p65. (<b>A</b>) HeLa cells were stimulated for the indicated periods with IL-1α (10 ng/mL) as shown. Extracts were prepared and equal amounts of proteins were analyzed by Western blotting for the occurrence and phosphorylation of the indicated proteins with specific antibodies. The position of molecular weight markers is indicated. The experiment is representative for three experiments performed in total. (<b>B</b>) The cells were stimulated with IL-1α (10 ng/mL) for the indicated periods and extracts were prepared. While one half of the extract was mixed with antibodies recognizing the IκBα protein, the other half was incubated with control IgG antibodies. Following the addition of True Blot anti rabbit Ig IP agarose beads, the IκBα protein, and the associated proteins were isolated by co-immunoprecipitation, followed by the analysis of proteins by Western blotting as shown. For p65, two different exposure times are shown. (<b>C</b>) Scheme of the modified Immuno-PLA procedure that allows discriminating p65/IκBα complex formation in unresponsive cells compared to (neighboring) cells that show nuclear translocation and thus activation of the canonical NF-κB pathway. (<b>D</b>) HeLa cells remained untreated or were stimulated for 30 min or 60 min with IL-1α (10 ng/mL) as shown. Cells were fixed and p65/IκBα complexes were revealed by PLA with specific antibodies. This PLA included an additional permeabilization step to improve access of the antibodies to the nuclear compartment. In parallel, the intracellular localization of p65 was analyzed by indirect immunofluorescence using a p65-specific antibody and DyLight 488-coupled secondary anti mouse (ms) antibody. Additionally, nuclear DNA was stained with Hoechst 33342. The cells were analyzed by microscopy, representative merged pictures are shown. (<b>E</b>–<b>G</b>) Quantification of data from three independent experiments was performed as described in the legend of <a href="#cancers-11-01199-f001" class="html-fig">Figure 1</a>. (<b>E</b>) Percentage of cells with nuclear translocation of p65. Bars show means +/− SD. (<b>F</b>,<b>G</b>) Evaluation and statistical analyses of PLA signals were performed as described for <a href="#cancers-11-01199-f001" class="html-fig">Figure 1</a>. Distribution of PLA signals is shown in (<b>F</b>). Note that for the IL-1α-activated situation, PLA signals were specifically counted in cells with nuclear translocation of p65. The summary and statistics of all relevant data are depicted in (<b>G</b>).</p>
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<p>Specificity of PLA in human cancer cells and its use to determine p65 protein levels. HeLa cells were transfected to express a p65-specific shRNA and were treated for three days with puromycin (1 µg/mL) to eliminate the non-transfected cells. These p65 knockdown cells (shp65), cells transfected with empty vector lacking the shRNA-encoding sequence (pSUPER.puro), and the parental non-transfected cells were treated for the indicated periods with IL-1α (10 ng/mL) and the p65/IκBα complex formation or expression of p65 only was revealed by PLA using incubation of fixed cells with antibodies recognizing the N-terminal and C-terminal part of p65. The p65 knockdown samples serve as an additional negative control to ensure the specificity of detected PLA signals. (<b>A</b>) A part of the cells was used to determine expression and RNAi-mediated suppression of p65 by immunoblotting. (<b>B</b>) Representative merged images of the PLA results assessing p65/IκBα complexes (left graphs) and total p65 protein expression (right graphs). (<b>C</b>–<b>F</b>) PLA data of cells from four (p65/IκBα PLA) or two (p65/p65 PLA) independent experiments were pooled. Evaluation and statistical analyses were performed as described for <a href="#cancers-11-01199-f001" class="html-fig">Figure 1</a>. Distribution of PLA signals is shown in (<b>C</b>,<b>E</b>) and the summary and statistics of all relevant data are depicted in (<b>D</b>,<b>F</b>).</p>
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<p>PLA-based analysis of IL-1α-induced translocation of p65/p50 heterodimers to study NF-κB functions in the nucleus. (<b>A</b>) HeLa cells were left untreated or stimulated with IL-1α (10 ng/mL) as shown. Cells were permeabilized using an adapted PLA protocol to increase the access of antibodies to the nuclear fraction, followed by PLAs using antibodies recognizing p50 and p65, respectively. Representative cells are displayed: Nuclei were visualized by Hoechst 33342 staining. (<b>B</b>,<b>C</b>) PLA data of cells from two independent experiments were pooled. Evaluation and statistical analyses were performed as described for <a href="#cancers-11-01199-f001" class="html-fig">Figure 1</a>. Distribution of PLA signals is shown in (<b>B</b>) and the summary and statistics of all relevant data are depicted in (<b>C</b>).</p>
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<p>PLA combined with RNA-FISH of the IL-1α target gene <span class="html-italic">NFKBIA</span> to study downstream NF-κB functions in the nucleus. (<b>A</b>) Scheme of the modified PLA procedure coupled to single molecule (sm)RNA-FISH that allows to discriminate p65/IκBα complex formation in unresponsive cells compared to (neighbouring) cells with IL-1α-induced mRNA expression of the <span class="html-italic">NFKBIA</span> gene that encodes the IκBα protein. (<b>B</b>) HeLa cells remained untreated or were stimulated for 30 or 60 min with IL-1α (10 ng/mL) as shown. Cells were fixed and p65/IκBα complexes were revealed by PLA with specific antibodies followed by <span class="html-italic">NFKBIA</span> smRNA-FISH. PLA signals appear in green, <span class="html-italic">NFKBIA</span> FISH signals in pink and nuclei in blue (stained with Hoechst 33342). The cells were analyzed by fluorescence microscopy, representative merged pictures and PLA negative controls (from untreated cells) are shown. (<b>C</b>) Both, PLA and smRNA-FISH signals were counted by the Duolink<sup>®</sup>ImageTool. The box plots show the distribution of PLA or smRNA-FISH signals. The table summarizes all relevant single cell data. (<b>D</b>) For each cell the number of p65/IκBα complexes was plotted against the <span class="html-italic">NFKBIA</span> smRNA-FISH signals. Data from untreated and IL-1α-stimulated conditions are depicted as separate scatter plots, red lines indicate mean signals.</p>
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<p>DCP1a is required for efficient nuclear translocation of p65 NF-κB. (<b>A</b>) HeLa cell lines with CRISPR/Cas9-mediated suppression of DCP1a (ΔDCP1a) or cells stably transfected with the control vector (px459) were incubated for the indicated times with IL-1α (10 ng/mL) or were left untreated. Cytosolic and nuclear extracts were prepared and the presence of p65 and DCP1a proteins was examined by immunoblotting. Anti-tubulin, β-actin, and P(S2)-RNA polymerase II antibodies were used to control for purity of the fractions and for equal loading. Shown is one out of two independent experiments with compatible results. (<b>B</b>) Shows relative quantification of mean p65 protein amounts +/− SD from two independent experiments. (<b>C</b>) The same cells were seeded at two different densities and cell numbers were assessed at the indicated time points. The graphs show cell numbers and corresponding fold changes.</p>
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<p>PLA for the validation of new p65 interactors: Analysis of endogenous NF-κB p65/DCP1a complexes. (<b>A</b>) Double-immunofluorescence analysis of DCP1 and p65 in HeLa cells stably transfected with pSUPER.puro. (<b>B</b>–<b>D</b>) PLAs were used to determine the interaction of endogenous DCP1a with p65 NF-κB in HeLa cells before and after stimulation with IL-1α (10 ng/mL) at different times. The specificity of the obtained PLA signals was validated by omitting one or both primary antibodies. (<b>E</b>–<b>G</b>) Additionally, specificity of PLA was assessed in cells transiently transfected for 24 h with a p65 shRNA construct or with empty vector (pSUPER.puro) control. Representative images, the summary of PLA quantification and statistical tests are shown. (<b>E</b>–<b>G</b>) Data were pooled from three independent experiments (50 cells each). Distribution of PLA signals and the summary and statistics of all relevant data are depicted graphically and as tables as before. (<b>H</b>) The p65 knockdown was also confirmed in cell extracts by immunoblotting.</p>
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<p>Summary: PLA to study multiple levels of NF-κB activation in individual tumor cells at the single cell level.</p>
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18 pages, 2937 KiB  
Technical Note
Clinical-Scale Production of CAR-T Cells for the Treatment of Melanoma Patients by mRNA Transfection of a CSPG4-Specific CAR under Full GMP Compliance
by Manuel Wiesinger, Johannes März, Mirko Kummer, Gerold Schuler, Jan Dörrie, Beatrice Schuler-Thurner and Niels Schaft
Cancers 2019, 11(8), 1198; https://doi.org/10.3390/cancers11081198 - 16 Aug 2019
Cited by 53 | Viewed by 8139
Abstract
Chimeric antigen receptor (CAR)-T cells already showed impressive clinical regressions in leukemia and lymphoma. However, the development of CAR-T cells against solid tumors lags behind. Here we present the clinical-scale production of CAR-T cells for the treatment of melanoma under full GMP compliance. [...] Read more.
