CN112578116A

CN112578116A - Applications of CLU (CLU), PRKD3 and down-regulation or inhibitor thereof in detection and typing, treatment and curative effect evaluation of triple negative breast cancer

Info

Publication number: CN112578116A
Application number: CN202011228037.1A
Authority: CN
Inventors: 陈礼明; 刘岩; 张俊
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2021-03-30

Abstract

The invention discloses CLU, PRKD3 and application of a down-regulation or inhibitor thereof in detection and typing, treatment and curative effect evaluation of triple negative breast cancer. The invention provides CLU and/or PRKD3 as a molecular marker for TNBC diagnosis, typing and prognosis efficacy evaluation, as a TNBC therapeutic drug target, and a CLU and/or PRKD3 down-regulator or inhibitor for preparing a drug for treating TNBC, and also provides a histological and serological molecular marker combination for diagnosing TNBC and typing and prognosis efficacy evaluation and a drug combination for treating TNBC; the invention discovers that CLU and PRKD3 are used as TNBC diagnosis, parting and prognosis curative effect evaluation molecular markers and therapeutic drug targets for the first time, CLU silencers and PRKDs inhibitors can respectively inhibit the activity of CLU expression and PRKD3, and the TNBC tumor growth inhibition effect is obvious.

Description

Applications of CLU (CLU), PRKD3 and down-regulation or inhibitor thereof in detection and typing, treatment and curative effect evaluation of triple negative breast cancer

Technical Field

The invention belongs to the field of breast cancer detection, typing, diagnosis, prognosis effect evaluation and treatment research, and particularly relates to an effect of CLU and PRKD3 in Triple Negative (TNBC) detection and treatment as molecular markers and drug targets, and an application of a CLU and/or PRKD3 down-regulator or inhibitor in preparation of a drug for inhibiting or treating TNBC.

Background

In women's cancer worldwide, the number of breast cancer attacks and deaths are located in the first two, and the incidence of disease is rising year by year, which is a major killer that endangers women's health. Triple Negative Breast Cancer (TNBC) is clinically manifested by low expression of Estrogen Receptor (ER), Progesterone Receptor (PR) and human epidermal growth factor receptor 2(HER 2). Unlike ER + and HER2+ positive breast cancer patients, TNBC patients currently undergo surgery, chemotherapy and radiotherapy only due to a lack of drug recognition targets. TNBC survival prognosis is the worst of all breast cancer subtypes. Therefore, further exploring the pathological mechanism of TNBC to explore the therapeutic target point is very important and urgent for TNBC clinical treatment and improvement of survival prognosis of TNBC patients.

Clusterin (CLU) has been studied and reported to be a stress-activated, ATP-independent chaperone that is secreted from cells after maturation. Studies have shown that CLU plays an important role in the development of many cancers, but the specific mechanism of action remains unclear. Protein kinase D3(PRKD3) belongs to the multigenic protein kinase D family of serine/threonine kinases and has been shown to regulate a variety of cancer progression.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides the application of CLU and/or PRKD3 as a molecular marker for TNBC diagnosis, typing and prognosis evaluation, and provides a new way and a product for TNBC diagnosis and treatment guidance.

The invention also provides application of a down-regulating agent or an inhibitor of CLU and/or PRKD3 in preparation of a medicament for inhibiting or treating TNBC.

The technical scheme is as follows: to achieve the above objects, the use of CLU and/or PRKD3 as defined in the present invention as a molecular marker for TNBC diagnosis, typing and prognosis evaluation.

Preferably, CLU and/or PRKD3 are used as histological and/or serological markers in the preparation of index reagents or kits for TNBC diagnosis, typing, curative effect and prognosis evaluation.

Preferably, the CLU and/or PRKD3 is used as a drug intervention target or a molecular marker in TNBC diagnosis, typing and prognosis evaluation and therapeutic drug preparation.

Wherein the CLU and PRKD3 comprise detecting the expression level of CLU and PRKD3 in a tumor/blood sample from a subject having breast cancer, wherein the appearance of an elevated expression level of CLU and PRKD3 is correlated with the presence of TNBC in the subject.

Wherein, the expression level of the CLU and PRKD3 in a normal tissue of a mammary gland is obviously lower than that of a cancer tissue, the expression level in a TNBC cancer tissue is obviously higher than that of a tissue beside the cancer, and the CLU in the serum of a TNBC patient is obviously higher than that of a healthy population.

The CLU and/or PRKD3 provided by the invention is applied as a TNBC key drug intervention target.

