CN114377132B

CN114377132B - Application of Ser129 locus phosphorylation of TSPAN8 protein in cancer cell proliferation regulation

Info

Publication number: CN114377132B
Application number: CN202210064792.3A
Authority: CN
Inventors: 王红霞; 李军建
Original assignee: Shanghai First Peoples Hospital
Current assignee: Shanghai First Peoples Hospital
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2024-02-20
Anticipated expiration: 2042-01-20
Also published as: CN114377132A

Abstract

The invention discloses an application of Ser129 locus phosphorylation of TSPAN8 protein in cancer cell proliferation regulation, which is an application in preparing products for regulating cancer cell proliferation and/or growth. The Ser129 phosphorylation site of the four transmembrane protein TSPAN8 discovered by the invention promotes the occurrence, development and metastasis of tumors: as the activation of EGF/EGFR signal path is accompanied with the activation of a series of protein kinases, the analysis of mass spectrum of Co-IP products of TSPAN8 shows that AKT kinase can interact with TSPAN8, and the analysis of mass spectrum shows that the possible phosphorylation sites of TSPAN8 comprise S129, T131 and T196, finally, the phosphorylation of the S129 site of TSPAN8 protein is obvious, and the preparation method can develop a tumor medicament for treating breast cancer aiming at the site and has great clinical application value.

Description

Application of Ser129 locus phosphorylation of TSPAN8 protein in cancer cell proliferation regulation

Technical Field

The invention relates to the biomedical field, in particular to an application of Ser129 locus phosphorylation of TSPAN8 protein in cancer cell proliferation regulation.

Background

Breast cancer is one of the major malignancies leading to female death. New diagnosis of breast cancer cases in china accounts for 12.2% of the world, and death cases account for 9.6% of the world. Invasive metastasis is the leading cause of death in breast cancer patients, and transmembrane proteins (Membrane Proteins, MPs) play an important role in the development of breast cancer. TSPAN8 is a four-time transmembrane protein which is widely expressed on the cell membrane of eukaryotic cells, has 4 highly hydrophobic transmembrane domains, has N-terminal and C-terminal at both sides of the protein located in cytoplasm, and plays an important role in tumor metastasis and angiogenesis by constituting TEMs with chaperones. Clinically, TSPAN8 is highly expressed in various tumor tissues such as breast cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, lung cancer, melanoma and the like, and is closely related to invasion and metastasis of tumors. TSPAN8 also promotes invasive metastasis of cells by either participating in cell signaling or antagonizing E-cadherein interactions with P120-catenin, resulting in reduced intercellular adhesion. In addition, TSPAN8 also contributes to the dry maintenance and drug-resistant response of breast cancer stem cells by maintaining the sustained activation of the Hedgehog pathway, making the patient's therapeutic response worse.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and develop medicaments for treating breast cancer aiming at phosphorylation of Ser129 (S129) site of TSPAN8 protein.

In order to achieve the above purpose, the invention provides an application of Ser129 locus phosphorylation of TSPAN8 protein in cancer cell proliferation regulation, wherein the application is an application in preparing products for regulating cancer cell proliferation and/or growth.

Preferably, the modulation refers to inhibiting Ser129 site phosphorylation of TSPAN8 protein, thereby inhibiting proliferation and/or growth of cancer cells.

Preferably, the cancer cells are any one of breast cancer cells, gastric cancer cells, colorectal cancer cells and pancreatic duct cancer cells.

Preferably, the TSPAN8 protein enters the nucleus by binding to cytoplasmic proteins 14-3-3 theta and importin beta 1.

Preferably, the Ser129 site is phosphorylated after interaction of TSPAN8 protein with AKT kinase in EGF-EGFR signaling pathway, so as to promote occurrence, development and metastasis of cancer cells.

Preferably, the product comprises any one of monoclonal antibody and polyclonal antibody prepared by Ser129 site phosphorylation of TSPAN8 protein.

The invention also provides an application of the Ser129 locus of the TSPAN8 protein as a target in screening, developing and/or designing a tumor product for preventing and/or treating.

Preferably, the tumor comprises any one of breast cancer, gastric cancer, colorectal cancer and pancreatic duct cancer.

The invention has the beneficial effects that:

the invention discovers a Ser129 phosphorylation site of a four-time transmembrane protein TSPAN 8: since activation of the EGF/EGFR signaling pathway is accompanied by a series of protein kinase activations, AKT kinase was found to be able to interact with TSPAN8 by mass spectrometry of the Co-IP product of TSPAN8, which indicated that the possible phosphorylation sites of TSPAN8 include S129, T131, T196, and finally it is precisely the S129 site phosphorylation of the TSPAN8 protein; an antitumor drug for treating breast cancer can be developed aiming at the site.

Drawings

Fig. 1: nuclear translocation of TSPAN8 in breast cancer is significantly correlated with progression of breast cancer.