Chimeric antigen receptor (CAR)-T cells already showed impressive clinical regressions in leukemia and lymphoma. However, the development of CAR-T cells against solid tumors lags behind. Here we present the clinical-scale production of CAR-T cells for the treatment of melanoma under full GMP compliance. In this approach a CAR, specific for chondroitin sulfate proteoglycan 4 (CSPG4) is intentionally transiently expressed by mRNA electroporation for safety reasons. The clinical-scale protocol was optimized for: (i) expansion of T cells, (ii) electroporation efficiency, (iii) viability, (iv) cryopreservation, and (v) potency. Four consistency runs resulted in CAR-T cells in clinically sufficient numbers, i.e., 2.4 × 109 CAR-expressing T cells, starting from 1.77x108 PBMCs, with an average expansion of 13.6x, an electroporation efficiency of 88.0% CAR-positive cells, a survival of 74.1% after electroporation, and a viability of 84% after cryopreservation. Purity was 98.7% CD3+ cells, with 78.1% CD3+/CD8+ T cells and with minor contaminations of 1.2% NK cells and 0.6% B cells. The resulting CAR-T cells were tested for cytolytic activity after cryopreservation and showed antigen-specific and very efficient lysis of tumor cells. Although our work is descriptive rather than investigative in nature, we expect that providing this clinically applicable protocol to generate sufficient numbers of mRNA-transfected CAR-T cells will help in moving the field of adoptive cell therapy of cancer forward. Full article
(This article belongs to the Special Issue CAR-T Cell Therapy-Novel Approaches and Challenges)
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Figure 1

Figure 1
<p>Schematic representation of the expansion and electroporation procedure. PBMCs were isolated from leukaphereses by density centrifugation using Lymphoprep<sup>TM</sup> and were expanded for nine days in X-vivo 15 medium in bags with a starting concentration of 1 × 10<sup>6</sup> cells/mL. OKT-3 (0.1 µg/mL) was added at day zero. IL-2 was added at day zero (1000 IU/mL), day two (1000 IU/mL), day five (500 IU/mL), and day seven (250 IU/mL). Cells were diluted at day two to 2 × 10<sup>5</sup> cells/mL, and at day five and seven, the volume of the X-vivo 15 medium is doubled. At day nine, the cells were harvested and 3.24 × 10<sup>9</sup> cells were electroporated with mRNA encoding the CSPG4-specific CAR. Four hours after that, the CAR-transfected T cells were cryoconserved in batches of 45 × 10<sup>6</sup> cells. For technical details of this protocol, please see <a href="#sec4-cancers-11-01198" class="html-sec">Section 4</a>.</p>
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<p>Phenotype of the cells during the expansion over nine days. PBMCs were expanded as described in <a href="#cancers-11-01198-f001" class="html-fig">Figure 1</a> in four consistency runs. At day zero, two, seven, and nine the phenotype of the cells was determined by measuring CD3, CD8, CD25, CD19, and CD56/CD16 expression using corresponding antibodies. The mean percentages of positive cells of the four consistency runs +/− SEM are shown.</p>
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<p>Phenotype of the chimeric antigen receptor (CAR)-transfected cells after expansion over nine days, electroporation and cryoconservation. PBMCs were expanded, electroporated, and cryoconserved as described in <a href="#cancers-11-01198-f001" class="html-fig">Figure 1</a> in four consistency runs. After several days, the cells were thawed, and 4 h after thawing the phenotype of the cells was determined by measuring CD3 (<b>a,b</b>), CD8 (<b>a</b>), CD25 (<b>a</b>), CD19 (<b>b</b>), and CD56/CD16 (<b>b</b>) expression using corresponding antibodies. The percentages of positive cells of all four consistency runs are shown.</p>
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<p>Transfection efficiency of mRNA encoding the chondroitin sulfate proteoglycan 4 (CSPG4)-specific CAR. PBMCs were expanded, electroporated, and cryoconserved as described in <a href="#cancers-11-01198-f001" class="html-fig">Figure 1</a> in four consistency runs. As a control mock-electroporated cells were generated. After several days, the cells were thawed, and 4 h after thawing the CAR expression on the cell surface of the T cells was determined using a PE-labeled goat anti-human IgG antibody, binding the Fc-part of the CAR (<b>a</b>,<b>b</b>). The percentages of positive cells of all four consistency runs are shown (<b>a</b>). Furthermore, histograms of the CAR expression are shown (<b>b</b>).</p>
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<p>Lysis of target cells by CSPG4-CAR-transfected T cells. PBMCs were expanded, electroporated, and cryoconserved as described in <a href="#cancers-11-01198-f001" class="html-fig">Figure 1</a> in four consistency runs. As a control mock-electroporated cells were generated. After several days, the cells were thawed and used in a cytotoxicity assay. As target cells the CSPG4-negative 293T cell line and the CSPG4-positive A375M melanoma cell line were used. 293T cells were labeled with 0.25 µM CFSE and A375M cells were labeled with 2.5 µM CFSE. These cells were mixed at a 1:1 ratio. Target cells and effector cells were co-incubated for 20 h at indicated target: effector ratios. Then cells were harvested and stained with 7-AAD. CFSE and 7-AAD staining was determined by flow cytometry. All surviving cells, i.e., 293T CFSE<sup>low</sup>, 7-AAD negative cells and A375M CFSE<sup>high</sup>, 7-AAD negative cells, were set to 100%. The proportion of A375M cells in all surviving cells are indicated in the dot blots. Shown is representative data of one consistency run. For gating strategy see <a href="#app1-cancers-11-01198" class="html-app">Supplementary Figure S6</a>.</p>
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<p>Lysis of A375M melanoma cells by mock- and CAR-transfected T cells. PBMCs were expanded, electroporated, and cryoconserved as described in <a href="#cancers-11-01198-f001" class="html-fig">Figure 1</a> in four consistency runs. As a control mock-electroporated cells were generated. After several days, the cells were thawed and used in a cytotoxicity assay. As target cells the CSPG4-negative 293T cell line and the CSPG4-positive A375M melanoma cell line were used. 293T cells were labeled with 0.25 µM CFSE and A375M cells were labeled with 2.5 µM CFSE. These cells were mixed at a 1:1 ratio. Target cells and effector cells were co-incubated for 20 h at indicated target: effector ratios. Then, cells were harvested and stained with 7-AAD. CFSE and 7-AAD staining was determined by flow cytometry. The percentage of lysis (i.e., cytotoxicity induced by T cells and background apoptosis) of A375M cells was calculated as follows, with A being: <div class="html-disp-formula-info" id="FD1-cancers-11-01198"> <div class="f"> <math display="block"><semantics> <mrow> <mi mathvariant="normal">A</mi> <mtext> </mtext> <mo>=</mo> <mfrac> <mrow> <mo>%</mo> <mtext> </mtext> <mi mathvariant="normal">l</mi> <mi mathvariant="normal">i</mi> <mi mathvariant="normal">v</mi> <mi mathvariant="normal">i</mi> <mi mathvariant="normal">n</mi> <mi mathvariant="normal">g</mi> <mtext> </mtext> <mi mathvariant="normal">A</mi> <mn>375</mn> <mi mathvariant="normal">M</mi> <mtext> </mtext> <mi mathvariant="normal">c</mi> <mi mathvariant="normal">e</mi> <mi mathvariant="normal">l</mi> <mi mathvariant="normal">l</mi> <mi mathvariant="normal">s</mi> </mrow> <mrow> <mo>%</mo> <mtext> </mtext> <mi mathvariant="normal">l</mi> <mi mathvariant="normal">i</mi> <mi mathvariant="normal">v</mi> <mi mathvariant="normal">i</mi> <mi mathvariant="normal">n</mi> <mi mathvariant="normal">g</mi> <mtext> </mtext> <mn>293</mn> <mi mathvariant="normal">T</mi> <mtext> </mtext> <mi mathvariant="normal">c</mi> <mi mathvariant="normal">e</mi> <mi mathvariant="normal">l</mi> <mi mathvariant="normal">l</mi> <mi mathvariant="normal">s</mi> </mrow> </mfrac> </mrow> </semantics></math></p>
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<p>Antigen-specific cytokine production by mock- and CAR-transfected T cells. PBMCs were expanded, electroporated, and cryoconserved as described in <a href="#cancers-11-01198-f001" class="html-fig">Figure 1</a> in four consistency runs. As a control mock-electroporated cells were generated. After several days, the cells were thawed and used in a cytometric bead array. As target cells the CSPG4-negative 293T cell line and the CSPG4-positive A375M melanoma cell line were used. Target cells and effector cells were co-incubated for 20 h at a 1:1 ratio. As control, effector cells alone were also incubated (ø). Then supernatants were harvested and cytokine concentration was determined with a cytometric bead array. The mean cytokine secretions by transfected cells from the four consistency runs +/− SEM are shown. Please note the different scales.</p>
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19 pages, 1610 KiB  
Review
Targeting Oncogenic BRAF: Past, Present, and Future
by Aubhishek Zaman, Wei Wu and Trever G. Bivona
Cancers 2019, 11(8), 1197; https://doi.org/10.3390/cancers11081197 - 16 Aug 2019
Cited by 158 | Viewed by 12248
Abstract
Identifying recurrent somatic genetic alterations of, and dependency on, the kinase BRAF has enabled a “precision medicine” paradigm to diagnose and treat BRAF-driven tumors. Although targeted kinase inhibitors against BRAF are effective in a subset of mutant BRAF tumors, resistance to the therapy [...] Read more.
