Application of CD44v5 in treatment of triple-negative breast cancer as a target

Abstract

The application belongs to the technical field of biological pharmacy, and provides application of CD44v5 in treatment of triple negative breast cancer target spot, through in vitro cell experiment, the application evaluates the effect of CD44v5 as a marker and CD44v5 inhibitor on triple negative breast cancer immunotherapy; the result shows that CD44v5 blocking significantly weakens IL-4 mediated triple negative breast cancer cell migration ability, colony formation ability and anti-apoptosis effect, and restores its sensitivity to BMS-1, restores T cell killing function, and establishes that CD44v5 is a core regulator of IL-4R / STAT3 pathway; CD44v5 can simultaneously regulate PD-L1 and PVR immune checkpoint molecules, and there is a positive feedback mechanism between CD44v5 and SAM68; blocking CD44v5 expression and combined with cisplatin treatment can inhibit tumor growth by inhibiting SLC7A11 expression and promoting ferroptosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the treatment of triple-negative breast cancer, specifically the application of CD44v5 as a target for the treatment of triple-negative breast cancer. Background Technology

[0002] Breast cancer (BC) is a common malignant tumor in women. Triple-negative breast cancer (TNBC) lacks the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), making endocrine and anti-HER2 targeted therapies ineffective. Chemotherapy is the core treatment, but cisplatin resistance is the primary challenge in clinical treatment.

[0003] Cisplatin is a first-generation platinum-based chemotherapy drug with broad-spectrum antitumor activity. However, some patients are prone to developing drug resistance when receiving platinum-based regimens, leading to poor treatment outcomes and becoming a major cause of their poor prognosis.

[0004] In recent years, immune checkpoint inhibitors (ICIs) such as PD-1 / PD-L1 inhibitors have brought new hope to TNBC, but most patients suffer from primary / secondary resistance. Synergistic inhibition of multiple immune checkpoints and dysregulation of the tumor immune microenvironment (TIME) are the main reasons. TNBC exhibits co-expression of PD-L1, CTLA-4, LAG-3, TIM-3, and other immune checkpoint molecules, forming a multi-pathway immunosuppressive network, thereby weakening the therapeutic effect of single PD-1 / PD-L1 inhibitors.

[0005] The tumor immune microenvironment (TIME) plays a central role in the progression and treatment of tumor-associated neoplasia (TNBC). Tumor immune cells (TAMs) are the most abundant immune cells in the TME. TAMs suppress T cell function and promote tumor invasion and metastasis by secreting immunosuppressive cytokines such as IL-4, IL-10, and TGF-β. Previous studies have found that TNBC cells themselves can also secrete IL-4, enhancing the local immunosuppressive microenvironment. However, whether IL-4 is involved in TNBC immune evasion remains unclear.

[0006] CD44v5 is a variant splicing subtype of CD44 that is highly expressed in TNBC. Previous studies have confirmed that it can activate the IL-4R / STAT3 signaling axis and promote tumor progression. This invention aims to reveal the role and mechanism of CD44v5 in TNBC immunotherapy resistance and cisplatin chemotherapy resistance, and to verify its feasibility as a therapeutic target. Summary of the Invention

[0007] The purpose of this invention is to provide the application of CD44v5 as a target for the treatment of triple-negative breast cancer. By using CD44v5 as a target in the prevention and treatment of triple-negative breast cancer, a new approach and means for the treatment of triple-negative breast cancer is provided, which solves the technical problems of limited treatment options, poor response to immune checkpoint inhibitor therapy and chemotherapy resistance in triple-negative breast cancer (TNBC).

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] This invention provides the application of CD44v5 inhibitors in promoting immunotherapy for triple-negative breast cancer and overcoming cisplatin-resistant chemotherapy.

[0010] Preferably, the inhibitor comprises a substance that reduces CD44v5 protein expression or CD44v5 protein content, or a substance that inhibits CD44v5 gene expression, wherein the substance comprises one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses.

[0011] This invention also provides the application of CD44v5 inhibitors in combination with active ingredients in the preparation of drugs for treating triple-negative breast cancer.