The invention relates to an application of a CLU and/or PRKD3 down-regulating agent or inhibitor in preparing a drug for inhibiting or treating TNBC.

Wherein the downregulator comprises a downregulator (silencer) of a CLU and a PRKDs inhibitor.

Preferably, the down-regulator (silencer) of the CLU is OGX-011, and the PRKDs inhibitor is CRT 0066101.

Further, the silencing agent OGX-011 and the inhibitor CRT0066101 can be used alone or in combination with other TNBC-treating drugs.

The invention relates to a group of molecular markers and therapeutic drug combinations applied to TNBC diagnosis, typing, treatment and curative effect evaluation, which comprise CLU and/or PRKD3 as molecular markers, and CLU and/or PRKD3 as down-regulating agents or inhibitors as therapeutic drugs.

The present study found that PRKD3 can modulate CLU protein levels, but does not affect CLU mRNA levels. Further experiments show that PRKD3 can regulate the stability of CLU protein, and PRKD3 can be used as a key regulator of CLU. Cell and mouse experiments found that PRKD3 and CLU promote TNBC tumor growth, and CLU and PRKD3 protein levels were found to be significantly and positively correlated in tumor samples of TNBC patients. More importantly, evidence from in vitro and in vivo clinical trials suggests that treatment with CLU silencers (OGX-011) and PRKDs inhibitors (CRT0066101) can inhibit TNBC tumor growth. CLU (sCLU) secreted in the serum of TNBC patients is obviously increased compared with that of a normal group, and OGX-011 and/or CRT0066101 treatment causes the sCLU in the serum of TNBC mice to be reduced and the growth of TNBC tumors is obviously inhibited. Based on the above research results, it is feasible to use CLU and PRKD3 as drug targets in TNBC treatment, and the effect of drug combination against CLU and PRKD3 is obvious.

According to the invention, cell growth is inhibited by knocking out PRKD3 in TNBC cells, and cell growth can be recovered by over-expression of CLU or PRKD3 in PRKD3-KO TNBC cells. The above findings were further confirmed in a xenograft murine model using TNBC cancer cell line. Consistent with in vitro cell experiments, tumors formed by PRKD3-KO TNBC cells showed significantly reduced tumor volume and weight compared to tumors formed by parental cells in mice, and overexpression of CLU or PRKD3 in PRKD3-KO TNBC cells restored TNBC tumor growth. The above data indicate that PRKD 3-stable CLU is required for TNBC tumor growth.

Another aspect of the invention also addresses the clinical relevance of PRKD3 to CLU. The invention shows that the expression levels of CLU and PRKD3 protein in clinical TNBC tumor tissues are in positive correlation through immunohistochemistry. The quantification and analysis of the expression levels of CLU and PRKD3 proteins in TNBC tumor tissues showed that the levels of CLU and PRKD3 proteins in TNBC cancer tissues were significantly increased on average and in a significant positive correlation with those in paraneoplastic tissues. The above data indicate that CLU and PRKD3 show a positive clinical correlation in TNBC.

The invention also provides that targeting PRKD3 and CLU shows effective clinical treatment effect. The effect on TNBC treatment was examined by targeting CLU and its modulator PRKD3 using CLU silencing agent (OGX-011) and PRKDs inhibitor (CRT 0066101). Treatment with OGX-011(OGX) and CRT0066101(CRT) in a TNBC cancer cell line xenograft mouse model can significantly inhibit TNBC tumor growth. Further using a mouse model of TNBC tumor derived from TNBC patients, treatment with OGX and CRT alone or in combination significantly inhibited TNBC tumor growth. The above data indicate that TNBC treatment is significantly effective by targeting CLU and PRKD 3.