Identifying recurrent somatic genetic alterations of, and dependency on, the kinase BRAF has enabled a “precision medicine” paradigm to diagnose and treat BRAF-driven tumors. Although targeted kinase inhibitors against BRAF are effective in a subset of mutant BRAF tumors, resistance to the therapy inevitably emerges. In this review, we discuss BRAF biology, both in wild-type and mutant settings. We discuss the predominant BRAF mutations and we outline therapeutic strategies to block mutant BRAF and cancer growth. We highlight common mechanistic themes that underpin different classes of resistance mechanisms against BRAF-targeted therapies and discuss tumor heterogeneity and co-occurring molecular alterations as a potential source of therapy resistance. We outline promising therapy approaches to overcome these barriers to the long-term control of BRAF-driven tumors and emphasize how an extensive understanding of these themes can offer more pre-emptive, improved therapeutic strategies. Full article
(This article belongs to the Special Issue Oncogenic Forms of BRAF as Cancer Driver Genes)
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Figure 1
<p>Pan-cancer BRAF alterations from The Cancer Genome Atlas (TCGA). TCGA-generated pan-cancer alteration frequency of BRAF was extracted from cBioportal (<a href="https://www.cbioportal.org" target="_blank">https://www.cbioportal.org</a>). Data are represented as a stacked histogram plot. Colors represent different types of alterations as indicated in the legend.</p>
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<p>BRAF mutation spectrum in cancer. TCGA pan-cancer studies from cBioportal (<a href="https://www.cbioportal.org" target="_blank">https://www.cbioportal.org</a>) were used for BRAF mutation lollipop plot. This plot summarizes 32 studies from TCGA that constitute 10,953 patients/10,967 samples. Somatic BRAF mutation frequency is 7.0% in these samples. The diagram shows the backbone of BRAF protein containing 766 amino acids (aa) with three main domains: (1) Raf-like RAS-binding domain (RBD, that spans 156–227 amino acids green), (2) phorbol esters/diacylglcerol-binding domain (C1 domain, 235–280aa, red), and (3) protein tyrosine kinase domain (Pkinase_Tyr, 458–712 aa, blue). The circles with different colors represent types of mutations: dark green, missense mutations; black, truncating mutations (including nonsense, nonstop, frameshift deletion, frameshift insertion, splice site mutations); dark red, in-frame deletion, in-frame insertion; pink, other mutations. <span class="html-italic">Y</span>-axis shows the frequency of particular BRAF mutations.</p>
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<p>BRAF-mediated signaling in normal and cancer cells. In normal cells, external growth stimuli activate receptor tyrosine kinase (RTK) and RAS, which relays growth signals to the mitogen-activated protein kinase (MAPK) pathway kinase cascade. In BRAF-driven cancers, mutant BRAF (BRAF *) can either act RAS independently as a monomer (Class 1) and as a dimer (Class 2) or act RAS dependently (Class 3) to hyperactivate cellular growth. Class 1 and Class 2 tumors respond to BRAF inhibitor-targeted therapy. However, various intrinsic or adaptive resistance mechanisms attenuate response to targeted BRAF inactivation. For example, preexisting NF1 loss, CDK4 mutations, and increased COT and YAP expression may specify intrinsic resistance. Therapy-induced HGF secretion and PI3K-AKT pathway activation are examples of some of the adaptive resistance mechanisms. On the other hand, EGFR-amplified population gives rise to acquired resistance to BRAF inhibitors.</p>
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<p>BRAF alterations concurrent with other genetic aberrations in non-small cell lung cancer (NSCLC). Targeted tumor exome sequencing using 403 cancer gene panel was performed by Foundation medicine. In all, 236 samples with BRAF alterations, such as structural variation (SV), copy number variation (CV), and rearrangement (RE), were stratified from 30,000 non-small cell lung cancer samples. Oncoprint of BRAF-mutant tumor shows coexistence of other genetic alterations with BRAF gene alteration. The different frequency of gene alterations is indicated on the left bar plot.</p>
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16 pages, 1145 KiB  
Article
Development, Implementation and Assessment of Molecular Diagnostics by Next Generation Sequencing in Personalized Treatment of Cancer: Experience of a Public Reference Healthcare Hospital
by Javier Simarro, Rosa Murria, Gema Pérez-Simó, Marta Llop, Nuria Mancheño, David Ramos, Inmaculada de Juan, Eva Barragán, Begoña Laiz, Enrique Cases, Emilio Ansótegui, José Gómez-Codina, Jorge Aparicio, Carmen Salvador, Óscar Juan and Sarai Palanca
Cancers 2019, 11(8), 1196; https://doi.org/10.3390/cancers11081196 - 16 Aug 2019
Cited by 16 | Viewed by 5757
Abstract
The establishment of precision medicine in cancer patients requires the study of several biomarkers. Single-gene testing approaches are limited by sample availability and turnaround time. Next generation sequencing (NGS) provides an alternative for detecting genetic alterations in several genes with low sample requirements. [...] Read more.
The establishment of precision medicine in cancer patients requires the study of several biomarkers. Single-gene testing approaches are limited by sample availability and turnaround time. Next generation sequencing (NGS) provides an alternative for detecting genetic alterations in several genes with low sample requirements. Here we show the implementation to routine diagnostics of a NGS assay under International Organization for Standardization (UNE-EN ISO 15189:2013) accreditation. For this purpose, 106 non-small cell lung cancer (NSCLC) and 102 metastatic colorectal cancer (mCRC) specimens were selected for NGS analysis with Oncomine Solid Tumor (ThermoFisher). In NSCLC the most prevalently mutated gene was TP53 (49%), followed by KRAS (31%) and EGFR (13%); in mCRC, TP53 (50%), KRAS (48%) and PIK3CA (16%) were the most frequently mutated genes. Moreover, NGS identified actionable genetic alterations in 58% of NSCLC patients, and 49% of mCRC patients did not harbor primary resistance mechanisms to anti-EGFR treatment. Validation with conventional approaches showed an overall agreement >90%. Turnaround time and cost analysis revealed that NGS implementation is feasible in the public healthcare context. Therefore, NGS is a multiplexed molecular diagnostic tool able to overcome the limitations of current molecular diagnosis in advanced cancer, allowing an improved and economically sustainable molecular profiling. Full article
(This article belongs to the Special Issue Application of Next-Generation Sequencing in Cancers)
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Figure 1
<p>Distribution of gene alterations in NSCLC (green) and mCRC patients (blue). Column chart in the upper part represents the total number of mutations for each sample. Left column indicates the percentage of samples with specific gene alteration. Dark grey—Not tested. R—Rearrangements.</p>
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<p>Circos diagram. Associations among the most prevalently mutated genes in NSCLC patients.</p>
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<p>Circos diagram. Associations among the most prevalently mutated genes in mCRC patients.</p>
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<p>Percentage of NSCLC patients with actionable alterations detected by NGS. Fifty-eight percent of patients included in the study were susceptible to being treated with targeted drugs approved in advanced cancers or in clinical trials.</p>
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<p>Classification of mCRC patients according to clinically relevant alterations detected by NGS.</p>
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17 pages, 2558 KiB  
Review
Targeting Heat Shock Protein 27 in Cancer: A Druggable Target for Cancer Treatment?
by Seul-Ki Choi, Heejin Kam, Kye-Young Kim, Suk In Park and Yun-Sil Lee
Cancers 2019, 11(8), 1195; https://doi.org/10.3390/cancers11081195 - 16 Aug 2019
Cited by 89 | Viewed by 9447
Abstract
Heat shock protein 27 (HSP27), induced by heat shock, environmental, and pathophysiological stressors, is a multi-functional protein that acts as a protein chaperone and an antioxidant. HSP27 plays a significant role in the inhibition of apoptosis and actin cytoskeletal remodeling. HSP27 is upregulated [...] Read more.
Heat shock protein 27 (HSP27), induced by heat shock, environmental, and pathophysiological stressors, is a multi-functional protein that acts as a protein chaperone and an antioxidant. HSP27 plays a significant role in the inhibition of apoptosis and actin cytoskeletal remodeling. HSP27 is upregulated in many cancers and is associated with a poor prognosis, as well as treatment resistance, whereby cells are protected from therapeutic agents that normally induce apoptosis. This review highlights the most recent findings and role of HSP27 in cancer, as well as the strategies for using HSP27 inhibitors for therapeutic purposes. Full article
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<p>Major roles of heat shock protein 27 (HSP27). HSP27 has important functions, including protein folding regulation by chaperone activity, immune response, cancer promotion, inducing resistance to anticancer drugs, aging, biomarkers of several diseases, aggravation of neurodegenerative disease, development, and differentiation [<a href="#B5-cancers-11-01195" class="html-bibr">5</a>,<a href="#B6-cancers-11-01195" class="html-bibr">6</a>,<a href="#B7-cancers-11-01195" class="html-bibr">7</a>,<a href="#B8-cancers-11-01195" class="html-bibr">8</a>].</p>
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<p>The structure of heat shock protein 27 (HSP27). The structure of human HSP27 consists of the N-terminal domain, the α-crystallin domain, and the C-terminal domain. The N-terminal domain contains a WDPF motif which is essential for large oligomerization. The C-terminal domain includes an α-crystallin motif that is highly conserved between species and is involved in the formation of small oligomerization. HSP27 phosphorylation sites S15, S78, S82, and T143 are indicated. S15 can be phosphorylated by p38 mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) and 3 (MK3), and protein kinase C (PKC). S78 can be phosphorylated by MK2, MK3, ribosomal S6 kinase (p70RSK), PKC, and protein kinase G (PKG). S82 can be phosphorylated by MK2, MK3, p70RSK, protein kinase B (PKB), protein kinase D (PKD), and PKG. T143 can be phosphorylated by PKG.</p>
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<p>Phosphorylation induced conformational structural switching between different states. Heat shock protein 27 (HSP27) exists as large oligomers when unphosphorylated. At specific serine residues in the mitogen-activated protein kinase (MAPK) pathway, HSP27 switches to smaller oligomers. HSP27 conformational structure changes actively and contributes to maintaining proteostasis.</p>
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<p>Role of heat shock protein 27 (HSP27) in different cellular apoptotic processes. HSP27 inhibits apoptosis by integrating with different signaling pathways, including the extrinsic and intrinsic apoptosis pathway. HSP27 inhibits Bcl-2-associated X protein (BAX) by directly binding to death domain associated protein (DAXX) or apoptosis signal-regulating kinase-1 (Ask-1) to inhibit its function, which enhances AKT activity inhibiting BH3 interacting-domain death agonist (BID) or protein kinase C delta type (PKC δ) function. HSP27 inhibits caspase 3, which directly functions in cellular apoptosis. HSP27 contributes to cell survival.</p>
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<p>Kaplan–Meier (KM) curves for <span class="html-italic">HSPB1</span> (gene name of heat shock protein 27 (HSP27)) in the overall survival of various cancers. Gastric cancer, lung cancer, hepatocellular carcinoma, breast cancer, clear cell renal carcinoma, and rectum adenocarcinoma show high survival rates associated with low HSPB1 expression. <span class="html-italic">p</span>-values were calculated using the log-rank test. The Hazard Ratio (HR) is the ratio of the hazard rates corresponding to the conditions described by two levels of an explanatory variable (HR &gt; 1 was considered a higher hazard of death from the <span class="html-italic">HSPB1</span>-High group). An independent univariate survival analysis of overall survival (OS) was analyzed based on a merged data set from the Kaplan–Meier Plotter [<a href="#B76-cancers-11-01195" class="html-bibr">76</a>]. Data were derived from <a href="http://kmplot.com/analysis/" target="_blank">http://kmplot.com/analysis/</a> and survival curves were drawn using PRISM software [<a href="#B77-cancers-11-01195" class="html-bibr">77</a>].</p>
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<p>Strategies of heat shock protein 27 (HSP27) inhibitors. (<b>1</b>) Three small molecule inhibitors (Brivudine (RP101), Quercetin, and cross-linker) bind directly to the HSP27 protein and inhibit the activity of the HSP27 protein. (<b>2</b>) Peptide aptamers (PA11 and PA50) bind directly to the HSP27 protein and inhibit oligomerization or dimerization. (<b>3</b>) Antisense oligonucleotide (OGX-427) binds to HSP27 mRNA and prevents translation of the HSP27 protein. As a result, the amount of HSP27 protein is reduced.</p>
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<p>Altered cross-linking of heat shock protein 27 (HSP27) using small molecules for HSP27 inhibition. Scheme for the mechanism of HSP27-cross-linking by small molecules. Normal dimerization of HSP27 contributes to cancer cell survival, but abnormal dimerization of HSP27 using small molecules causes cancer cell death.</p>
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36 pages, 14714 KiB  
Review
The Multiple Roles and Therapeutic Potential of Molecular Chaperones in Prostate Cancer
by Abdullah Hoter, Sandra Rizk and Hassan Y. Naim
Cancers 2019, 11(8), 1194; https://doi.org/10.3390/cancers11081194 - 16 Aug 2019
Cited by 49 | Viewed by 8589
Abstract
Prostate cancer (PCa) is one of the most common cancer types in men worldwide. Heat shock proteins (HSPs) are molecular chaperones that are widely implicated in the pathogenesis, diagnosis, prognosis, and treatment of many cancers. The role of HSPs in PCa is complex [...] Read more.