[0012] Preferably, the CD44v5 exerts a dual regulatory effect through the IL-4R / STAT3 axis, the dual regulatory effect including:

[0013] (1) It regulates the expression of multiple immune checkpoint molecules such as PD-L1 and PVR, promotes immune escape in triple-negative breast cancer and enhances its resistance to PD-1 / PD-L1 therapy.

[0014] (2) Upregulate the expression level of SLC7A11 and stabilize its cell membrane localization, thereby enhancing the chemotherapy resistance of triple-negative breast cancer to cisplatin by inhibiting the process of tumor cell ferroptosis.

[0015] Preferably, the application uses CD44v5 expression level as an evaluation index, and then uses CD44v5 inhibitors in combination with PD-1 / PD-L1 and cisplatin chemotherapy drugs for sensitization in the treatment of triple-negative breast cancer.

[0016] Preferably, the drug is used to promote tumor cell apoptosis and ferroptosis and restore T cell function.

[0017] Preferably, the drug comprises a CD44v5 inhibitor and a medically approved excipient.

[0018] Preferably, the drug comprises various acceptable dosage forms, such as injections, pills, capsules, granules, tablets, or oral liquids.

[0019] The present invention also provides a diagnostic model for triple-negative breast cancer, wherein the CD44v5 gene expression level or CD44v5 protein expression level is used as one of the effective indicators of the diagnostic model.

[0020] The beneficial effects of this invention are:

[0021] This invention provides the application of CD44v5 as a target for the treatment of triple-negative breast cancer. Compared with existing technologies, the key achievements are reflected in two aspects: First, it confirms that CD44v5 can regulate immune checkpoint molecules such as PD-L1 and PVR, mediating TNBC immune escape and PD-1 / PD-L1 inhibitor resistance. Blocking this target can reverse the above effects, and the anti-tumor effect is better than that of immune checkpoint inhibitor monotherapy. At the same time, it also reveals the positive feedback mechanism between CD44v5 and SAM68, providing a new theory for understanding the TNBC immune microenvironment. Second, it confirms that CD44v5 regulates ferroptosis through the IL-4R / STAT3 signaling axis, mediating TNBC cisplatin resistance. Knocking down this target in combination with cisplatin can significantly inhibit tumor growth.

[0022] In summary, CD44v5 can simultaneously mediate resistance to immunotherapy and cisplatin chemotherapy in TNBC, making it a potential core target for improving the treatment efficacy of TNBC and providing a new approach for the clinical treatment of this disease. Attached Figure Description

[0023] Figure 1 This is a graph showing the results of PD-L1 in TNBC and its association with type II immune response-related proteins in this invention (A shows the expression differences of CD163, IL-4, CD44v5, IL-4Rα, and PD-L1 in TNBC and non-TNBC patient tissues; scale bar: 100 μm; B shows the expression differences of CD44v5 and PD-L1 between TNBC and non-TNBC cell lines in qPCR experiments; C shows the protein expression results of CD44v5, IL-4R, and PD-L1 in TNBC and non-TNBC cell lines in Western blot experiments; D shows the correlation analysis of PD-L1 and KHDRBS1 (SAM68) expression levels in TNBC samples; *P<0.05, **P<0.01, intergroup comparison).

[0024] Figure 2 The figure shows the effect of IL-4 secreted by TAMs on promoting immune escape by inducing PD-L1 expression in this invention (A is the effect of IL-4 on the expression of CD69 and CD44 on the surface of T cells; B is the effect of IL-4 on T cell activation without TNBC cell co-culture; C is the result of PD-L1 protein expression in TNBC cells after IL-4 stimulation; *P<0.05, compared with the control group).

[0025] Figure 3This is a diagram showing the results of partial IL-4 enhancing CD44v5 expression and inducing PD-1 / PD-L1 resistance through the IL-4R / STAT3 / PD-L1 pathway in this invention (AB represents the effect of IL-4 pretreatment on BMS-1's inhibition of TNBC cell malignancy and promotion of apoptosis; **P<0.01, ***P<0.001, intergroup comparison).