The invention provides application of Clusterin (CLU) and protein kinase D3(PRKD3) as diagnosis and treatment markers of Triple Negative Breast Cancer (TNBC), and application of the Clusterin (CLU) and the protein kinase D3(PRKD3) as single and combined medicines of target-targeted medicines OGX-011 and CRT0066101 for treating TNBC medicines. In vitro and in vivo experiments show that over-expression of CLU and PRKD3 in a knockout PRKD3 cell can obviously restore proliferation of breast tumors, and meanwhile, in a mouse and a TNBC patient xenograft tumor model, a CLU silencing agent (OGX-11) and a PRKDs inhibitor (CRT0066101) can be used singly or jointly to obviously inhibit growth of TNBC tumors in vitro and in vivo, and the tumor remission rate can reach more than 95 percent at most. Clinical tumor sample research finds that PRKD3 and CLU can be used as further typing indexes of triple negative breast cancer, and serological data of TNBC patients indicate that CLU in blood can be used for detecting triple negative breast cancer and evaluating the curative effect of the related drug treatment. The above experimental results show that CLU and PRKD3 as molecular markers can be used as the diagnosis, typing and curative effect evaluation indexes of TNBC; OGX-011 and CRT0066101 are respectively targeted to CLU and PRKD3 as drug targets, and can effectively treat triple negative breast cancer singly and jointly.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention discovers that CLU and PRKD3 are used as TNBC diagnosis, parting and prognosis evaluation molecular markers and treatment targets for the first time, CLU silencing agent (OGX-011) and PRKDs inhibitor (CRT0066101) can respectively inhibit CLU expression and PRKD3 activity, have obvious effect on TNBC tumor growth inhibition, provide the application of CLU and/or PRKD3 down-regulating agent or inhibitor in the preparation of TNBC inhibition or treatment drugs, and simultaneously provide the molecular marker and treatment drug combination applied to TNBC diagnosis, parting, treatment and curative effect evaluation.

According to the invention, experimental research shows that PRKD3 can regulate CLU stability, is in positive correlation clinically, and can inhibit the growth phenomenon of TNBC tumor by targeted inhibition of CLU and PRKD 3. The experimental results show that CLU and PRKD3 as molecular markers can be used as the diagnosis, typing and curative effect evaluation indexes of TNBC; as drug targets, OGX-011 and CRT0066101 target silencing or inhibiting drugs of CLU and PRKD3, respectively, alone and in combination can effectively treat triple negative breast cancer.

Drawings

FIG. 1 is a graph of the results of a stable CLU with PRKD 3;

among them, (A-B) is a Western blot showing that the knock-out of PRKD3(PRKD3-KO) resulted in a decrease in both pCLU and sCLU protein levels and in cell culture medium in TNBC cells MDA-MB-468 and MDA-MB-231. (C-D) is a real-time fluorescent quantitative PCR map, and the results show that the knockout of PRKD3(PRKD3-KO) does not cause the mRNA level of CLU to change in TNBC cells MDA-MB-468 and MDA-MB-231. (E-F) is a Cycloheximide (CHX) experimental graph, and results show that the knockout of PRKD3(PRKD3-KO) leads to the reduction of protein stability of pCLU and sCLU in cell culture medium in TNBC cells MDA-MB-468 and MDA-MB-231.

FIG. 2 is a graph showing the results of PRKD3 and CLU promoting TNBC tumor growth;

wherein (A-B) is a cell proliferation experimental graph, and the result shows that the deletion of PRKD3(PRKD3-KO) in TNBC cells MDA-MB-468 and MDA-MB-231 inhibits cell proliferation, and the over-expression of PRKD3 and CLU can restore the cell proliferation capacity. (C-D) is a clone formation experiment chart, and the result shows that the deletion of PRKD3(PRKD3-KO) in TNBC cells MDA-MB-468 and MDA-MB-231 inhibits the formation of cell clones, and the over-expression of PRKD3 and CLU can restore the cell clone formation capability. (E-F) is a statistical plot of the size of the mouse tumorigenic tumors, and the results show that the knockout of PRKD3(PRKD3-KO) inhibits the growth of the tumors, while over-expression of PRKD3 and CLU can restore the growth of the tumors. (G-H) is a solid map of the mouse tumorigenic tumors. (I-J) is a statistical graph of tumor weights of mice, and the result shows that the knockout of PRKD3(PRKD3-KO) can inhibit the reduction of tumor weights, and the over-expression of PRKD3 and CLU can recover the tumor weights.

FIG. 3 is a graph of the results of the clinical correlation of PRKD3 with CLUs;

where (a) is an Immunohistochemistry (IHC) profile, the results show elevated PRKD3 and CLU expression in TNBC tumor tissue. (B) The results are a statistical plot of PRKD3 and CLU expression in tissues of clinical patients and show that the ratio of PRKD3 to CLU expression is highest in TNBC tumors. (C) For immunohistochemical analysis of the statistical figures, the results showed that PRKD3 was significantly positively correlated with CLU in TNBC tumors. (D) For semi-quantitative immunohistochemical analysis statistical figures, PRKD3 and CLU expression were elevated in TNBC cancer tissues compared to paracancerous tissues. (E) And (4) a correlation analysis chart shows that the expression of PRKD3 and CLU is in positive correlation in the TNBC tumor.