Prostate cancer (PCa) is one of the most common cancer types in men worldwide. Heat shock proteins (HSPs) are molecular chaperones that are widely implicated in the pathogenesis, diagnosis, prognosis, and treatment of many cancers. The role of HSPs in PCa is complex and their expression has been linked to the progression and aggressiveness of the tumor. Prominent chaperones, including HSP90 and HSP70, are involved in the folding and trafficking of critical cancer-related proteins. Other members of HSPs, including HSP27 and HSP60, have been considered as promising biomarkers, similar to prostate-specific membrane antigen (PSMA), for PCa screening in order to evaluate and monitor the progression or recurrence of the disease. Moreover, expression level of chaperones like clusterin has been shown to correlate directly with the prostate tumor grade. Hence, targeting HSPs in PCa has been suggested as a promising strategy for cancer therapy. In the current review, we discuss the functions as well as the role of HSPs in PCa progression and further evaluate the approach of inhibiting HSPs as a cancer treatment strategy. Full article
(This article belongs to the Special Issue Prostate Cancer: Past, Present, and Future)
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<p>Induction of HSR by HSF1. Various factors including environmental, non-stress and pathological conditions can trigger HSP expression via HSF1 pathway. In non-activated state, HSF1 is sequestered in the cytoplasm due to its binding to chaperone complex including HSP70 and HSP90, thus prevented from performing its transcriptional activity. Upon activation, the chaperone complex dissociates and HSF1 is liberated, homotrimerizes and translocated to the nucleus. In the nucleus, the HSF1 homotrimer binds to the heat shock elements (HSEs) that are located upstream to heat shock gene promotors to initiate the transcription of its target genes including HSP genes.</p>
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<p>Domain architecture of common chaperones involved in PCa. From top to bottom, human HSPs including HSP90α/β, HSP70, HSP60, HSP27, are displayed relative to their relative length where the number of amino acids constituting each chaperone is written as superscripts in their C-terminals. HSPs have N- and C-terminal domains in addition to middle domain like HSP90 and HSP70 while linear representation of human HSP60 domains is still unclear due to its oligomerization and complex association with HSP10. HSP27 has a middle highly conserved α-crystalline domain. Phosphorylation sites of HSP27 are designated as yellow spheres representing either phosphorylated serine or threonine amino acid residues. Human clusterin (CLU) exists initially as polypeptide precursor which undergoes proteolytic cleavage of its first 22 amino acid secretory signal in addition to its cleavage at Arg227–Ser228 to yield two chains; namely α and β chains. The two chains are arranged in anti-parallel orientation to constitute a heterodimeric molecule. Pink boxes represent cysteine-rich centers that are linked by five disulfide bridges. Yellow ovals point to predicted amphipathic α-helices while green tetragonal stars refer to the N-glycosylation sites.</p>
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<p>HSPs regulate AR signaling. A complex of chaperones including HSP90 associates with the AR to expedite and maintain its high affinity binding conformation, thus allowing for DHT interaction. As a consequence, the AR forms a dimer and the chaperone complex dissociates. Thereafter, HSP27 binds to the AR homodimer enabling its nuclear translocation, subsequent binding to the ARE, and activation of transcription of AR-dependent genes.</p>
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<p>Common small inhibitors of HSP90.</p>
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42 pages, 3648 KiB  
Review
Epigenetic Dysregulation at the Crossroad of Women’s Cancer
by Rakesh Kumar, Aswathy Mary Paul, Pranela Rameshwar and M. Radhakrishna Pillai
Cancers 2019, 11(8), 1193; https://doi.org/10.3390/cancers11081193 - 16 Aug 2019
Cited by 10 | Viewed by 5688
Abstract
An increasingly number of women of all age groups are affected by cancer, despite substantial progress in our understanding of cancer pathobiology, the underlying genomic alterations and signaling cascades, and cellular-environmental interactions. Though our understanding of women’s cancer is far more complete than [...] Read more.
An increasingly number of women of all age groups are affected by cancer, despite substantial progress in our understanding of cancer pathobiology, the underlying genomic alterations and signaling cascades, and cellular-environmental interactions. Though our understanding of women’s cancer is far more complete than ever before, there is no comprehensive model to explain the reasons behind the increased incidents of certain reproductive cancer among older as well as younger women. It is generally suspected that environmental and life-style factors affecting hormonal and growth control pathways might help account for the rise of women’s cancers in younger age, as well, via epigenetic mechanisms. Epigenetic regulators play an important role in orchestrating an orderly coordination of cellular signals in gene activity in response to upstream signaling and/or epigenetic modifiers present in a dynamic extracellular milieu. Here we will discuss the broad principles of epigenetic regulation of DNA methylation and demethylation, histone acetylation and deacetylation, and RNA methylation in women’s cancers in the context of gene expression, hormonal action, and the EGFR family of cell surface receptor tyrosine kinases. We anticipate that a better understanding of the epigenetics of women’s cancers may provide new regulatory leads and further fuel the development of new epigenetic biomarkers and therapeutic approaches. Full article
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<p>Incidence and mortality of women’s cancer. (<b>a</b>,<b>b</b>) Global incidence and mortality of breast, cervical, endometrial, and ovarian cancers [<a href="#B1-cancers-11-01193" class="html-bibr">1</a>,<a href="#B2-cancers-11-01193" class="html-bibr">2</a>,<a href="#B3-cancers-11-01193" class="html-bibr">3</a>,<a href="#B4-cancers-11-01193" class="html-bibr">4</a>,<a href="#B5-cancers-11-01193" class="html-bibr">5</a>]. (<b>c</b>,<b>d</b>) Incidence and mortality of breast, cervical, endometrial, and ovarian cancer in North America [<a href="#B1-cancers-11-01193" class="html-bibr">1</a>,<a href="#B2-cancers-11-01193" class="html-bibr">2</a>,<a href="#B3-cancers-11-01193" class="html-bibr">3</a>,<a href="#B4-cancers-11-01193" class="html-bibr">4</a>,<a href="#B5-cancers-11-01193" class="html-bibr">5</a>].</p>
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<p>Women’s cancer incidence in North America since 1975. (<b>a</b>,<b>b</b>) Incidence rates of breast and endometrial cancer as per age-groups. Right inserts, partial enlargements of incidence for two age-groups from 2000–2015. (<b>c</b>,<b>d</b>) Incidence rates of cervical and ovarian cancers as per age-groups.</p>
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<p>Changing trends in women’s cancer incidence. (<b>a</b>) Simplistic view for modifying the growth of cancer cells by epigenetic regulators with or without influencing genetic controls. (<b>b</b>) Number of publications for indicated years in PubMed accessed on the 12 May 2019, and searched for “epigenetics” and “epigenetic” with “breast cancer”, “ovarian cancer”, “cervical cancer”, or “endometrial cancer” for indicated years.</p>
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<p>Modules of chemical modifications on cytosine, adenosine, and lysine residues. (<b>a</b>) Illustrations showing the DNA methylation at the fifth residue in cytosine (5mC) by DNMTs and demethylation by oxidation of 5mC into 5-hydroxymethylcytosine (5hmC) by TETs, and recycling back to cytosine. (<b>b</b>) Illustrations showing the modulation of RNA methylation and demethylation at the sixth position in adenosine, N6-methyladenosine (m<sup>6</sup>A) by writers and erasers, respectively, and recognition of modified base by readers. (<b>c</b>) Illustrations showing histone acetylation on ε-N-acetyl lysine and deacetylation by HATs and HDACs, respectively, and recognition of modified base by readers. Ac, acetylation; Me, methylation. Refer to the main test for details.</p>
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<p>Breast cancer development and DNA-demethylation. (<b>a</b>) Illustrations showing breast cancer development and breast cancer—tumor microenvironment (TME) interactions. See <a href="#app1-cancers-11-01193" class="html-app">Supplementary Figure S1</a> for description of TME. Reduced levels of TETs and 5hmC and increased levels of DNMT and 5mC in aggressive breast cancer. (<b>b</b>) TET activity regulation by ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG). (<b>c</b>) Inflammation regulation of TET activity, (<b>d</b>) MicroRNA regulation of TET expression and function. (<b>e</b>) HMGA regulation of HOXA7/9 via TET. (<b>f</b>) TET regulation of WNT signaling via DKK. (<b>g</b>) Left, hypoxia regulation of TET activity and expression; Right, hypoxia regulation of HIF1-alpha-regulated genes.</p>
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<p>DNA 5mC status and cancer aggressiveness. (<b>a</b>) Illustrations showing the tumor and tumor microenvironment interactions (TME). See <a href="#app1-cancers-11-01193" class="html-app">Supplementary Figure S1</a> for description of TME. (<b>b</b>) Reduced levels of TETs and 5hmC and increased levels of DNMT and 5mC in aggressive ovarian and endometrial cancers. (<b>c</b>) TET regulation of WNT signaling via controlling the transcription of negative regulators DKK and SFPR2. (<b>d</b>) TET1 regulation of ovarian cancer growth via tumor suppressor RASSF5 expression. (<b>e</b>) Insulin stimulation of TET1 expression in TME cells and upregulation of non-genomic GPER signaling in endometrial cancer cells. (<b>f</b>) Macrophage infiltration linked released of IL17A cytokine in re-expression of ER in endometrial cancer cells via modulating the level of TET1 expression.</p>
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<p>Epigenetic regulation of cervical cancer development. (<b>a</b>) Illustrations showing HPV-driven cervical cancer development and interaction with tumor microenvironment (TME). See <a href="#app1-cancers-11-01193" class="html-app">Supplementary Figure S1</a> for description of TME. (<b>b</b>) Reduced levels of TETs and 5hmC and increased levels of DNMT and 5mC in cervical cancer. (<b>c</b>) Regulation of DNMT expression by E6/E7 oncoproteins. (<b>d</b>) Regulation of TET expression by DNA methylation pathway. (<b>e</b>) TET regulation of EMT via modifying the expression of EMT master regulator ZEB1. (<b>f</b>) Potential role of TET1 mutations in acquired resistance to radiotherapy. (<b>g</b>) E6/E7 regulation of target gene expression via modifying the activity of HATs and p53 acetylation. (<b>h</b>) E6/E7 regulation of gene expression via HDACs, NuRD complexes, and PRC2/EZH2 complexes.</p>