[0026] Figure 4 This is a partial diagram showing the results of IL-4 enhancing CD44v5 expression and inducing PD-1 / PD-L1 resistance through the IL-4R / STAT3 / PD-L1 pathway in this invention (A shows the effect of IL-4 pretreatment on BMS-1-induced inhibition of TNBC cell-related protein expression; B shows the effect of IL-4 on BMS-1-induced T cell CD69 and CD44 RNA expression; C shows the effect of S3I-201 on IL-4-mediated activation of the IL-4R / STAT3 / PD-L1 pathway; D shows the effect of S3I-201 on IL-4-induced SAM68 protein expression; *P<0.05, **P<0.01, ***P<0.001, within-group comparison).

[0027] Figure 5 This is a partial result diagram of the effects of blocking CD44v5 on reversible IL-4-induced immune escape and PD-1 / PD-L1 inhibitor resistance in this invention (AB represents the verification results of the knockdown efficiency of CD44v5 knockdown TNBC cell lines; CD represents the effect of blocking CD44v5 on IL-4-mediated TNBC cell migration, colony formation, anti-apoptosis ability, and the restoration of BMS-1 ​​sensitivity; *P<0.05, **P<0.01, ***P<0.001, intergroup comparison).

[0028] Figure 6 This is a partial result diagram of the effects of CD44v5 blockade on reversible IL-4-induced immune escape and PD-1 / PD-L1 inhibitor resistance in this invention (A shows the expression of related proteins; B shows the effect of CD44v5 blockade on the expression of CD44 and CD69 on the surface of T cells; C shows the effect of CD44v5 knockdown on the basal expression of IL-4R, p-STAT3, PD-L1 and the ability to induce IL-4 upregulation; D shows the effect of CD44v5 knockdown on SAM68 protein expression; *P<0.05, **P<0.01, ***P<0.001, within-group comparison).

[0029] Figure 7This is a graph showing the results of the stronger anti-tumor effect of blocking CD44v5 compared to PD-1 / PD-L1 inhibitors in this invention (AB represents the effect of blocking CD44v5, BMS-1 ​​monotherapy, and combination therapy on T cell-mediated TNBC cell apoptosis; C represents the PVR protein expression results in breast cancer cell lines; D represents the effect of S3I-201 on IL-4-induced PVR expression; E represents the effect of CD44v5 knockdown on basal PVR expression and IL-4 induction; *P<0.05, **P<0.01, ***P<0.001, intergroup comparison).

[0030] Figure 8 This invention presents partial results showing that CD44v5 upregulates SLC7A11 expression and enhances its membrane stability by activating the IL-4 / IL-4Rα / STAT3 pathway, thereby inhibiting TNBC ferroptosis (A shows the upregulation of IL-4Rα, p-STAT3, CD44v5, sam68, and SLC7A11 / GPX4 expression by TAMs co-culture; B shows the effect of IL-4-derived treatment on STAT3 and SLC7A11 / GPX4 protein expression; C shows the effect of STAT3 inhibitor S3I-201 treatment on IL-4-induced p-STAT3, SLC7A11, and GPX4 proteins; D shows the construction of CD44v5 knockdown cell lines; E shows the effect of anti-CD44v5 monoclonal antibody and dupilumab on SLC7A11 protein expression).

[0031] Figure 9 This is a partial result of the invention showing that CD44v5 upregulates SLC7A11 expression and enhances its membrane stability by activating the IL-4 / IL-4Rα / STAT3 pathway, thereby inhibiting ferroptosis in TNBC (A is the result of correlation analysis with the TCGA database; B is the result of breast cancer subtyping analysis with the TCGA database; C is the result of immunohistochemical staining of SLC7A11 in TNBC and non-TNBC tissues; scale bar: 100 μm).

[0032] Figure 10 This invention presents partial results showing that CD44v5 upregulates SLC7A11 expression and enhances its membrane stability by activating the IL-4 / IL-4Rα / STAT3 pathway, thereby inhibiting ferroptosis in TNBC (A shows AlphaFold3 prediction and PyMOL three-dimensional visualization analysis results; B shows confocal microscopy staining results of CD44v5 and SLC7A11 on the TNBC cell membrane, scale bar: 5 µm; C shows the effect of CD44v5 monoclonal antibody on SLC7A11 protein expression after paclitaxel treatment).