Fig. 4 is a graph of the results of targeting PRKD3 with CLU showing effective clinical treatment.

Wherein (A) is OGX and/or CRT0066101 in the mouse experiment process diagram. (B-E) shows that the TNBC cell line MDA-MB-468 xenografted mice had tumors, and the results show that OGX and/or CRT has good effect on inhibiting the growth of TNBC tumors, and the weight of the mice is not affected. (F-I) shows that the TNBC cell line MDA-MB-231 xenografted mice have tumors, and the results show that OGX and/or CRT have good effect on inhibiting the growth of TNBC tumors and do not influence the body weight of the mice. (J) The TNBC tumor tissue immunofluorescence image shows that the expression of ER, PR and HER2 is deleted, and the expression of PRKD3 and CLU in the tissue is increased. (K) A map is generated for the TNBC tumor tissue organoids, and the results show that TNBC tumor tissue organoids formation is reduced after OGX and/or CRT treatment. (L) is a TNBC tumor tissue organoid proliferation map, and the results show that OGX and/or CRT effectively inhibited TNBC organoid proliferation. (M) shows the immunofluorescence chart of the patient tumor tissue xenograft tumor, and the result shows that the expression of ER, PR and HER2 is deficient, and the expression of PRKD3 and CLU in the tissue is increased. (N) is a statistical plot of the volume size of the patient's tumor tissue xenografts, showing that OGX and/or CRT effectively inhibited the proliferation of the patient's tumor tissue xenografts. (O) is a statistical plot of mouse body weight, and the results show that OGX and/or CRT were used without affecting mouse body weight. (P) is the solid map of the mouse tumorigenic tumors. (Q) is a mouse tumorigenic tumor weight histogram, and the results show that OGX and/or CRT effectively inhibited the proliferation of patient tumor tissue xenografts.

Detailed Description

The invention is further illustrated by the following figures and examples.

Materials, reagents and the like used in examples are commercially available unless otherwise specified.

The biological materials, reagents, kits and the like used in the invention can be obtained by conventional commercial purchase, and the experimental techniques are all routine operations in the field or operations according to the instruction of corresponding commodities.

The sources or types of the biological materials used in the invention are as follows:

TNBC cell MDA-MB-231(

HTB-26^TM)；

TNBC cell MDA-MB-468(

HTB-132^TM)。

PRKD3-KO cell source and construction, PRKD3-KO + PRKD3 and PRKD3-KO + CLU cell construction are all referred to in Liu, Yan et al, "Protein Kinase D3 proteins the cell promotion by activation the ERK1/c-MYC axis in Breast cancer, Journal of cellular and molecular medium.vol.24 (2020), 2135-2144.doi:10.1111/jcmm.14772, TNBC cell-MB-231 (MDA-M A-M-D-M

HTB-26^TM) And TNBC cells MDA-MB-468(

HTB-132^TM) Construction was performed for the original wild-type cells.

The sources of the biological and chemical reagents used in the invention are shown in table 1:

TABLE 1 sources of biological and chemical reagents

Example 1

PRKD3 stabilizes CLUs

Western blot experiment: collecting TNBC wild type cells (MDA-MB-468 and MDA-MB-231) and TNBC cells (PRKD3-KO) knocked out by PRKD3 in a 60mm cell culture dish in logarithmic growth phase, adding precooled 500 mul of RIPA lysate (5 mul of protease inhibitor is added) to lyse the cells, collecting the cell lysate to quantify the protein concentration, adding 5 x loading buffer after quantification, boiling for 10 minutes at 95 ℃, taking 10 mul of protein sample to load in SDS-PAGE gel, carrying out electrophoresis, and then transferring the membrane. After the completion of the membrane transfer, the PVDF membrane was taken out and blocked with 50ml of 5% skim milk. After the blocking is finished, a target band is cut out and incubated with different primary antibodies, namely PRKD3, CLU and beta-actin. After gentle shaking at 4 ℃ overnight, the column was washed 5 times with TBST buffer for 5 minutes each. After washing, the target bands bound to the primary antibody were incubated with a secondary antibody corresponding to horseradish peroxidase: anti-mouse IgG and anti-rabbit IgG (purchased from Santa Cruz Biotechnology), antibodies were diluted as per instructions, secondary antibody was incubated with the membrane for 1 hour at room temperature, and after incubation of secondary antibody was completed, washed 5 times with TBST buffer for 5 minutes each. And taking out the membrane, adding a developing solution, and detecting the change of the target protein by using a biomolecule imager analysis system. Experimental results figures 1A and 1B show that PRKD3 knockdown significantly reduced CLU protein levels.