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<p>RNA m<sup>6</sup>A methylation and women’s cancer. (<b>a</b>) Illustrations showing a progressive loss of m6A modification and increase FTO status during cancer progression. (<b>b</b>) Effect of increased expression of exemplified RNA-demethylating enzymes in modifying the stability of indicated target mRNAs and resulting functions in breast cancer cells. Also shown is the effect of conditional <span class="html-italic">Mettl3</span> depletion or overexpression in mesenchymal stem cells on osteoporosis in mice. (<b>c</b>). Effect of reduced m<sup>6</sup>A expression on coordinated regulation of negative and positive regulators of AKT signaling in endometrial cancer cells. (<b>d</b>) Examples of modifying effects of METTL3 levels in glioblastoma cancer cells, lung cancer cells, and myocardiocytes. Status of these cellular effects of METTL3 in women’s cancer remains unknown.</p>
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18 pages, 3879 KiB  
Article
Lambda-Carrageenan Enhances the Effects of Radiation Therapy in Cancer Treatment by Suppressing Cancer Cell Invasion and Metastasis through Racgap1 Inhibition
by Ping-Hsiu Wu, Yasuhito Onodera, Frances C. Recuenco, Amato J. Giaccia, Quynh-Thu Le, Shinichi Shimizu, Hiroki Shirato and Jin-Min Nam
Cancers 2019, 11(8), 1192; https://doi.org/10.3390/cancers11081192 - 16 Aug 2019
Cited by 10 | Viewed by 5824
Abstract
Radiotherapy is used extensively in cancer treatment, but radioresistance and the metastatic potential of cancer cells that survive radiation remain critical issues. There is a need for novel treatments to improve radiotherapy. Here, we evaluated the therapeutic benefit of λ-carrageenan (CGN) to enhance [...] Read more.
Radiotherapy is used extensively in cancer treatment, but radioresistance and the metastatic potential of cancer cells that survive radiation remain critical issues. There is a need for novel treatments to improve radiotherapy. Here, we evaluated the therapeutic benefit of λ-carrageenan (CGN) to enhance the efficacy of radiation treatment and investigated the underlying molecular mechanism. CGN treatment decreased viability in irradiated cancer cells and enhanced reactive oxygen species accumulation, apoptosis, and polyploid formation. Additionally, CGN suppressed radiation-induced chemoinvasion and invasive growth in 3D lrECM culture. We also screened target molecules using a gene expression microarray analysis and focused on Rac GTPase-activating protein 1 (RacGAP1). Protein expression of RacGAP1 was upregulated in several cancer cell lines after radiation, which was significantly suppressed by CGN treatment. Knockdown of RacGAP1 decreased cell viability and invasiveness after radiation. Overexpression of RacGAP1 partially rescued CGN cytotoxicity. In a mouse xenograft model, local irradiation followed by CGN treatment significantly decreased tumor growth and lung metastasis compared to either treatment alone. Taken together, these results suggest that CGN may enhance the effectiveness of radiation in cancer therapy by decreasing cancer cell viability and suppressing both radiation-induced invasive activity and distal metastasis through downregulating RacGAP1 expression. Full article
(This article belongs to the Special Issue Role of Natural Bioactive Compounds in the Rise and Fall of Cancers)
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<p>λ-carrageenan (CGN) treatment decreases cell viability and increases apoptosis in irradiated cancer cell lines. Cells were treated with 4 Gy IR, followed by CGN on the next day, and then analyzed 72 h after IR. (<b>A</b>) Cell viability was quantified by cell counting in MDA-MB-231, FaDu, PANC-1, and 4T1 cell lines. (<b>B</b>) The percentage of dead cells was measured by PI staining, followed by flow cytometry. The proportion of dead cells was quantified by gating the population of PI-positive cells. (<span class="html-italic">p</span>-values: MDA-MB-231, 0.0056; FaDu, 0.0129; PANC-1, 0.0489; 4T1, 0.0468.) (<b>C</b>) Apoptotic cells were measured by Annexin V-FITC staining, followed by flow cytometry. Mean fluorescence intensity of FITC was calculated and normalized to the untreated group. Columns, mean (<span class="html-italic">n</span> ≥ 3); bars, SE. *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>IR exposure in combination with CGN increases ROS accumulation in MDA-MB-231 cells. Cells were treated with 4 Gy IR, followed by CGN on the next day, and then analyzed 72 h after IR. (<b>A</b>) ROS was measured by DCFDA. Columns, mean (<span class="html-italic">n</span> = 5); bars, SE. *, <span class="html-italic">p</span> &lt; 0.05. (<b>B</b>) Caspase-3, caspase-8, and caspase-9 activities were detected by microplate reader at specific wavelengths: caspase-3 excitation (Ex)/emission (Em) = 535/620 nm; caspase-8 Ex/Em = 490/525 nm; caspase-9 Ex/Em = 370/450 nm. Columns, mean (<span class="html-italic">n</span> = 5); bars, SE. **, <span class="html-italic">p</span> &lt; 0.01; ns, not significant. (<b>C</b>) Cells stained with α-tubulin (green) and PI (red) after treatments. Bar, 25 μm. (<b>D</b>) To measure polyploid populations, cells were treated with staining solution and PI and analyzed by flow cytometry. Columns, mean (<span class="html-italic">n</span> = 3); bars, SE. *, <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>CGN inhibits the IR-induced invasive activity and 3D lrECM growth in breast cancer cells. (<b>A</b>, <b>B</b>) The invasive activity was measured by Matrigel chemoinvasion assay after IR and/or CGN treatments in MDA-MB-231 (<b>A</b>) and 4T1 (<b>B</b>) cells. Columns, mean (<span class="html-italic">n</span> = 4); bars, SE. **, <span class="html-italic">p</span> &lt; 0.01; ***, <span class="html-italic">p</span> &lt; 0.001. (<b>C</b>) MDA-MB-231 cells were cultured in 3D lrECM. Bar, 50 µm.</p>
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<p>Upregulation of RacGAP1 is involved in cancer cell survival and invasion after IR. (<b>A</b>) Gene expression was analyzed by cDNA microarray. RacGAP1 expression level is shown by the heat map. The values were normalized to the untreated group. (<b>B</b>) RacGAP1 protein expressions with different doses of IR were analyzed in MDA-MB-231, FaDu, PANC-1, and 4T1 cell lines. (<b>C</b>) MDA-MB-231 cells were transfected with siRNA duplexes targeting RacGAP1 (# 1 or # 2) or the control sequence, as indicated. Cell lysates were subjected to western blot two days after transfection. (<b>D</b>–<b>H</b>) MDA-MB-231 cells were transfected with siRNAs and incubated for two days, and then treated with 4 Gy IR. Each experiment was performed 24 h after IR treatment. Cell viability was quantified by cell counting (<b>D</b>). Apoptotic cells were measured by Annexin V-FITC staining and flow cytometry (<b>E</b>). ROS was measured by DCFDA (<b>F</b>). Cells were treated with staining solution and PI, and then the cell cycle was analyzed by flow cytometry. The percentage of polyploid cells in each group was normalized with control group (<b>G</b>). The invasive activity was measured by Matrigel chemoinvasion assay (<b>H</b>). Columns, mean (<span class="html-italic">n</span> ≥ 3); bars, SE. *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01. Data shown in (<b>D</b>–<b>H</b>) were normalized to that of MDA-MB-231 cells transfected with siRNA control. For the continuous full length-image of Western blot signals, please refer to <a href="#app1-cancers-11-01192" class="html-app">Supplementary Materials Figure S7</a>.</p>
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<p>RacGAP1 expression is suppressed by CGN in MDA-MB-231 cells. (<b>A</b>) Protein expression of RacGAP1 was analyzed after treatment with different concentrations of CGN in MDA-MB-231 cells. Total cell lysates were subjected to western blot. (<b>B</b>) RacGAP1 protein expression was analyzed using cell lysates after IR and/or CGN treatment in MDA-MB-231 cells. (<b>C</b>) Immunofluorescence images show RacGAP1 (green), α-tubulin (red) and nuclei (blue). Bar, 25 μm. (<b>D</b>) RacGAP1-mVenus expression was induced by doxycycline in MDA-MB-231 cells. RacGAP1 expression was analyzed by western blot using anti-RacGAP1 antibody. Dox, doxycycline. (<b>E</b>) Cell number was quantified by cell counting. Columns, mean (<span class="html-italic">n</span> = 3); bars, SE. *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01. For the continuous full length-image of Western blot signals, please refer to <a href="#app1-cancers-11-01192" class="html-app">Supplementary Materials Figure S7</a>.</p>
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<p>Radiation followed by CGN treatment decreases tumor size and metastasis in a 4T1 mouse xenograft model. (<b>A</b>) Treatment schedule of each group. Mouse 4T1 cells were injected into Balb/c mice (<span class="html-italic">n</span> = 6–7 in each group). (<b>B</b>) 4T1 tumor sizes were measured and normalized to size at day 12 in each group. (<b>C</b>) Relative tumor size for each group was measured on Day 25. Scatter plot; mean ± SE. *, <span class="html-italic">p</span> &lt; 0.05. (<b>D</b>) Representative H&amp;E images of lung sections. Bar, 500 µm. Arrow, metastatic lung nodules. The number of metastatic lung nodules was counted (right panel). Scatter plot; mean ± SE. *, <span class="html-italic">p</span> &lt; 0.05. (<b>E</b>) Sections from 4T1 tumors were subjected to IHC staining with antibodies against RacGAP1. Bar, 100 µm. RacGAP1 expression level was determined by scoring, as described in the methods (right panel). Scatter plot; bars, mean (<span class="html-italic">n</span> = 5). *, <span class="html-italic">p</span> &lt; 0.05.</p>
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20 pages, 845 KiB  
Review
Reactive Oxygen Species in the Tumor Microenvironment: An Overview
by Frank Weinberg, Nithya Ramnath and Deepak Nagrath
Cancers 2019, 11(8), 1191; https://doi.org/10.3390/cancers11081191 - 16 Aug 2019
Cited by 326 | Viewed by 19395
Abstract
Reactive oxygen species (ROS) are important signaling molecules in cancer. The level of ROS will determine physiological effects. While high levels of ROS can cause damage to tissues and cell death, low levels of ROS can have a proliferative effect. ROS are produced [...] Read more.