Representative immunohistochemical patterns of TSPAN8 staining for a.95 cases of breast cancer;

b. the nuclear localization of TSPAN8 is significantly increased in breast cancer;

c. the results of one-factor analysis of variance and t-test show that the nuclear TSPAN8 level of breast cancer patients is positively correlated with TNM stage;

d. the single factor analysis of variance and t-test results showed that nuclear TSPAN8 levels were specifically elevated in TNBC patients in breast cancer patients of different molecular subtypes (luminela/B, her2+, TNBC).

e-g. TSPA 8 nuclear translocation is positively correlated with tumor progression. Unpaired t-test analysis results show that nuclear TSPAN8 is associated with tumor size, lymph node metastasis or distant metastasis;

h. the unpaired t-test analysis results show that nuclear TSPAN8 is positively correlated with the mesenchymal phenotype;

i. pairing t-test analysis of nuclear TSPAN8 cells to cytoplasmic TSPAN8 cells;

j. high expression of nuclear TSPAN8 is significantly associated with a poor prognosis for BrCa.

Fig. 2: the EGF-EGFR signaling pathway triggers TSPAN8 nuclear translocation.

Wherein, EGF stimulation promotes TSPAN8 nuclear translocation. The TSPAN8 subcellular localization was detected by immunofluorescence by treatment with EGF to stimulate MCF-7 (a) or MDA-MB-231 (b) for 60 minutes. Scale = 10 μm;

EGF promotes TSPAN8 nuclear translocation in a dose dependent manner. MCF-7 cells were treated with a prescribed dose of EGF and subjected to immunofluorescence analysis;

d. immunoblots were analyzed for time dependence of nuclear TSPAN8 after EGF stimulation;

EGF promotes nuclear translocation of TSPAN8;

f. immunofluorescence analysis showed the localization of Mem-TruboID in MDA-MB-231;

g. schematic representation of nuclear translocation under EGF in MDA-MB-231 cells using a Mem-turboID tag and tracking membranous TSPAN8;

EGF stimulated, nuclear TSPAN8 was derived from plasma membrane. Immunoblots were analyzed for the relative abundance of biotinylated TSPAN8 in the membrane and nucleus at various time points after 100ng/ml EGF treatment.

Fig. 3: upon EGF stimulation, AKT kinase phosphorylates directly the Ser129 site of TSPAN8.

Wherein, a Tyrosine Kinase Inhibitors (TKIs) inhibit EGF-induced nuclear translocation of TSPAN8;

b. identifying AKT as a potential TSPAN8 interacting protein by BioID;

EGF treatment promotes TSPAN8-AKT interactions;

akt interacts with TSPAN8 in the cytoplasm;

akt phosphorylates TSPAN8 in vitro;

f. mass spectrometry of phosphorylated proteins revealed that AKT phosphorylates a key residue of TSPAN8;

akt phosphorylates Ser129 site of TSPAN8 in vitro;

h. alignment of TSPAN8 Ser129 sequences of different species;

akt inhibitors attenuate EGF-stimulated phosphorylation of the TSPAN8 Ser129 site.

Fig. 4: s129 site phosphorylation enhances nuclear translocation of TSPAN8 and is associated with breast cancer progression;

wherein, s129 site phosphorylation enhances TSPAN8 nuclear localization;

tspan8 nuclear translocation promotes breast cancer cell proliferation;

EGF passage through pTSPAN8 ^S129 The site enhances the clonogenic capacity of breast cancer cells;

e-f.pTSPAN8 ^S129 up-regulation of expression in breast cancer tissue;

g-h.pTSPAN8 ^S129 positively correlated with tumor progression.

Fig. 5: s129 site phosphorylation was associated with EGF-induced cytoskeletal remodeling;

the immunofluorescence analysis results show that EGF can induce cytoskeletal remodeling, but requires phosphorylation at the TSPAN8S129 site;

b. paired t-test was used to quantitatively analyze cytoskeletal remodelled cells.

Fig. 6: a. immunoblot analysis of commercial TSPAN8 antibodies using cell lysates of MDA-MB-231 treated with control siRNA or siRNA against TSPAN8;

b. co-staining with Flag antibody and commercial TSPAN8 antibody in MDA-MB-231 cells overexpressing TSPAN8;

the tspan8 antibody is unable to recognize other four transmembrane protein members;

d. immunohistological fluorescent staining of tumor tissue expressing Flag-TSPAN8 was performed using commercial TSPAN8 antibodies.

Fig. 7: a-d. typical TSPAN8 immunohistochemical images at breast (n=95), gastric (n=60), colorectal (n=60) and pancreatic (n=60) cancers;

e. nuclear TSPAN8 quantitative analysis.

Fig. 8: EGF stimulation does not affect the palmitoylation status of TSPAN8;

EGF stimulation did not affect TSPAN8 membrane extraction;

tkis inhibited EGF-stimulated TSPAN8 nuclear translocation;

effects of tkis treatment on TSPAN8 localization in different cell compartments;

e. nuclear AKT does not interact with TSPAN8.

Fig. 9: EGF promotes predominantly membraneless TSPAN8 phosphorylation;

b. after EGF treatment, the TSPAN8S 129A mutant was partially enriched in the cytosol at S100;

EGF promotes TSPAN8-Importin interactions;

d.TSPAN8 ^S129 phosphorylation promoting TSPAN8-Importin phaseInteraction;

e. knocking down the Importin or 14-3-3 theta protein inhibits EGF-stimulated TSPAN8 nuclear translocation;

f. knocking down the Importin or 14-3-3 theta protein reduces nuclear accumulation of TSPAN8.

Fig. 10: TSPA 8 ^S129D Promoting clone formation;

b.TSPAN8 ^S129D promoting invasion of MCF-7 cells;

c. nuclei TSPAN8 and pTSPAN8 ^S129 Positive correlation is presented;

pTSPAN8 in patients with Ecad+ or Ecad-BrCa ^S129 And (5) quantitatively analyzing.