[0033] Figure 11This invention demonstrates that blocking CD44v5 promotes ferroptosis and reverses IL-4-induced partial cisplatin resistance (A and B are results from scratch assays; C and D are results from colony formation assays; E and F are results from flow cytometry analysis of apoptosis; **P<0.01, ***P<0.001, intergroup comparisons).

[0034] Figure 12 This invention demonstrates that blocking CD44v5 promotes ferroptosis and reverses IL-4-induced partial cisplatin resistance (A and B are flow cytometry results; C and D are protein expression results of SLC7A11, GPX4, and IL-4 after CD44v5 blocking; *P<0.05, **P<0.01, ***P<0.001, intergroup comparison).

[0035] Figure 13 This is part of the results of CD44v5 knockdown enhancing in vivo cisplatin sensitivity in this invention (A is a schematic diagram of the in vivo experimental design and process; B is a photograph of representative xenografts in each treatment group; C is the weight statistics of xenografts in each group at the experimental endpoint; D is the growth curve of xenograft volume over time; *P<0.05, **P<0.01, ***P<0.001, intergroup comparison).

[0036] Figure 14 This is part of the results of CD44v5 knockdown enhancing in vivo cisplatin sensitivity in this invention (A is the curve of mouse body weight change during the experiment; B is the expression results of CD44v5, IL-4Rα, SLC7A11 and GPX4 proteins in the CD44v5 knockdown group; C is the immunohistochemical staining results of transplanted tumor tissue; scale bar: 100 μm). Detailed Implementation

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] Example

[0041] 1. Experimental Materials

[0042] In this embodiment, triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468), non-triple-negative breast cancer cell line (MCF-7), and human mononuclear cell line THP-1 were selected. Each cell line was prepared with the corresponding culture medium according to conventional methods and cultured at 37°C and 5% CO2. Before the experiment, the cells were tested for mycoplasma and verified by STR, and the number of passages was strictly controlled.

[0043] 2 Experimental Methods

[0044] 2.1 Immunoblot Analysis

[0045] Total cellular protein was extracted and its concentration was determined. After SDS-PAGE electrophoresis, transfer, and blocking, primary antibodies against target proteins such as CD44v5 and PD-L1 were added and incubated. After binding with secondary antibodies, the protein was developed using ECL chemiluminescent substrate to detect the expression level of the target proteins.

[0046] 2.2 Co-culture Experiment

[0047] THP-1 cells were induced by PMA and polarized into M2 macrophages with the addition of IL-4. The tumor-associated macrophage (TAMs) model was constructed by co-culturing TNBC cells with THP-1 cells through a porous plug.

[0048] 2.3 Cell migration assay

[0049] After TNBC cells reached 80%-90% confluence, they were scratched to remove detached cells and an initial image was taken. After culturing for another 24 hours, another image was taken to analyze the cell migration distance.

[0050] 2.4 Cloning experiment

[0051] Cells from the control group and the experimental group were seeded into six-well plates and cultured for 10-15 days. After fixation and staining, the number of cell clones with a diameter ≥50 μm was counted to detect cell proliferation capacity.

[0052] 2.5 Primary T cell co-culture

[0053] After ethical approval and obtaining informed consent, peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated and activated T cells were obtained by induction with IL-2 and CD3 / CD28 activators. Tumor cells and activated T cells were co-cultured in a certain ratio. BMS-1 ​​was added to the PD-L1 blockade group. After 48 hours of culture, the cells and culture medium were collected for later use.

[0054] 2.6 Flow cytometry

[0055] TNBC cells and PBMCs were collected, stained with fluorescent dyes and specific antibodies, and then flow cytometry was used to detect cell apoptosis and the expression of T cell activation markers.

[0056] 2.7 Lentiviral transfection

[0057] Lentiviral vectors and packaging plasmids were co-transfected into 293T cells. After collecting viral supernatant, TNBC cells were infected. Stable cell lines were obtained by puromycin selection. The CD44v5 knockdown efficiency was verified by qPCR and Western blot.