Reactive oxygen species (ROS) are important signaling molecules in cancer. The level of ROS will determine physiological effects. While high levels of ROS can cause damage to tissues and cell death, low levels of ROS can have a proliferative effect. ROS are produced by tumor cells but also cellular components that make up the tumor microenvironment (TME). In this review, we discuss the mechanisms by which ROS can affect the TME with particular emphasis on tumor-infiltrating leukocytes. Greater insight into ROS biology in this setting may allow for therapeutic manipulation of ROS levels in order to remodel the tumor microenvironment and increase anti-tumor activity. Full article
(This article belongs to the Special Issue Metabolic Reprogramming and Vulnerabilities in Cancer)
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<p>The mitochondria are the major contributor to cellular reactive oxygen species (ROS) levels while oxidative enzymes (e.g., NAPDH oxidases, cyclooxygenases, lipooxygenases and thymidine phosphorylase) also contribute to cellular ROS pooles. Mitochondrial ROS have many effects on cellular biology including, Mitogen-activated protein kinase (MAPK) (e.g., extracellular-signal-regulated kinase (ERK), p38 MAPK, Jun N-terminal kinase (JNK)), induction of transcription factors (e.g., nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ), hypoxia-inducible transcription factors (HIF), activator protein 1 (AP-1), nuclear respiratory factor (NRF), heat shock factor 1 (HSF-1)) and deregulation of protein phosphatases (e.g., phosphatase and tensin homolog (PTEN)). This leads to enhancement of angiogenesis in the case of HIF, survival, growth, altered metabolism and other cellular processes through MAPKs, transcriptional factors and protein phosphatase and immune cell function and regulation.</p>
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<p>(<b>A</b>) Reactive oxygen species (ROS) generated by the mitochondria and/or exogenous sources within a tumor cell affect tumor immunity to promote a more tumorigenic environment. Mitochondrial ROS (mROS) can stimulate differentiation of cancer-associated fibroblasts (CAFs) and ROS produced by the tumor cell can facilitate uptake of exosomes through caveolin-1 inhibition leading to metabolic reprogramming of certain CAFs. ROS can also affect the function of tumor-infiltrating T-cells depending on the level of mROS. Myeloid-derived suppressor cells (MDSCs) and tumor-associated Macrophages (TAMs) also produce ROS that can affect the function of other immune cells and ROS can affect regulatory T-cell function as well. (<b>B</b>) The amount of ROS corresponds to differing effects on biological function. While cytostatic levels of ROS lead to maintenance of biological processes, cytotoxic levels of ROS lead to cell death as well as immune deregulation. Tumor promotion through ROS occurs when ROS reach super-physiological or cytostatic levels while avoiding levels conducive to cell death. As mentioned previously, oxidative stress can arise from tumor cells.</p>
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13 pages, 9316 KiB  
Perspective
FDG-PET/CT for Response Monitoring in Metastatic Breast Cancer: Today, Tomorrow, and Beyond
by Malene Grubbe Hildebrandt, Jeppe Faurholdt Lauridsen, Marianne Vogsen, Jorun Holm, Mie Holm Vilstrup, Poul-Erik Braad, Oke Gerke, Mads Thomassen, Marianne Ewertz, Poul Flemming Høilund-Carlsen and The Centre for Personalized Response Monitoring in Oncology (PREMIO)
Cancers 2019, 11(8), 1190; https://doi.org/10.3390/cancers11081190 - 15 Aug 2019
Cited by 13 | Viewed by 6810
Abstract
While current international guidelines include imaging of the target lesion for response monitoring in metastatic breast cancer, they do not provide specific recommendations for choice of imaging modality or response criteria. This is important as clinical decisions may vary depending on which imaging [...] Read more.
While current international guidelines include imaging of the target lesion for response monitoring in metastatic breast cancer, they do not provide specific recommendations for choice of imaging modality or response criteria. This is important as clinical decisions may vary depending on which imaging modality is used for monitoring metastatic breast cancer. FDG-PET/CT has shown high accuracy in diagnosing metastatic breast cancer, and the Positron Emission Tomography Response Criteria in Solid Tumors (PERCIST) have shown higher predictive values than the CT-based Response Evaluation Criteria in Solid Tumors (RECIST) for prediction of progression-free survival. No studies have yet addressed the clinical impact of using different imaging modalities or response evaluation criteria for longitudinal response monitoring in metastatic breast cancer. We present a case study of a patient with metastatic breast cancer who was monitored first with conventional CT and then with FDG-PET/CT. We retrospectively applied PERCIST to evaluate the longitudinal response to treatment. We used the one-lesion PERCIST model measuring SULpeak in the hottest metastatic lesion on consecutive scans. This model provides a continuous variable that allows graphical illustration of disease fluctuation along with response categories. The one-lesion PERCIST approach seems able to reflect molecular changes and has the potential to support clinical decision-making. Prospective clinical studies addressing the clinical impact of PERCIST in metastatic breast cancer are needed to establish evidence-based recommendations for response monitoring in this disease. Full article
(This article belongs to the Special Issue Role of Medical Imaging in Cancers)
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<p>FDG-PET/CT performed for the first time in February 2010. Left (<b>a</b>): Maximum-intensity projection image showing an FDG-positive lesion in the left trochanter major, outlined by the blue square, suspicious of bone metastasis. High FDG uptake is seen in activated physiological brown fat tissue, but FDG uptake could not be seen in the primary tumor in the right breast. Right: Axial images of the pelvic region: (<b>b</b>) CT alone, (<b>c</b>) FDG-PET alone), and (<b>d</b>) fused FDG-PET/CT.</p>
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<p>Maximum intensity projection images of a patient with metastatic breast cancer monitored longitudinally with FDG-PET/CT. Baseline scan (<b>A</b>) and pretreatment scans (<b>B</b>–<b>J</b>). Blue squares outline metastatic lesions. Red circles outline the hottest lesion representing a shifting target lesion for which SULpeak was measured using PERCIST 1.0.</p>
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<p>Graphical illustration of changes in PERCIST one-lesion SULpeak in a patient with metastatic breast cancer monitored for more than six years with FDG-PET/CT. Colored response categories: complete metabolic response (CMR), partial metabolic response (PMR), stable metabolic disease (SMD), and progressive metabolic disease (PMD). Grey-toned months represent time points for change of treatment (CoT). Conventional CT was performed Aug 11, Oct 11, and Jan 12. * Indicates scans with detection of new lesions.</p>
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11 pages, 959 KiB  
Review
How to Deal with Second Line Dilemma in Metastatic Colorectal Cancer? A Systematic Review and Meta-Analysis
by Antonio Galvano, Lorena Incorvaia, Giuseppe Badalamenti, Sergio Rizzo, Aurelia Guarini, Stefania Cusenza, Luisa Castellana, Nadia Barraco, Valentina Calò, Sofia Cutaia, Giuseppe Currò, Nicola Silvestris, Giordano Domenico Beretta, Viviana Bazan and Antonio Russo
Cancers 2019, 11(8), 1189; https://doi.org/10.3390/cancers11081189 - 15 Aug 2019
Cited by 5 | Viewed by 3735
Abstract
Monoclonal antibodies targeting epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF) have demonstrated efficacy with chemotherapy (CT) as second line treatment for metastatic colorectal cancer (mCRC). The right sequence of the treatments in all RAS (KRAS/NRAS) wild type (wt) patients [...] Read more.