Detailed Description

The technical scheme of the invention is further described below with reference to the accompanying drawings and examples.

Experimental materials and methods

1.1 Experimental cells

Primary human breast cancer cells, gastric cancer cells, colorectal cancer cells, pancreatic ductal cancer cells.

1.2 Experimental drugs and antibodies

Fluorescent-labeled antibody solution, PBS, RPMI-1640, 3%H ₂ O ₂ Alcohol, liquid paraffin, citrate Buffer, mceery antibody (Takara, 632496), tspan8 antibody (Abcam, ab 70007), histone H3 antibody (CST, 4499), alpha-Tubulin antibody (Sigma, T6199), beta-actin antibody (Sigma, A2228), flag antibody (Sigma, M3165), AKT antibody (CST, 9272), pSer/Thr antibody (Abcam, ab 17464), secondary antibody, RIPABuffer, IP Buffer, BCA protein concentration kit, SDS-PAGE electrophoresis Buffer, SDS-PAGE protein loading Buffer, PBST Buffer, tris-HCl Buffer, chromogenic solution, tyrosine kinase inhibitor, freund's Complete Adjuvant (FCA), incomplete adjuvant (FIA) and the like.

1.3 laboratory glassware and consumptive material

Image-Pro Plus Version 6.2, fluorescence microscope, 37 ℃ incubator, slicer, centrifuge, enzyme label instrument, electrophoresis apparatus, eppendorf pipette, constant temperature shaking bed, nitrocellulose NC membrane, cell culture plate, etc.

1.4 Experimental methods

The experimental methods not mentioned in the present specification are all experimental methods commonly used in the art.

1.4.1 immunohistochemistry

Adding liquid paraffin into the mould, placing the tissue to be embedded into the paraffin after slightly cooling, covering the mould box cover, and freezing to enable the paraffin to be solid;

taking the embedded tissue out of the mould, placing the tissue on a paraffin slicer, cutting the tissue by the slicer, and placing a glass slide containing the whole tissue into warm water at 40 ℃ by using small forceps;

placing the glass slide in a 37 ℃ incubator for drying, and then sequentially placing the glass slide in xylene-dimethylbenzene-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol for dewaxing;

dewaxing, rinsing in clear water, adding 3%H ₂ O ₂ Soaking for 10min, and pouring out H ₂ O ₂ Washing three times in clear water, adding a citric acid buffer solution, heating for 3min to expose antigen sites, cooling to room temperature, pouring the citric acid buffer solution, washing 3 times, placing a glass slide in PBS for 5min, washing 2 times, adding serum to seal non-specific sites, and then placing in a 37 ℃ incubator for standing for half an hour;

after the slide glass is taken out of the incubator, adding the primary antibody, and placing the slide glass in a refrigerator at 4 ℃ for storage overnight;

after taking out the slide glass from the refrigerator, putting the slide glass into PBS for washing 3 times, adding the secondary antibody, and putting the slide glass into a constant temperature box at 37 ℃ for half an hour;

washing the slide glass in PBS for 3 times after the slide glass is taken out, adding SABC, and standing in a constant temperature box at 37 ℃ for half an hour;

washing the slide glass in PBS for 3 times after the slide glass is taken out, adding a color developing agent, washing the developed slide glass for 5min by using clear water, soaking the slide glass in hematoxylin for dyeing for counterstaining, and finally dehydrating, transparentizing and sealing the slide glass.

1.4.2 Co-immunoprecipitation

Washing the cells twice with pre-chilled PBS, and finally blotting the PBS;

add pre-chilled IP Buffer;

the cells were blown off with a pipette, the suspension was transferred to a 1.5mLEP tube, slowly shaken for 15min at 4 ℃ (EP tube was inserted on ice, placed on a horizontal shaker);

centrifuging at 14000rpm for 15min at 4℃and immediately transferring the supernatant to a new centrifuge tube;

ProteinAaagarose was prepared, the beads were washed twice with PBS, and then formulated to 50% concentration with PBS;

add 100. Mu.L of ProteinA agarose beads (50%) per 1mL total protein, shake 10min at 4 ℃ (EP tube on ice, put on horizontal shaker);

centrifugation at 14000rpm for 15min at 4℃and transfer supernatant to a new centrifuge tube to remove ProteinA beads;

preparing a protein curve, and measuring the protein concentration;

diluting the total protein to about 1. Mu.g/. Mu.L with PBS;

add a volume of rabbit antibody to 500 μl total protein;

slowly shaking the antigen-antibody mixture overnight at 4 ℃;

add 100. Mu.L of ProteinA agarose beads to capture antigen-antibody complexes, slowly shake antigen-antibody mixture at 4℃for 1h at room temperature;

instantaneous centrifugation at 14000rpm for 5s, collecting agarose bead-antigen antibody complexes, removing supernatant, washing 3 times with pre-chilled IPbuffer;

the agarose bead-antigen antibody complex was suspended in 60. Mu.L of 2 Xloading buffer and gently mixed;

the sample was boiled for 5min, centrifuged, and the supernatant was subjected to agarose gel electrophoresis.