[0058] 2.8 Real-time quantitative PCR

[0059] Total RNA was extracted from cells and reverse transcribed into cDNA. Specific primer pairs were designed for RT-qPCR amplification of CD44v5 and PD-L1. GAPDH was used as an internal control, and the relative gene expression levels were calculated using the 2⁻ΔΔCt method.

[0060] 2.9 Immunohistochemistry and confocal microscopy:

[0061] After ethical approval and obtaining informed consent, tumor tissue from patients was collected to prepare slides. After dewaxing, hydration, antigen retrieval, and blocking, primary and secondary antibodies were added for incubation. After DAB staining and hematoxylin counterstaining, the target protein in the tissue sample was detected.

[0062] 2.10 Calculation and Statistical Analysis

[0063] All experiments were independently repeated at least twice. Experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPadPrism 9.0 and SPSS 23 software. *P < 0.05 was considered statistically significant.

[0064] 3. Experimental Results

[0065] 3.1 Study on the mechanism of CD44v5 regulation of immune checkpoint molecules mediating immune escape and PD-1 / PD-L1 therapy resistance in triple-negative breast cancer

[0066] 3.1.1 PD-L1 is positively correlated with type II immune response-related proteins in TNBC.

[0067] Clinical tissue samples and cytological experiments showed that ( Figure 1 In the AC (anti-inflammatory mediator) of TNBC, TAMs infiltration is closely related to high PD-L1 expression and participates in PD-L1-mediated immune escape. Meanwhile, the expression of the key CD44v5 splicing factor SAM68 (KHDRBS1) is positively correlated with CD274 mRNA levels. Figure 1 D).

[0068] 3.1.2 IL-4 secreted by TAMs promotes immune escape by inducing PD-L1 expression.

[0069] Previous studies have confirmed that TAMs promote the migration and proliferation of TNBC cells and facilitate TNBC immune escape. Furthermore, TAMs secrete the Th2 cytokine IL-4, exerting an immunosuppressive function. This example demonstrates that IL-4 can reduce the expression of CD69 and CD44 on the surface of T cells (…). Figure 2 A). The results showed that IL-4 could not directly inhibit T cell activation in the absence of TNBC cell co-culture. Figure 2 B); while IL-4 stimulation can significantly upregulate the expression of PD-L1 protein on the surface of TNBC cells (B); Figure 2 C).

[0070] 3.1.3 IL-4 enhances CD44v5 expression and induces PD-1 / PD-L1 inhibitor resistance through the IL-4R / STAT3 / PD-L1 pathway.

[0071] In this embodiment, the addition of the PD-1 / PD-L1 inhibitor BMS-1 ​​(1 nM) to the TNBC-T cell co-culture system can inhibit the malignant phenotype of TNBC cells and promote their apoptosis. However, pretreatment of TNBC cells with IL-4 can reverse the antitumor effect of BMS-1. Figure 3 AB and Figure 4 A). Simultaneously, IL-4 eliminated BMS-1-induced upregulation of CD69 and CD44 expression, weakening its tumor-killing effect. Figure 4 B).

[0072] Previous studies have shown that IL-4 promotes the malignant phenotype of TNBC cells by activating the IL-4R / STAT3 pathway. This example further demonstrates that IL-4 activates STAT3 phosphorylation through the type II IL-4 receptor and upregulates CD44v5 and PD-L1 expression. Treatment with the STAT3-specific inhibitor (S3I-201) not only reduced the basal expression levels of IL-4R, p-STAT3, and PD-L1, but also weakened IL-4-mediated activation of this pathway. Figure 4 C). Simultaneously, IL-4 stimulation upregulated SAM68 protein levels, while S3I-201 treatment eliminated this effect, indicating that SAM68 is regulated by the type II IL-4R / STAT3 signaling axis (C). Figure 4 D).

[0073] These results indicate that IL-4 promotes tumor immune escape by upregulating the expression of CD44v5, type II IL-4R, and PD-L1, and directly induces resistance to PD-1 / PD-L1 inhibitors in triple-negative breast cancer. This finding suggests that IL-4 can drive immune escape through a mechanism independent of PD-L1.