RNA extraction, reverse transcription experiment: OMEGA Total RNA extraction kit was used, operating according to the product instructions. After elution, the RNA concentration was measured by Nanodrop 2000, and reverse transcription was performed. Reverse transcription reaction system: a reaction volume of 50. mu.L contained 2500ng RNA, 10. mu.L of 5 XPrimeScript RT Master Mix and double distilled water. The reaction was carried out in a PCR apparatus according to the following set-up procedure: 15min at 37 ℃; 85 ℃ for 5 sec.

Real-time PCR experiment: using the reverse transcription product cDNA as a template, and carrying out PCR amplification by using the following primers: mu.L of the reaction system contained 1. mu.L of cDNA template, 0.2. mu.L (10nmol) of forward primer, 0.2. mu.L (10nmol) of sequence reverse primer, 5. mu.L of 2 XSSYBR Mix, 3.6. mu.L of double distilled water. The specific primer sequences are shown in the following table 2:

TABLE 2

The reaction was carried out for 40 cycles on a CFX96TMReal-Time System fluorescent quantitative PCR instrument according to the following set program: pre-denaturation at 95 ℃ for 30 sec; denaturation, 95 ℃ for 5 sec; annealing at 60 ℃ for 30 sec. Experimental results figures 1C and 1D show that PRKD3 knock-out did not affect CLU mRNA levels.

Cycloheximide (CHX) treatment experiment: cycloheximide is an organic substance that inhibits protein synthesis. TNBC wild-type cells and PRKD3 knock-out TNBC cells were plated in six-well plates and treated 24 hours later with CHX (final concentration 100 μ g/ml) for 0 hour, 6 hours, and 18 hours, respectively, and cell pellets were collected and assayed for change in CLU protein level by western blot. Experimental results fig. 1E and 1F show that protein stability of CLU is decreased following PRKD3 knockdown.

Example 2

PRKD3 and CLU promote TNBC tumor growth

Cell proliferation assay:

collecting wild TNBC cells MDA-MB-468 and MDA-MB-231 in logarithmic growth phase, digesting with 0.25% trypsin to obtain single cell suspension, counting with hemacytometer, adding 5000 cells and 100 μ L of culture medium DMEM (Gibco), 37 deg.C, 5% CO in each well of six-well plate₂Culturing in a cell culture box, and arranging 3 multiple wells for each of the experimental group and the control group. The control group is wild type TNBC cell (WT), the experimental group is PRKD3 knockout cell (PRKD3-KO), the PRKD3 knockout over-expresses PRKD3 cell (PRKD3-KO + PRKD3) and the PRKD3 knockout over-expresses CLU cell (PRKD3-KO + CLU). The old medium in 96 wells was discarded every 24 hours and replaced with fresh medium. According to the Cell Counting Kit-8 Kit protocol, 90. mu.L of complete medium plus 10. mu.L of CCK-8 solution were added to each well and the 96-well plate was returned to the incubator for incubation for 2 h. The mixed culture solution with the original 96-well plate changed in color is transferred to a new 96-well plate, and the absorbance (A) value at 450nm is measured by a microplate reader.The experimental results (fig. 2A and 2B) show that PRKD3 knock-out significantly inhibited TNBC cell growth, and overexpression of PRKD3 and CLU restored TNBC cell growth.

Plate cloning experiment:

collecting cells MDA-MB-468 and MDA-MB-231 in logarithmic growth phase, digesting with 0.25% trypsin to obtain single cell suspension, counting with hemacytometer, adding 500 cells and 2mL culture medium DMEM (Gibco) into each well of six-well plate, 37 deg.C, and 5% CO₂Culturing in a cell culture box, and arranging 3 multiple wells for each of the experimental group and the control group. The control group is wild type TNBC cell (WT), the experimental group is PRKD3 knockout cell (PRKD3-KO), the PRKD3 knockout over-expresses PRKD3 cell (PRKD3-KO + PRKD3) and the PRKD3 knockout over-expresses CLU cell (PRKD3-KO + CLU). Cultured in a 5% CO2 cell culture box at 37 ℃ for one week. The supernatant was discarded and carefully rinsed 3 times with PBS. Cells were fixed with 500. mu.L of 4% paraformaldehyde PFA for 30 min at room temperature. Then the fixative solution is removed, a proper amount of GIMSA is added to apply the staining solution for staining for 30 minutes, then the staining solution is slowly washed away by running water, and the air is dried. The six-well plate was inverted over a sheet of A4 white paper, photographed to record the results, and finally counted for clones using ImageJ. Experimental results fig. 2C and 2D show that PRKD3 knock-out significantly inhibited TNBC cell growth, and overexpression of PRKD3 and CLU restored TNBC cell growth.