1.4.3 immunofluorescence

Dropwise adding 0.01moL/L PBS with pH of 7.4 into a sample to be detected, and discarding after 10min;

dropping a properly diluted solution of the fluorescent-labeled antibody so as to completely cover the specimen;

remove slide, place on slide rack, rinse with 0.01moL/L, PBS with pH 7.4;

taking out the slide, sucking the excess moisture with filter paper, but not drying the specimen, adding a drop of buffer glycerol, and covering with a cover slip;

immediately observe with fluorescence microscope.

1.4.4 phosphorylation assays

EGF treatment of MDA-MB-231 cells stably overexpressing Flag-TSPAN8 for 1hr;

washing the cells twice with pre-chilled PBS, and finally blotting the PBS;

adding pre-chilled IP Buffer, scraping off cells with a cell scraper;

repeatedly sucking and beating on ice for incubation for 30min, and blowing and beating 1 time every 10min;

centrifuging at 14000g for 15min at 4 ℃, and taking a supernatant;

add 10. Mu.LPBS washed Flag-beads, mix incubation overnight at 4 ℃;

instantaneous centrifugation at 14000rpm for 5s, taking the supernatant;

IP Buffer wash 3 times;

60 μL of 2 Xloading buffer resuspended beads and boiled for 10min;

agarose gel electrophoresis of 10. Mu.L of the supernatant;

sealing the defatted milk powder at room temperature for 1hr;

detection of TSPAN8 phosphorylation using pSer/Thr antibodies.

1.4.5pTSPAN8 ^S129 Polyclonal antibody preparation of (C)

Preparing a KLH-coupled phosphorylated polypeptide according to the LLS129ATGESEKQFQ sequence, and purifying;

diluting the phosphorylated polypeptides with PBS and with the corresponding adjuvants 1:1, mixing to form a stable emulsion;

4 New Zealand rabbits, serum is taken as a negative control for standby;

injections were performed subcutaneously around the shoulders of rabbits and the muscles of the legs;

basic immunization with FCA 1-fold, 3-fold booster immunization with FIA;

serum was collected from the auricular artery 7 days after the last boost;

serum was separated by centrifugation at 1000rpm for 10min, naN3 was added at a final concentration of 0.02%, and the mixture was stored in aliquots at-20 ℃.

Purifying the antibody using affinity;

ELISA to detect serum and antibody titers, serum titers reached 1:10000 (against protein antigen) and purified antibodies were as follows: and the OD value is more than 0.3 when the diluent is diluted by 10000, so that the diluent is qualified.

1.4.6 Mass Spectrometry of proximity markers

Constructing a Tspan-birA fusion protein expression plasmid;

when MCF-7 cells grow to 80% -90% of a 10cm dish, 10ug Tspan8-birA is transiently transformed, and liquid is changed after 6 hr;

48hr post transfection with EGF containing 50. Mu.M Biotin and 100ng/mL for 1hr;

discard medium, pre-chilled PBS wash twice;

add 1mL of IPbuffer on ice for 30min;

100 μLBiotin labeled Beads pre-chilled PBS wash twice;

1mL of protein supernatant was added to Biotin-beads and incubated overnight in suspension at 4 ℃;

And (3) cutting glue, beating a Biotin modification mass spectrum, and judging the binding distance of the interactive protein according to the modified amount of the protein Biotin.

Example 1 clinical and pathological relevance of TSPAN8 nuclear translocation to tumorigenesis

According to the related studies, it was found that TSPAN8 could be detected in the nuclei of multiple cell lines (from Huang, y. Et al, nuclear translocation of the-pass transmembrane protein tspan8.Cell Res (2021)), and the present invention began to investigate the pathological significance of TSPAN8 nuclear expression in order to evaluate the biological effects and importance of TSPAN8 nuclear translocation. To this end, as shown in fig. 6, the present invention uses a commercial TSPAN8 antibody (Abcam, ab 70007) to specifically verify it in vivo and in vitro, and immunohistochemical staining (IHC) analysis was performed on clinical specimens of primary human breast cancer (BrCa, n=95), gastric cancer (GC, n=60), colorectal cancer (CRC, n=60) and pancreatic ductal carcinoma (PDAC, n=60).

The experimental results show that TSPAN8 nuclear expression is significantly up-regulated in various tumor tissues compared to Paired Normal Tissues (PNT), as shown in fig. 1 b and fig. 7 e. As shown in fig. 1 c, fig. 1 d, fig. 1 e, fig. 1 f, fig. 1 g, fig. 1h, nuclear TSPAN8 protein levels were positively correlated with advanced breast cancer invasion features, triple negative subtype (TNBC), tumor size, lymph node infiltration, distant tumor metastasis and compartment She Biaoxing. Furthermore, as shown in i of fig. 1, the ratio of TSPAN8 nuclear stained cells (TSPAN 8 Nuc) to TSPAN8 cytoplasmic stained cells (TSPAN 8 Cyto) was significantly increased in BrCa tissue compared to the paired normal tissue.

The BrCas patients were then divided into a nuclear TSPAN8 high expression group (IOD > median) and a TSPAN8 nuclear low expression group (iod+.median). Analysis using Image-Pro Plus Version 6.2 software, as shown in j of fig. 1, the overall survival of BrCa patients with high TSPAN8 nuclear expression was significantly shortened (hr= 0.4803, 95% ci= 0.3101-0.7439). Furthermore, as shown in table 1, multivariate analysis of BrCa patient prognostic factors showed statistically significant differences in the variable nuclear TSPAN8, indicating that nuclear TSPAN8 is an independent prognostic factor for BrCa patients. Taken together, these results confirm nuclear localization of TSPAN8 and further reveal the pathological association of nuclear TSPAN8 with tumorigenesis.