[0074] 3.1.4 Blocking CD44v5 can reverse IL-4-induced immune escape and PD-1 / PD-L1 inhibitor resistance.

[0075] Constructing a CD44v5 knockdown TNBC cell line ( Figure 5 The study investigated the inhibition of CD44v5 function through both gene knockout and monoclonal antibody blockade. Results showed that CD44v5 blockade significantly reduced IL-4-mediated migration, colony formation, and anti-apoptotic effects in triple-negative breast cancer cells, while restoring their sensitivity to BMS-1 ​​(BMS-1). Figure 5 CDs and Figure 6 A). CD44v5 blockade also increased the expression of CD44 and CD69 on T cells ( Figure 6 B), restoring T cell killing function. Notably, CD44v5 knockdown reduces basal expression of IL-4R, p-STAT3, and PD-L1, and inhibits their upregulation response to IL-4 (B). Figure 6 C). These results establish CD44v5 as a core regulator of the IL-4R / STAT3 / PD-L1 pathway.

[0076] Furthermore, CD44v5 depletion leads to a decrease in SAM68 expression. Figure 6 (D) reveals a positive feedback mechanism between the two, which can sustain high CD44v5 expression and mediate PD-L1-dependent immunosuppression.

[0077] 3.1.5 Compared to PD-1 / PD-L1 inhibitors, blocking CD44v5 has a stronger anti-tumor effect.

[0078] Comparing the effects of CD44v5 blockade, BMS-1, and their combined treatment, it was found that CD44v5 blockade was superior to BMS-1 ​​treatment in inducing T cell-mediated TNBC cell killing; while the combined use of the two showed the strongest anti-tumor response and significantly promoted tumor cell apoptosis. Figure 7 (AB in the middle).

[0079] 3.1.6 CD44v5 participates in the regulation of PVR immune checkpoint proteins other than PD-L1 along the IL-4R / STAT3 axis.

[0080] Cellular experiments have shown that the TIGIT ligand PVR (CD155) is specifically highly expressed in TNBC. Figure 7 C). IL-4 treatment significantly upregulated PVR expression, while S3I-201 pretreatment inhibited this effect. Figure 7 D). Furthermore, CD44v5 knockdown simultaneously reduces both basal levels and IL-4-induced PVR expression (D). Figure 7 E).

[0081] The above findings confirm that CD44v5 can simultaneously regulate multiple immune checkpoint molecules, including PD-L1 and PVR, through the IL-4R / STAT3 pathway, and is a key regulatory factor in the immunosuppressive microenvironment of TNBC.

[0082] In summary, the above results systematically confirm that PD-L1 is highly expressed in TNBC and is closely related to type II immune response. CD44v5, as a core regulator of the IL-4R / STAT3 pathway, can simultaneously regulate PD-L1 and PVR immune checkpoint molecules, mediating IL-4-induced TNBC immune escape and PD-1 / PD-L1 inhibitor resistance. Blocking CD44v5 can reverse the above effects, and its anti-tumor effect is superior to PD-1 / PD-L1 inhibitor monotherapy. At the same time, the positive feedback mechanism between CD44v5 and SAM68 provides a new perspective for understanding the formation of the TNBC immune microenvironment, confirming that CD44v5 can serve as a potential target for improving the efficacy of TNBC immunotherapy.

[0083] 3.2 Study on CD44v5-mediated cisplatin resistance in triple-negative breast cancer and its mechanism

[0084] 3.2.1 CD44v5 upregulates SLC7A11 expression and enhances its membrane stability by activating the IL-4 / IL-4Rα / STAT3 pathway, thereby inhibiting TNBC ferroptosis.

[0085] Both TAMs and exogenous IL-4 can activate the IL-4Rα / STAT3 pathway, upregulate SLC7A11 and GPX4, and inhibit ferroptosis. This effect can be blocked by Dupilumab or the STAT3 inhibitor S3I-201. Figure 8 (A–C). Knockdown of CD44v5 or use of anti-CD44v5 antibody can inhibit IL-4Rα / STAT3 pathway activation and reduce SLC7A11 and GPX4 levels ( Figure 8 DE in the middle.