Mouse tumorigenesis experiments:

female nude mice, 3-4 weeks old, were purchased from Shanghai Su commercial Biotechnology, Inc. Will be 5X 10⁶Each TNBC cell (MDA-MB-231, MDA-MB-468) was injected into the mammary fat pad of the mice. The control group was wild-type TNBC cells (WT), the experimental group was PRKD3 knock-out cells (PRKD3-KO), PRKD3 knock-out cells overexpressing PRKD3 cells (PRKD3-KO + PRKD3) and PRKD3 knock-out cells overexpressing CLU cells (PRKD3-KO + CLU), and the body weight of nude mice was observed and weighed once a week after cell injection and the size of tumors was measured with a vernier caliper. At about 60 days, the nude mice were transferred to a dissecting room, sacrificed uniformly, the subcutaneous tumors were dissected out and weighed, the length and width of the tumors were measured, and the relevant data were recorded. According to the formula 1/2 (length) x (width)²Calculating the volume of tumor. Fig. 2E and 2F show that PRKD3 knockout can be significantThe tumor body of TNBC tumor is increased, and the tumor body of TNBC tumor can be recovered by over-expressing PRKD3 and CLU. Fig. 2G and 2H are solid maps of tumors, and fig. 2I and 2J show that PRKD3 knockout significantly inhibits tumor weight gain of TNBC tumors, and that overexpression of PRKD3 and CLU restores tumor weight gain of TNBC tumors. In summary, PRKD3 knock-out significantly inhibited TNBC tumor growth, and overexpression of PRKD3 and CLU restored TNBC tumor growth.

Example 3

Clinical relevance of PRKD3 to CLU

79 primary breast cancer tumor tissues were obtained from the first subsidiary hospital of the suzhou university, and the studies were reviewed and approved by the ethical review committee of the study at the first subsidiary hospital of the suzhou university, and patients were provided with written informed consent and clinical samples were collected according to approved guidelines.

Immunohistochemical experiments:

primary breast tumor tissue was fixed with 4% paraformaldehyde, embedded in paraffin blocks and then microdissected into several slices. The sections were then deparaffinized and antigen extraction was performed in citrate buffer (pH 3.5) for 15 minutes. Sections were then incubated in 1% catalase for 10 min, then incubated overnight at 4 ℃ with HRP-conjugated antibodies against ER, PR, HER2, PRKD3 or CLU, respectively, and stained using the HRP-IHC kit (CST 8114) according to the manufacturer's instructions. Photographs were taken with an optical microscope and analyzed using Image-Pro Plus 6.0 software. FIG. 3A shows that ER, PR and HER2 expression was absent in patient tumor tissues and PRKD3 and CLU were highly expressed.

Tumor tissue analysis of clinical TNBC patients:

results were evaluated using a double blind method of clinical pathology and clinical course. The staining intensity of PRKD3 or CLU was 0 (negative, -), 1 (weak, +), 2 (medium, + +) and 3 (strong, + ++), respectively. The degree of staining was scored from 0 to 1.0(0 to 100%). The final staining score (0-3) was calculated as the product of the intensity score and the degree score. A final score of ≧ 1 is defined as high expression < 1 score is defined as low expression. And (4) counting the immunohistochemical experiment results and the staining scores according to the judgment standards, and grouping the tumor samples into a PRKD3 low expression group, a PRKD3 high expression group, a CLU low expression group and a CLU high expression group. Figure 3B shows that high PRKD3 and CLU expression were more predominantly compared in TNBC tumors. Fig. 3D shows elevated PRKD3 and CLU expression in TNBC cancer tissues compared to paracancerous tissues. The results of the experiment (FIGS. 3C and 3E) show that RKD3 and CLU are in clinically positive correlation. In summary, PRKD has clinical relevance to CLU and is positively correlated.