TABLE 1 cox multivariate analysis of BrCa patient prognosis factors

Table S1 Cox multivariate analysis of the prognostic factor for Brca patients

HR，hazard ratio；95％Cl，95％confidence interval

Example 2 EGF-EGFR Signal mediates nuclear translocation of TSPAN8

Based on the findings of TSPAN8 nuclear localization and its potential role in tumorigenesis in example 1 above, the present invention further investigated the mechanism of TSPAN8 translocation from the plasma membrane to the nucleus. Thus, the present inventors studied the upstream signal triggering TSPAN8 nuclear translocation, and speculated that EGF-EGFR signaling pathway might be one of the factors regulating TSPAN8 subcellular structure, given that excessive activation of EGF-EGFR signaling pathway is more common in more aggressive malignancies (from Yang, Y.C.et al. Circulating Proteoglycan Endocan Mediates EGFR-Driven Progression of Non-Small Cell Lung cancer Res 80,3292-3304 (2020)). To verify this hypothesis, brCa cells were treated for EGF and immunofluorescence analysis was performed for TSPAN8 expression in the nucleus. The human breast cancer cells used in this example were MCF-7 and MDA-MB-231.

The experimental results show that the nuclear accumulation of TSPAN8 in human breast cancer cells MCF-7 and MDA-MB-231 is significantly increased under EGF stimulation, as shown in FIG. 2 a and FIG. 2 b. As shown in FIG. 1 d, this result is consistent with increased TSPAN8 nuclear expression in TNBC subtype BrCa tissue, comparing FIG. 2 a with FIG. 2 b, and experiments have found that TSPAN8 nuclear staining is significantly more in MDA-MB-231 cells than in MCF-7 cells. Considering that the physiological concentration of EGF ranges from 10 to 100ng/mL, the present invention further determines the dose-dependent effect of EGF in promoting TSPAN8 nuclear translocation. As shown in fig. 2 c, immunofluorescence analysis results showed that EGF stimulation dose-dependently enhanced nuclear translocation of TSPAN8. Furthermore, as shown in fig. 2 d, the present invention observed that the level of intracellular TSPAN8 protein increased significantly after 15min of EGF (100 ng/mL) stimulation, peaked at 60min, and then decreased to basal level at 120 min. Next, in order to determine the relative abundance of TSPAN8 in the nuclei after EGF stimulation, the present invention employs a cell separation experiment to separate the cell membrane, cytoplasmic and nuclear fractions, and quantitates the relative abundance of TSPAN8 in each component of MDA-MB-231 cells by western blotting. Although nuclear TSPAN8 represents only a small fraction of the total protein of basal level TSPAN8, this fraction of nuclear TSPAN8 protein increased significantly from 9% to near 22% and cytoplasmic TSPAN8 protein decreased from 61% to 50% with little change in plasma membrane associated TSPAN8 after EGF treatment, as shown in fig. 2 e.

According to the related studies, TSPAN8 site-specific palmitoylation was shown to be necessary for its binding to cholesterol and subsequent membrane extraction (from Huang, y.et al, nuclear translocation of the 4-pass transmembrane protein tspan8.Cell Res (2021)). However, as shown in fig. 8 a, the present invention found that EGF stimulation did not alter the palmitoylation status of TSPAN8, indicating that EGF primarily promotes translocation of TSPAN8 from the cytoplasm to the nucleus. Considering that TSPAN8 undergoes palmitoylation in cells, the present invention next seeks to determine whether TSPAN8, nuclear translocation under EGF stimulation, is derived from the cytoplasmic membrane or a newly synthesized protein in the cytoplasm. For this purpose, the present invention employs a plasma membrane inhibited, proximity labeling technique (Mem-TurboID) as shown in fig. 2 f, pre-labeling membranous TSPAN8 with biotin modification prior to EGF stimulation. As shown in FIG. 2 g, cell membrane and nuclear fractions for streptavidin immunoprecipitation were collected and the presence of biotinylated TSPAN8 in each fraction was detected by western blotting.

The experimental results showed that, as shown in h of fig. 2, the membrane-partially biotinylated TSPAN8 protein was gradually decreased after epidermal growth factor treatment, while the nuclear biotinylated TSPAN8 protein was increased in time-dependent manner, indicating that nuclear TSPAN8 originated from the cell membrane. In contrast, as shown in fig. 8 b, although EGF can dose-dependently promote the accumulation of TSPAN8 nuclei after 1h of treatment, membrane-associated biotinylated TSPAN8 remaining between the different dose treatment groups was comparable, consistent with the results observed earlier (e of fig. 2).

Taken together, these results demonstrate the insight of the present invention that EGF treatment does not alter TSPAN8 membrane extraction, but promotes translocation of TSPAN8 on the plasma membrane into the nucleus.