Monoclonal antibodies targeting epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF) have demonstrated efficacy with chemotherapy (CT) as second line treatment for metastatic colorectal cancer (mCRC). The right sequence of the treatments in all RAS (KRAS/NRAS) wild type (wt) patients has not precisely defined. We evaluated the impact of aforementioned targeted therapies in second line setting, analyzing efficacy and safety data from phase III clinical trials. We performed both direct and indirect comparisons between anti-EGFR and anti-VEGF. Outcomes included disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS), overall survival (OS) and G3-G5 toxicities. Our results showed significantly improved OS (HR 0.83, 95% CI 0.72–0.94) and DCR (HR 1.27, 95% CI 1.04–1.54) favouring anti-VEGF combinations in overall population; no statistically significant differences in all RAS wt patients was observed (HR 0.87, 95% CI 0.70–1.09). Anti-EGFR combinations significantly increased ORR in all patients (RR 0.54, 95% CI 0.31–0.96), showing a trend also in all RAS wt patients (RR 0.63, 95% CI 0.48–0.83). No significant difference in PFS and DCR all RAS was registered. Our results provided for the first time a strong rationale to manage both targeted agents in second line setting. Full article
(This article belongs to the Special Issue Colorectal Cancers)
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<p>Flow diagram (CONSORT) for the meta-analysis included studies (according to the PRISMA statement).</p>
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<p>Forest plot of anti-VEGF vs anti-EGFR combination therapy for clinical endpoints according to mutational status. Abbreviations: disease control rate (DCR); overall response rate (ORR); progression-free survival (PFS); overall survival (OS).</p>
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<p>Forest plot of anti-VEGF vs anti-EGFR combination therapy for most common toxicities.</p>
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<p>Plot for publication bias assessment (Egger’s test <span class="html-italic">p</span> &gt; 0.05).</p>
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<p>Bias summary: Review authors’ judgements about each risk of bias item for each included study.</p>
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13 pages, 1103 KiB  
Article
Anaplastic Thyroid Cancer: Clinical Picture of the Last Two Decades at a Single Oncology Referral Centre and Novel Therapeutic Options
by Joana Simões-Pereira, Ricardo Capitão, Edward Limbert and Valeriano Leite
Cancers 2019, 11(8), 1188; https://doi.org/10.3390/cancers11081188 - 15 Aug 2019
Cited by 23 | Viewed by 4360
Abstract
Anaplastic thyroid cancer (ATC) is a rare tumour but also one of the most lethal malignancies. Therapeutic modalities have usually been limited, but clinical trials with new drugs are now being implemented. The aims of this study were to analyse the clinical presentation, [...] Read more.
Anaplastic thyroid cancer (ATC) is a rare tumour but also one of the most lethal malignancies. Therapeutic modalities have usually been limited, but clinical trials with new drugs are now being implemented. The aims of this study were to analyse the clinical presentation, therapeutic modalities and independent prognostic factors for survival. We also reviewed the most recent literature on novel ATC therapies. We performed a retrospective analysis of 79 patients diagnosed between 2000 and 2018. Variables with impact on survival were identified using the Cox proportional-hazard regression model. At presentation, 6.3% had thyroid-confined disease, 30.4% evidenced extrathyroidal extension and 60.8% were already metastatic. Surgery was feasible in 41.8% and radiotherapy was applied to 35.4%, with those receiving >45 Gy having longer estimated survival (p = 0.020). Chemotherapy, either conventional or with tyrosine kinase inhibitors, was performed in 17.7% and 7.6%, respectively. Multimodality therapy with surgery, radiotherapy and chemotherapy/tyrosine kinase inhibitors (TKI) had the greatest impact on disease specific survival (DSS), providing a risk reduction of death of 96.9% (hazard ratio (HR) = 0.031, 0.005–0.210, p < 0.001). We concluded that most of these patients join reference centres at advanced stages of disease and multimodality treatment may offer the best chances for prolonging survival. Full article
(This article belongs to the Special Issue Thyroid Cancer)
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<p>Anaplastic thyroid cancer incidence.</p>
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<p>Survival functions regarding surgical margins (R0 vs. R1 vs. R2).</p>
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<p>Survival functions regarding radiotherapy dose (≤45 Gy vs. &gt; 45 Gy).</p>
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18 pages, 3220 KiB  
Article
4-Methylumbelliferone Inhibits Cancer Stem Cell Activation and Overcomes Chemoresistance in Ovarian Cancer
by Noor A. Lokman, Zoe K. Price, Emily K. Hawkins, Anne M. Macpherson, Martin K. Oehler and Carmela Ricciardelli
Cancers 2019, 11(8), 1187; https://doi.org/10.3390/cancers11081187 - 15 Aug 2019
Cited by 37 | Viewed by 6055
Abstract
We have recently shown that the extracellular matrix molecule hyaluronan (HA) plays a role in the development of ovarian cancer chemoresistance. This present study determined if HA production is increased in chemotherapy-resistant ovarian cancers and if the HA inhibitor 4-methylubelliferone (4-MU) can overcome [...] Read more.
We have recently shown that the extracellular matrix molecule hyaluronan (HA) plays a role in the development of ovarian cancer chemoresistance. This present study determined if HA production is increased in chemotherapy-resistant ovarian cancers and if the HA inhibitor 4-methylubelliferone (4-MU) can overcome chemoresistance to the chemotherapeutic drug carboplatin (CBP) and inhibit spheroid formation and the expression of cancer stem cell (CSC) markers. We additionally assessed whether 4-MU could inhibit in vivo invasion of chemoresistant primary ovarian cancer cells in the chicken embryo chorioallantoic membrane (CAM) assay. The expression of the HA synthases HAS2 and HAS3 was significantly increased in chemoresistant compared to chemosensitive primary ovarian cancer cells isolated from patient ascites. 4-MU significantly inhibited HA production, cell survival, and spheroid formation of chemoresistant serous ovarian cancer cells. In combination with CBP, 4-MU treatment significantly decreased ovarian cancer cell survival and increased apoptosis of chemoresistant primary cells compared to CBP alone. 4-MU significantly reduced spheroid formation, expression of CSC markers ALDH1A1 and ABCG2 in primary cell spheroid cultures, and ALDH1 immunostaining in patient-derived tissue explant assays following treatment with CBP. Furthermore, 4-MU was very effective at inhibiting in vivo invasion of chemoresistant primary cells in CAM assays. Inhibition of HA is therefore a promising new strategy to overcome chemoresistance and to improve ovarian cancer survival. Full article
(This article belongs to the Special Issue Cancer Stem Cells and Personalized Medicine for Gynecologic Cancers)
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<p>Serum hyaluronan (HA) is elevated in patients with chemoresistant disease. (<b>a</b>) HA serum levels (ng/mL) in serous ovarian cancer patients at initial diagnosis and following relapse with chemoresistant disease (<span class="html-italic">n</span> = 9). * significantly different from levels at diagnosis (<span class="html-italic">p</span> = 0.0039, Wilcoxon pair test). (<b>b</b>) HA serum levels (ng/mL) in serous ovarian cancer patients at initial diagnosis and following relapse with chemosensitive disease (<span class="html-italic">n</span> = 7, <span class="html-italic">p</span> = 0.219, Wilcoxon pair test).</p>
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<p>Hyaluonan (HA) synthase and hyaluronidase expression and HA production in chemosensitive and chemoresistant serous ovarian cancer cells. Expression in chemotherapy-resistant primary serous ovarian cancer cells compared to chemotherapy-sensitive cells and OV-90 cells made resistant to carboplatin (OV-90 CBPR). <span class="html-italic">HAS1</span> (<b>a</b>), <span class="html-italic">HAS2</span> (<b>b</b>) <span class="html-italic">HAS3</span> (<b>c</b>), <span class="html-italic">HYAL1</span> (<b>d</b>), and <span class="html-italic">HYAL2</span> (<b>e</b>) *, <span class="html-italic">HAS2</span> (<span class="html-italic">p</span> = 0.0218, Mann Whitney U test) and <span class="html-italic">HAS3</span> (<span class="html-italic">p</span> = 0.0107, Mann Whitney U test) but not <span class="html-italic">HAS1</span> expression (<span class="html-italic">p</span> = 0.879, Mann Whitney U test) was significantly increased in chemoresistant cells compared to chemosensitive cells. **, <span class="html-italic">HAS2</span> (<span class="html-italic">p</span> = 0.021, Student <span class="html-italic">t</span> test) and <span class="html-italic">HAS3</span> (<span class="html-italic">p</span> &lt; 0.0001, Student <span class="html-italic">t</span> test) were significantly increased in OV-90 CBPR compared to parental cells. <span class="html-italic">HYAL1</span> and <span class="html-italic">HYAL2</span> expression was not significantly different between the chemosensitive and chemoresistant primary cancer cells nor the OV-90 cell lines. The bars for the primary cells specify the median values in each group and are expressed as the mean fold change from RNA samples (<span class="html-italic">n</span> = 6–9) from three independent experiments. Data for OV-90 cells are expressed as the mean fold change ± SEM from 7–12 individual RNA samples from 2–3 independent experiments. (<b>f</b>) HA levels measured by ELISA assay in conditioned media. *, significantly increased in primary chemoresistant (<span class="html-italic">n</span> = 8) compared to chemosensitive (<span class="html-italic">n</span> = 10) serous ovarian cancer cells (<span class="html-italic">p</span> = 0.043, Mann Whitney U test). **, significantly increased in OV-90 CBPR conditioned media compared to parental cells (<span class="html-italic">p</span> = 0.0227, Mann Whitney U test).</p>