Example 4

Targeting PRKD3 and CLU shows effective clinical treatment effect

Drug treatment experiments:

female nude mice, 3-4 weeks old, were purchased from Shanghai Su commercial Biotechnology, Inc. Will be 5X 10⁶Each TNBC cell (MDA-MB-231, MDA-MB-468) was injected into the mammary fat pad of the mice. Two weeks later, mice were injected subcutaneously with CRT0066101(50mg/kg) and/or OGX-011(15mg/kg), DMSO as a control. For four weeks of continuous dosing, blood was taken from mice weekly, and the nude mice were weighed and the tumor size was measured with a vernier caliper. In the sixth week, the nude mice were transferred to an autopsy room, sacrificed in unison, the subcutaneous tumors were dissected out, and their weights were measured, the length and width of the tumors were measured, and the relevant data were recorded. According to the formula 1/2 (length) x (width)²Calculating the volume of tumor. Fig. 4A shows a schematic flow chart of OGX and/or CRT0066101 experiments in mice. FIGS. 4B-4E show the tumor formation in xenograft mice of TNBC cell line MDA-MB-468, which showed that OGX and/or CRT was effective in inhibiting TNBC tumor growth without affecting the body weight of the mice. FIGS. 4F-4I show the tumor formation in xenografted mice of TNBC cell line MDA-MB-231, showing that OGX and/or CRT was effective in inhibiting TNBC tumor growth without affecting the body weight of the mice. In general, the CLU silencing agent (OGX-011) and the PRKDs inhibitor (CRT0066101) can inhibit the growth of TNBC tumors, and the inhibition effect is more obvious when the CLU silencing agent and the PRKDs inhibitor are used in combination.

Detection experiment of tumor tissue organoid formation of patients:

TNBC tumor tissue was digested with collagenase I and counted. And (3) spreading the cells into a 24-well plate, culturing until organoid tissue samples are formed, and carrying out immunofluorescence staining. Throughout the process, the cells on the slide were washed with PBS. Cells were fixed with 4% Paraformaldehyde (PFA) for more than 30 minutes at Room Temperature (RT) and then permeabilized/blocked with PBS containing 0.1% Triton X-100/1% BSA at room temperature. Primary and secondary antibodies (Alexa Fluor 555-and 488-coupled secondary antibodies) to ER, PR, HER2, PRKD3 and CLU, respectively, were used to examine protein expression in cells according to the antibody instructions. DAPI (Solarbio) is used to label nuclei. The results of the experiment (fig. 4J) showed that PRKD3 and CLU are highly expressed in TNBC tumor tissue.

TNBC tumor tissue was digested with collagenase I and counted. Cells were plated in 24-well plates and cultured to form organoid tissue-like samples, and the effect of the drug on organoid tissue-like formation was examined using CRT0066101 (final concentration 1 μ M) and/or OGX-011 (final concentration 300nM), DMSO as control treatment. The experimental result (figure 4K) shows that the CLU silencer (OGX-011) and the PRKDs inhibitor (CRT0066101) can inhibit the organoid tissue-like formation, and the inhibition effect is more obvious when the CLU silencer and the PRKDs inhibitor are used in combination.

TNBC tumor tissue was digested with collagenase I and counted. 5000 cells were plated in 96-well plates containing 10mM HEPES (Sigma-Aldrich), 10. mu.g/mL Gentamycin (Euroclone), 2mM L-Glutamine (Euroclone), 1% Pennicilin/streptomycin (Euroclone), 2.5. mu.g/mL Amphostrin B (Euroclone), 5mM Nicotinam (Sigma), 1.25mM N-acetylcysteine (Sigma), 1 XB 35 supplementation (Gibco), 250ng/mL R-spondin 3(R & D), 5nM Heregulin (Peprotech), 5ng/mL KGF (Peprotech), 20ng/mL FGF10 (Petecproh), 5ng/mL EGF (Peprotech), 100 ng/Noggin (nM propyh), 500A 32-32 (Peprotec), 20ng/mL FGF 365 (Abecin), 20ng/mL FGF 36190. mu.g/mL (Abcrco) and 500. mu.g/mL of Sigma-GCM-GCE-, DMSO was used as a control treatment. Culturing at 37 ℃ for 7 days, removing old culture medium in 96 holes every 24 hours, replacing fresh culture medium, adding 90 mu of LDMEM complete culture medium and 10 mu of CCK-8 solution into each hole according to the operation flow of the Cell Counting Kit-8 Kit, and putting the 96-hole plate back into the incubator for incubation for 2 hours. The mixed culture solution with the original 96-well plate changed in color is transferred to a new 96-well plate, and the absorbance (A) value at 450nm is measured by a microplate reader. The experimental results (fig. 4L) show that PRKD3 knock-out significantly inhibited TNBC cell growth, and overexpression of PRKD3 and CLU restored TNBC cell growth.