EXAMPLE 3 direct phosphorylation of the Ser129 site of TSPAN8 by AKT kinase under EGF stimulation

Given that the EGF-EGFR signaling pathway triggers activation of a variety of cytoplasmic tyrosine kinases, the present invention speculates that tyrosine kinases may also play an important role in mediating TSPAN8 nuclear translocation. To confirm this hypothesis, the present invention treats MDA-MB-231 cells with a commercial Tyrosine Kinase Inhibitor (TKIs) such as Afatinib, erlotinib or AEF788 prior to EGF stimulation, and then detects nuclear localization of TSPNA8 by Immunofluorescence (IFA). Experimental results show that using these TKIs effectively blocks EGF-induced nuclear translocation of TSPAN8, as shown in fig. 3 a and 8 c. TSPAN8 may be present in the cytoplasm in membrane-associated (P100) and membrane-free (S100) forms. Then, the effect of TKIs on TSPAN8 distribution at different subcellular structures after EGF treatment was studied. Consistent with the view that EGF promotes cytoplasmic transnuclear of TSPAN8, as shown in d of fig. 8, TKIs treatment was found to significantly promote accumulation of TSPAN8 in S100 portion without affecting TSPAN8 localization in cell membrane and P100 portion.

Based on mass spectrometry analysis (BioID) of proximity labels, the method can effectively label protein-protein transient interactions after biotin substrate addition, so the invention applies the analysis method to detect the binding protein of TSPAN8, and the detection result shows that the binding capacity of TSPAN8 to AKT of an EGF treatment group is significantly higher than that of an EGF-free treatment group, as shown in b of FIG. 3, and the interaction between TSPAN8 and AKT is observed to be enhanced under EGF stimulation compared with a control group. Subsequently, the mass spectrometry results were verified by performing TSPAN8 immunoprecipitation (Co-IP) detection at various time points after EGF stimulation. As a result, as shown in fig. 3 c, the correlation between TSPAN8 and AKT was significantly enhanced after EGF treatment. Since AKT has also been reported to be capable of transferring to the nucleus, the present invention subsequently performed AKT Co-IP experiments at cytoplasmic and nuclear sites to determine where interactions occur. As shown in FIG. 3 d, the interaction of AKT-TSNA 8 in the cytoplasm was detected, further enhanced after EGF treatment. However, as shown in fig. 8 e, the present invention fails to detect this interaction in the nucleus.

Since AKT is a kinase downstream of the EGF-EGFR signaling pathway, the present invention investigated whether AKT could directly phosphorylate TSPAN8. To verify this possibility, the present invention performed an in vitro kinase assay with a ubiquitinated Tyr/Ser antibody.

The experimental results are shown in fig. 3e, which shows that activated AKT is indeed able to phosphorylate wild-type TSPAN8 in vitro, indicating that TSPAN8 is a true substrate for AKT. Next, specific sites of AKT phosphorylation of TSPAN8 under EGF stimulation were determined by mass spectrometry. Analysis of the protein sequence revealed that S129, T131 and T196 of TSPAN8 might be sites of phosphorylation following EGF treatment. As shown in f of fig. 3, mass spectrometry analysis showed that S129 site was phosphorylated after EGF treatment. The present invention further uses a point mutation strategy to verify the phosphorylation of the TSPAN8S129 site. As shown in g of FIG. 3, the TSPAN8S 129A mutant, but not the T131A and T196A mutants, abrogated in vitro the phosphorylation of TSPAN8 by AKT. As shown in FIG. 3, h, the TSPAN8 Ser129 sequence alignment shows that this residue is conserved in humans, mice, rabbits, cattle, dogs. For further investigation, the present invention treats MDA-MB-231 cells expressing TSPAN8 (WT and mutant) with EGF. As shown in FIG. 3 i, elevated levels of TSPAN8 were observed in cells expressing WT-, T131A-and S196A-, and the levels of TSPAN8 were greatly reduced after further incubation with AKT inhibitor MK 2206. Nevertheless, the S129A mutation almost blocked EGF-induced phosphorylation of TSPAN8, whether MK2206 treatment or not.

Taken together, these results indicate that AKT kinase directly interacts with TSPAN8 at the Ser129 site of the EGF-EGFR signaling pathway and phosphorylates TSPAN8.

Example 4S129 phosphorylated TSPAN8 enters the nucleus by binding to cytoplasmic proteins 14-3-3 theta and importin beta 1

To investigate Ser129 phosphorylation of TSPAN8 (hereinafter referred to as pTSPAN 8) ^S129 ) Potential role in nuclear translocation of EGF-EGFR signaling TSPAN8 the present invention constructs TSPAN8 that is no longer phosphorylated by AKT ^S129A Mutant and TSPAN8 mimicking sustained phosphorylation of AKT ^S129D Wild-type and mutant TSPAN8 were overexpressed in MCF-7 cells and alterations in the nucleus and cytoplasm of TSPAN8 were detected by immunofluorescence. If phosphorylation of TSPAN8 at the S129 site is critical for nuclear translocation, it is expected that it is important to hybridize with wild-type TSAPN8 (hereinafter TSAPN8 ^WT ) In contrast, TSPAN8 ^S129A Nuclear translocation of the mutant was reduced, while TSPAN8 ^S129D The nuclear translocation of the mutant is increased.

The experimental results are shown in FIG. 4 a, TSPAN8 ^S129A The mutant was barely detectable in the nucleus, while TSPAN8 ^S129D Mutants are distributed throughout the cell, including the nucleus.