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<p>Effects of 4-methylubelliferone (4-MU) and carboplatin on ovarian cancer cell survival and HA production. (<b>a</b>) Ovarian cancer cell survival following 72 h of 4-MU (1 mM) treatment. The black and grey bars represent chemosensitive and chemoresistant cells, respectively. Data are expressed as percentage of control, mean ± SEM from 2–3 independent experiments performed in triplicate. *, significantly different from control media treatment (<span class="html-italic">p</span> &lt; 0.05, independent <span class="html-italic">t</span> test). (<b>b</b>). Effect of 4-MU (1 mM) 72 h on HA production in conditioned media measured by HA ELISA. Data are expressed as ng/mL from 4–6 determinations. *, significantly different from control treatment (<span class="html-italic">p</span> &lt; 0.05, independent <span class="html-italic">t</span> test). Effect of 4-MU on survival of OV-90 (<b>c</b>), chemosensitive primary cells (<span class="html-italic">n</span> = 2) (<b>d</b>), SKOV3 (<b>e</b>), and chemoresistant primary cells (<span class="html-italic">n</span> = 8), (<b>f</b>) assessed by MTT assay. Cells were treated with phosphate buffered saline (PBS), control, 4-MU (1 mM), carboplatin (CBP, 100 µM), and 4-MU (1 mM) + CBP (100 µM) for 72 h. Data are expressed as % of control from 2–5 independent experiments performed in quadruplicate. Effects of 4-MU (1 mM) and/or CBP (100 µM) treatment on apoptosis measured by caspase 3/7 cleavage in primary serous ovarian cancer cells. (<b>g</b>) chemosensitive (<span class="html-italic">n</span> = 3), and (<b>h</b>) chemoresistant (<span class="html-italic">n</span> = 5) cells. (<b>a</b>–<b>h</b>) *, significantly different from control, **, significantly different from CBP treatment, ***, significantly different from 4-MU treatment, (<span class="html-italic">p</span> &lt; 0.05, one way ANOVA, Tukey’s multiple comparisons test).</p>
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<p>Effects of 4-methylubelliferone (4-MU) on spheroid formation. Representative images of spheroids formed by (<b>a</b>) OV-90 and (<b>b</b>) one of the chemoresistant primary ovarian cancer cells treated with control, 4-MU (1 mM), carboplatin (CBP, 100 µM), and 4-MU (1 mM) + CBP (100 µM) for 72 h. Data are expressed as median area of spheroids &gt;200 µm in diameter (95% confidence interval, CI, <span class="html-italic">n</span> = 34–106) in five fields/treatment groups from 3–4 independent experiments. *, significantly different from control, **, significantly different from CBP treatment (<span class="html-italic">p</span> &lt; 0.05, Kruskal–Wallis test, Dunn’s multiple comparison test). Scale bar = 1000 µm.</p>
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<p>Effects of 4-methylubelliferone (4-MU) on stem cell marker expression in spheroids. Expression of stem cell markers in spheroids from a chemotherapy-resistant primary serous ovarian cancer and OV-90 cells treated with control media or 4-MU (1 mM) for 72 h. <span class="html-italic">HAS2</span> (<b>a</b>), <span class="html-italic">HAS3</span> (<b>b</b>), <span class="html-italic">PROM1</span> (<b>c</b>), <span class="html-italic">ALDH1A1</span> (<b>d</b>), <span class="html-italic">ABCG2</span> (<b>e</b>), and <span class="html-italic">CD44</span> (<b>f</b>). Data are expressed as the median from RNA samples (<span class="html-italic">n</span> = 5–6) from three independent experiments. *, significantly different from control, (<span class="html-italic">p</span> &lt; 0.05, Mann Whitney U test).</p>
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<p>Effect of chemotherapy and 4-methylubelliferone (4-MU) on ALDH1 immunostaining in ovarian cancer tissues. (<b>a</b>) Quantitation of ALDH1 immunostaining in serous ovarian cancer explants tissues (<span class="html-italic">n</span> = 5) treated with control media, carboplatin alone (100 µM), 4-MU alone (1 mM), and CBP + 4-MU in combination. Data are expressed as percentage positive area (% POS) measured by video image analysis. *, Significantly different from control treatment (<span class="html-italic">p</span> = 0.0087, Friedman test, Dunn’s multiple comparison test). Representative images of ALDH1 immunostaining in the explant tissues treated with control media (<b>b</b>), CBP (<b>c</b>), 4-MU (<b>d</b>), and CBP+4-MU (<b>e</b>). (<b>f</b>) Quantitation of ALDH1 immunostaining in serous ovarian cancer tissues at diagnosis (<span class="html-italic">n</span> = 15), following neoadjuvant chemotherapy (<span class="html-italic">n</span> = 20) and at recurrence (<span class="html-italic">n</span> = 5). Data are expressed as % POS area. *, Significantly reduced from level at diagnosis (<span class="html-italic">p</span> = 0.0002, Kruskal–Wallis test, Dunn’s multiple comparison test). ALDH1 immunostaining in matched patient tissues at diagnosis (<b>g</b>) and following neoadjuvant chemotherapy (<b>h</b>). ALDH1 immunostaining in matched patient tissues at diagnosis (<b>i</b>) and at relapse with chemoresistant disease (<b>j</b>). All images are the same magnification. Scale bar = 100 µm.</p>
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<p>4-MU and carboplatin treatment inhibit in vivo invasion of chemoresistant primary cells. Representative images of the invasion of chemoresistant primary ovarian cancer cells shown by CD44 immunostaining. Treatment includes (<b>a</b>) control media, (<b>b</b>) 4-MU (1 mM), (<b>c</b>) carboplatin (CBP 100 µM, and (<b>d</b>) CBP 100µM + 4-MU (1 mM). Paraffin sections were immunostained with a CD44 mouse monoclonal antibody. Asterisks show examples of CD44 positive cancer cells that have invaded into the mesoderm (MES). (<b>e</b>) Quantitation of chorioallantoic membrane (CAM) invasion into the mesoderm. Data are of the CD44-positive area (µm<sup>2</sup>/mm<sup>2</sup> of mesoderm) from 5–9 chick embryos implanted with chemoresistant primary cells from one patient. *, Significantly different from control (<span class="html-italic">p</span> = 0.0272, Kruskal–Wallis test, Dunn’s multiple comparison test). CM = cancer cells in matrigel implant, ECT = ectoderm, MES = mesoderm, END = endoderm. Scale bar = 100 µm.</p>
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14 pages, 2561 KiB  
Article
Clinical Relevance of Collagen Protein Degradation Markers C3M and C4M in the Serum of Breast Cancer Patients Treated with Neoadjuvant Therapy in the GeparQuinto Trial
by Malgorzata Banys-Paluchowski, Sibylle Loibl, Isabell Witzel, Christoph Mundhenke, Bianca Lederer, Christine Solbach, Thomas Karn, Frederik Marmé, Valentina Nekljudova, Christian Schem, Elmar Stickeler, Nicholas Willumsen, Morten A. Karsdal, Michael Untch and Volkmar Müller
Cancers 2019, 11(8), 1186; https://doi.org/10.3390/cancers11081186 - 15 Aug 2019
Cited by 11 | Viewed by 4488
Abstract
Background: Remodeling of extracellular matrix through collagen degradation is a crucial step in the metastatic cascade. The aim of this study was to evaluate the potential clinical relevance of the serum collagen degradation markers (CDM) C3M and C4M during neoadjuvant chemotherapy for [...] Read more.
Background: Remodeling of extracellular matrix through collagen degradation is a crucial step in the metastatic cascade. The aim of this study was to evaluate the potential clinical relevance of the serum collagen degradation markers (CDM) C3M and C4M during neoadjuvant chemotherapy for breast cancer. Methods: Patients from the GeparQuinto phase 3 trial with untreated HER2-positive operable or locally advanced breast cancer were enrolled between 7 November 2007, and 9 July 2010, and randomly assigned to receive neoadjuvant treatment with EC/docetaxel with either trastuzumab or lapatinib. Blood samples were collected at baseline, after four cycles of chemotherapy and at surgery. Cutoff values were determined using validated cutoff finder software (C3M: Low ≤9.00 ng/mL, high >9.00 ng/mL, C4M: Low ≤40.91 ng/mL, high >40.91 ng/mL). Results: 157 patients were included in this analysis. At baseline, 11.7% and 14.8% of patients had high C3M and C4M serum levels, respectively. No correlation was observed between CDM and classical clinical-pathological factors. Patients with high levels of CDM were significantly more likely to achieve a pathological complete response (pCR, defined as ypT0 ypN0) than patients with low levels (C3M: 66.7% vs. 25.7%, p = 0.002; C4M: 52.7% vs. 26.6%, p = 0.031). Median levels of both markers were lower at the time of surgery than at baseline. In the multivariate analysis including clinical-pathological factors and C3M levels at baseline and changes in C3M levels between baseline and after four cycles of therapy, only C3M levels at baseline (p = 0.035, OR 4.469, 95%-CI 1.115–17.919) independently predicted pCR. In a similar model including clinical-pathological factors and C4M, only C4M levels at baseline (p = 0.028, OR 6.203, 95%-CI 1.220–31.546) and tumor size (p = 0.035, OR 4.900, 95%-CI 1.122–21.393) were independent predictors of pCR. High C3M levels at baseline did not correlate with survival in the entire cohort but were associated with worse disease-free survival (DFS; p = 0.029, 5-year DFS 40.0% vs. 74.9%) and overall survival (OS; p = 0.020, 5-year OS 60.0% vs. 88.3%) in the subgroup of patients randomized to lapatinib. In the trastuzumab arm, C3M did not correlate with survival. In the entire patient cohort, high levels of C4M at baseline were significantly associated with shorter DFS (p = 0.001, 5-year DFS 53.1% vs. 81.6%) but not with OS. When treatment arms were considered separately, the association with DFS was still significant (p = 0.014, 5-year DFS 44.4% vs. 77.0% in the lapatinib arm; p = 0.023, 5-year DFS 62.5% vs. 86.2% in the trastuzumab arm). Conclusions: Collagen degradation markers are associated with response to neoadjuvant therapy and seem to play a role in breast cancer. Full article
(This article belongs to the Special Issue New Biomarkers in Cancers)
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<p>Serum collection in the HER2-positive cohort in the GeparQuinto trial.Abbreviations: EC–epirubicin/cyclophosphamide, T–docetaxel, H–trastuzumab, L–lapatinib.</p>
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<p>Flow diagram showing the number of patients with a blood sample collected at different time points.</p>
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<p>Kaplan–Meier plots of disease-free and overall survival stratified by C3M levels at baseline.</p>
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<p>Kaplan–Meier plots of disease-free and overall survival stratified by C4M levels at baseline.</p>
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<p>Kaplan–Meier plots of disease-free and overall survival stratified by changes in C3M levels between baseline and after four cycles of therapy (‘no change’ is defined as an increase or decrease &lt;20%).</p>
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