Patient tumor tissue xenograft model experiments:

the method was approved by the medical ethics committee of the institute of biomedical engineering and technology, Suzhou. Human TNBC tumour tissue was taken from the first subsidiary hospital of the suzhou university, and all patients signed an authorization book.

Immunofluorescence experiment of patient tumor tissue xenograft tumor: xenograft tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin blocks, and then microdissected into several slices. The sections were then deparaffinized and antigen extraction was performed in citrate buffer (pH 3.5) for 15 minutes. The sections were then incubated in 1% catalase for 10 minutes and then permeabilized/blocked with 0.1% Triton X-100/1% BSA in PBS at room temperature. Primary and secondary antibodies (Alexa Fluor 555-and 488-coupled secondary antibodies) to ER, PR, HER2, PRKD3 and CLU, respectively, were used to examine protein expression in cells according to the antibody instructions. DAPI (Solarbio) is used to label nuclei. The results of the experiment (fig. 4M) showed that PRKD3 and CLU were highly expressed in xenograft tumor tissues.

Drug treatment experiment of patient tumor tissue xenograft tumor: female nude mice, 3-4 weeks old, were purchased from Shanghai Su commercial Biotechnology, Inc. Tumor tissue of TNBC patients was digested into single cells and counted, and 5X 10 cells were added⁶Individual cells were injected into mammary fat pads of mice. Two weeks later, mice were injected subcutaneously with CRT0066101(50mg/kg) and/or OGX-011(15mg/kg), DMSO as a control. For four weeks of continuous dosing, blood was taken from mice weekly, and the nude mice were weighed and the tumor size was measured with a vernier caliper. In the sixth week, the nude mice were transferred to an autopsy room, sacrificed in unison, the subcutaneous tumors were dissected out, and their weights were measured, the length and width of the tumors were measured, and the relevant data were recorded. According to the formula 1/2 (length) x (width)²Calculating the volume of tumor. FIG. 4N is a statistical plot of the volume size of the patient's tumor tissue xenografts showing that OGX and/or CRT effectively inhibited the proliferation of the patient's tumor tissue xenografts. Fig. 4O is a statistical graph of mouse body weight showing that OGX and/or CRT were used without affecting mouse body weight. Fig. 4P is a solid map of mouse neoplasia. FIG. 4Q is a mouse tumorigenic tumorThe results show that OGX and/or CRT effectively inhibited the proliferation of xenograft tumors in tumor tissues of patients. The general CLU silencing agent (OGX-011) and the PRKDs inhibitor (CRT0066101) can inhibit the growth of the xenograft tumor, and the inhibition effect is more obvious when the general CLU silencing agent and the PRKDs inhibitor are used in combination.

Claims

Use of CLU and/or PRKD3 as molecular markers for the diagnosis, typing, efficacy and prognostic evaluation of TNBC for triple negative breast cancer.
Application of CLU and/or PRKD3 as a histological and/or serological marker in preparation of an index reagent or a kit for TNBC diagnosis, typing, curative effect and prognosis evaluation.
3. The use of claim 1 or 2, wherein the CLU and PRKD3 comprise detecting the expression level of CLU and PRKD3 in a tumor/blood sample from a subject having breast cancer.
4. The use according to claim 1 or 2, wherein CLU and PRKD3 are expressed in significantly lower amounts in normal breast tissue than in cancer tissue, in TNBC cancer tissue than in paracancerous tissue, and in serum of TNBC patients significantly higher than in healthy people.
The use of CLU and/or PRKD3 as a TNBC key drug intervention target.
Use of a down-regulator or inhibitor of CLU and/or PRKD3 in the manufacture of a medicament for the inhibition or treatment of TNBC.
7. The use of claim 6, wherein the down-regulator or inhibitor comprises a down-regulator or inhibitor targeted to CLUs and/or PRKDs.
8. Use according to claim 7, wherein the CLU down-regulator is preferably OGX-011 and the PRKD3 inhibitor is preferably CRT 0066101.
9. The use according to claim 8, wherein the down-regulator is OGX-011 and the inhibitor is CRT0066101 alone or in combination with other TNBC-treating agents.
10. A molecular marker and therapeutic drug combination applied to TNBC diagnosis, typing, treatment and curative effect evaluation is characterized in that CLU and/or PRKD3 are used as molecular markers, and CLU and/or PRKD3 down-regulation agents or inhibitors are used as therapeutic drugs.