Previous studies by the inventors have shown that TSPAN8 in the nucleus is not associated with any membrane structure or vesicle, but rather exists as a cytoplasmic TSPAN 8-cholesterol complex. In this case, the hydrophobic transmembrane domain of TSPAN8 may be protected by cholesterol, while the Ser129 site of TSPAN8 may be phosphorylated by AKT. To confirm this hypothesis, the present invention further examined the level of cytoplasmic localized TSPAN8 after 30min of EGF stimulation, as shown in FIG. 9a, found S100 enriched pTSPAN8 ^S129 Significantly increased after EGF stimulation. For further demonstration, pTSPAN8 ^S129 TSPAN8, which affects mainly membraneless morphology, the present invention compares TSPAN8 with Flag tags in S100 and P100 fractions after EGF treatment ^WT And TSPAN8 ^S129A Is a relative abundance of (c). Westernblotting analysis as in FIG. 9 b shows that with TSAPN8 ^WT In contrast, TSPAN8 ^S129A More was identified in the S100 part, as shown in d of fig. 8, which demonstrates the effect of TKIs on TSPAN8 in the S100 part. These results indicate that EGF-AKT-pTSPAN8 ^S129 The axis mediates mainly TSPAN8 cytoplasmic transnuclei.

The present invention next investigated how AKT-mediated phosphorylation at the Ser129 site promotes nuclear translocation of TSPAN8 protein. The 14-3-3 protein plays a key role in protein subcellular localization and nuclear import. According to previous studies by the inventors, 14-3-3 inhibitor R18 could block translocation of TSPAN8 to the nucleus, whereas a 14-3-3 theta knock down could block nuclear translocation of TSPAN8 (from Huang, Y.et al Nuclear translocation of the 4-pass transmembrane protein Tpan8. Cell Res (2021)). Consistent with the observation that EGF induces nuclear translocation of TSPAN8, as shown in FIG. 9 c, this experiment found that EGF treatment could dose-dependently promote interactions between TSPAN8 and 14-3-3 theta and importin beta 1. The present invention then explores whether TSPAN8 phosphorylation may affect its association with 14-3-3 theta, thereby participating in nuclear translocation of TSPAN8. As shown in FIG. 9 d, co-IP interaction experiments showed that MDA-MB-231 in cells, TSPAN8 ^S129D Has stronger binding ability to 14-3-3 theta, while TSPAN8 ^S129A The binding ability to 14-3-3 theta is severely impaired.

The present invention then uses the siRNA approach to specifically knock down 14-3-theta and Importin beta 1 in MDA-MB-231 cells, and then re-analyzes TSPAN8 expression in the nucleus and cytoplasm by immunofluorescence or immunoblotting assays. As shown in fig. 9 e, fig. 9 f, consumption of 14-3-theta or Importin β1 specifically inhibited accumulation of EGF-stimulated TSPAN8 at the core site.

Taken together, these experimental results indicate that AKT mediated phosphorylation at the Ser129 site promotes nuclear translocation of TSPAN8 by promoting TSPAN8 binding to cytoplasmic proteins 14-3-3 theta and importin beta 1.

Example 5S129 phosphorylated TSPAN8 is more correlated with an aggressive cancer phenotype than wild-type TSPAN8

The present invention evaluates pTSPAN8 ^S129 Pathological importance in tumor progression. First, stable transition TSPAN8WT and TSPAN8 are detected ^S129A Or TSPAN8 ^S129D Is a proliferative capacity of MCF-7 cells. As shown in FIG. 4 b, the present invention found that TSPAN8 was overexpressed ^S129D Cell ratio of mutant overexpressing TSPAN8 ^WT Or TSPAN8 ^S129A The cell proliferation rate of the mutant was significantly improved and showed a strong positive correlation with its nuclear localization pattern (as shown in a of fig. 4). With pTSPAN8 ^S129 As shown in FIG. 10 a and FIG. 10 b, the present invention found that TSPAN8 was overexpressed ^S129D Cells of the mutant and overexpression of TSPAN8 ^WT Has a higher clonogenic and invasive capacity than the cells of the same. Next, to further determine pTSPAN8 ^S129 Mediated function, the invention treats MDA-MB-231 cells expressing different mutant TSPAN8 with EGF or without EGF. As shown in FIG. 4 c, the results demonstrate that MDA-MB-231 overexpresses TSPAN8 ^S129D Cell clonality of the mutant is higher than that of TSPAN8 ^WT And TSPAN8 ^S129A . Notably, EGF treatment significantly promoted TSPAN8 ^WT But express TSPAN8 ^S129A The cells of the mutant had no promoting effect.

To further investigate the pathological function of AKT phosphorylated TSPAN8, the present invention prepared a specific recognition pTSPAN8 ^S129 Is described. As shown in FIG. 4 d, the MDA-MB-231 cells are endogenous to pTSPAN8 ^S129 The level gradually increased after EGF stimulation and reached a peak after 60min of EGF treatment. Using S100 fractionation, as shown in fig. 9a, S100 enriched TSPAN8 was found to exhibit predominantly Ser129 phosphorylation status and increased significantly after EGF treatment, which suggests that phosphorylation events occurred prior to TSPAN8 nuclear translocation.

Then, the present invention detects pTSPAN8 in tissues of 95 BrCa patients by using the polyclonal antibody ^S129 Horizontal. As a result, pTSPAN8 was found to be higher in comparison with the paired normal tissues as shown in FIG. 4 e and FIG. 4 f ^S129 Is significantly elevated in BrCa tissue. In addition, pTSPAN8 ^S129 Is mainly located in the nucleus and is positively correlated with TSPAN8 nuclear expression (e.g., c of fig. 10). In addition, pTSPAN8 is shown in FIG. 10 d, FIG. 4 g and FIG. 4 h ^S129 Elevated levels are clearly associated with mesenchymal cell characteristics, progressive breast cancer stage and TNBC subtypes. As shown in FIG. 10 e, pTSPAN8 ^S129 The expression level of (2) was correlated with a shorter overall survival time for BrCa patients (p=0.0003). It will be appreciated that pTSPAN8 is specifically recognized ^S129 The monoclonal antibodies of (2) can also achieve the above effects.

Taken together, these results demonstrate pTSPAN8 ^S129 Positive correlation with its nuclear localization and preparation of specific recognition pTSPAN8 ^S129 Has an important role in the tumor progression research process.

Example 6S129 phosphorylated TSPAN8 is critical for EGF-mediated cytoskeletal remodeling

To study pTSPAN8 ^S129 Effect on tumor invasive Capacity the present invention examined stable expression of TSPAN8 ^WT 、TSPAN8 ^S129A And TSPAN8 ^S129D Cytoskeletal changes. As shown in fig. 5 a and 5 b, the tumor cell skeleton was remodeled under EGF stimulation. And express TSPAN8 ^WT Is expressing TSPAN8 ^S129A Mutant cell inhibitionEGF remodeling of cytoskeleton was produced, while TSPAN8 ^S129D Mutations significantly enhance cytoskeletal remodeling even in the absence of EGF stimulation.

Emerging evidence suggests that some protein receptors, originally thought to be located only on the plasma membrane, may shuttle into the nucleus (from Sardi, s.p., murtie, j., kortala, s., patten, B.A) depending on the particular cellular environment.&Corfas, G.presentin-dependent ErbB4 nuclear signaling regulates the timing of astrogenesis in the developing brain. Cell 127,185-197 (2006). Thus, coordinated control of the dual roles of these receptors in the cell surface and nucleus may contribute to tumor development. Nuclear localized TSPAN8 can be detected in a variety of human tumor tissues. And nuclear translocation of TSPAN8 is an independent indicator, which can indicate patient progression, advanced and poor prognosis. The transport of TSPAN8 from the cell membrane to the cytoplasm is associated with endocytic internalization and endosomal sorting. However, one confusing problem is with respect to the specific cellular environment and mechanisms that regulate TSPAN8 nuclear translocation from the cytoplasm. In the present invention' S mechanism that attempts to determine TSPAN8 nuclear localization, experiments have found that EGF stimulation can induce AKT to phosphorylate TSPAN8 at the S129 site. Thus, the present invention proposes that EGF-EGFR signaling pathway activates AKT-phosphorylated TSPAN8, promoting the binding of TSPAN8 to 14-3-3 theta and importin beta 1, and thus transporting TSPAN8 into the nucleus. Previous studies have shown that NPCs and importin beta are involved in the transport of EGFR, erbB-2 and FGF receptor-1 (FGFR-1) from the cell surface to the nucleus (from reily, J.F).&Maher, P.A. importin beta-mediated nuclear import of fibroblast growth factor receptor: role in Cell pro-file.J Cell Biol 152,1307-1312 (2001). In this model, phosphorylation of TSPAN8 at the S129 site plays a crucial role in its nuclear transport. Thus, TSPAN8 ^S129A Mutations block S129 phosphorylation, not only abrogating nuclear translocation of TSPAN8, but also reduce to some extent the proliferation, clonogenic and invasive capacity of cancer cells. In contrast, TSPAN8 mimicking S129 constitutive phosphorylation ^S129D Mutations strongly promote nuclear translocation of TSPAN8, as well as invasive phenotypes of cancer cells. More importantly, in a variety of human malignancies, including BrCaIn pTSPAN8 ^S129 Such an increase in phosphorylation levels is positively correlated with both nuclear levels and invasive phenotypes of TSPAN8, including inter She Biaoxing, advanced tumor size, TNBC subtype and distant metastasis. In particular, pTSPAN8 ^S129 The level is inversely related to the overall survival of cancer patients. IHC score, pTSPAN8, expressed by TSPAN8 after adjustment of related clinical covariates in a Cox multivariate model ^S129 Or nuclear TSPAN8 expression is an independent predictor of poor patient survival.

In summary, the present invention provides a four transmembrane protein TSPAN8 Ser129 phosphorylation site, i.e., AKT kinase interacts with TSPAN8 at Ser129 site of EGF-EGFR signaling pathway and phosphorylates TSPAN8, promoting tumor initiation, development and metastasis. Designing the phosphorylation site, such as developing a drug capable of inhibiting Ser129 site phosphorylation of TSPAN8; commercial kits for preparing monoclonal or polyclonal antibodies to phosphorylate the site; the antibody and the drug are connected to carry out tumor guiding treatment, and the antibody and the radioactive label are connected to carry out radioimmunoimaging to assist diagnosis of tumors, etc.

While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.

Claims

1. A method for screening an anti-breast cancer drug, characterized in that the inhibition of serine 129 site phosphorylation of TSPAN8 protein by the drug is detected.