[0086] It is worth noting that, compared with dupilumab, CD44v5 monoclonal antibody has a stronger inhibitory effect on SLC7A11. Figure 8 E), suggesting that CD44v5 may have a regulatory mechanism independent of the IL-4Rα / STAT3 pathway. TCGA data analysis showed a positive correlation between CD44, sam68, and SLC7A11 expression ( Figure 9 A), and SLC7A11 was most highly expressed in TNBC, which was further confirmed by immunohistochemical results. Figure 9 BC in the middle). Structural prediction and confocal microscopy results showed that CD44v5 interacts with SLC7A11 and co-localizes on the cell membrane ( ). Figure 10Mechanistically, treatment with the microtubule inhibitor paclitaxel no longer affected SLC7A11 expression (AB in the text). Figure 10 (C) indicates that CD44v5 may maintain its stability on the cell membrane by inhibiting the endosome transformation pathway of SLC7A11.

[0087] 3.2.2 Blocking CD44v5 can promote ferroptosis and reverse IL-4-induced cisplatin resistance.

[0088] To verify the function of CD44v5, the promoting effect of IL-4 on TNBC cell migration and colony formation was significantly inhibited after antibody blockade or gene knockdown of CD44v5. Figure 11 In the presence of cisplatin, blocking CD44v5 further enhanced apoptosis (AD). Figure 11 EF in the middle), and induces ROS accumulation, downregulates SLC7A11 and GPX4 expression ( Figure 12 The presence of AD in the blood indicates that ferroptosis is activated. Therefore, blocking CD44v5 can both reverse the inhibition of apoptosis by IL-4 and promote ferroptosis, thereby enhancing the antitumor effect of cisplatin, indicating that CD44v5 plays a key role in IL-4-mediated cisplatin resistance in TNBC.

[0089] 3.2.3 CD44v5 knockdown significantly enhances in vivo cisplatin sensitivity.

[0090] To further verify the regulatory role of CD44v5 in cisplatin sensitivity, we established a subcutaneous xenograft model in BALB / c nude mice using CD44v5 knockdown MDA-MB-231 cells. Figure 13 A). Experimental results show that CD44v5 knockdown significantly enhances the antitumor effect of cisplatin. Figure 13 BD and Figure 14 A). Western blot analysis showed that the protein expression of IL-4Rα, SLC7A11, and GPX4 was significantly downregulated in the CD44v5 knockdown group. Figure 14 B). Immunohistochemical staining further showed that the proportion of Ki67-positive cells was reduced in the CD44v5 knockdown group, and the expression of IL-4Rα, SLC7A11, and GPX4 was simultaneously downregulated. Figure 14 C).

[0091] The above results systematically demonstrate the crucial role of CD44v5 in cisplatin resistance in TNBC. Blocking CD44v5 significantly enhances the efficacy of cisplatin, providing a new treatment strategy for overcoming chemotherapy resistance in TNBC.

[0092] In summary, this invention systematically elucidates the core driving role and dual molecular mechanism of CD44v5 in cisplatin resistance in triple-negative breast cancer (TNBC). The results show that high expression of CD44v5 is significantly positively correlated with tumor-associated macrophage (TAM) infiltration in TNBC and is closely linked to poor patient prognosis. Mechanistically, TAMs activate the IL-4Rα / STAT3 signaling pathway in TNBC cells by secreting IL-4, and CD44v5 plays a crucial upstream regulatory role in this process. It is not only an effector molecule of this pathway but also forms a positive feedback loop with IL-4, continuously enhancing pathway activation. More importantly, this invention reveals that CD44v5 also directly interacts with the cysteine ​​transporter SLC7A11 through its variable region, anchoring and stabilizing it on the cell membrane, thereby independently inhibiting cisplatin-induced ferroptosis. Therefore, CD44v5 promotes cisplatin resistance in TNBC through two parallel and synergistic pathways: "activation of IL-4R / STAT3 signaling" and "stabilization of SLC7A11 membrane protein." In vitro and in vivo experiments have confirmed that targeting and blocking CD44v5 can simultaneously block the above-mentioned mechanisms, effectively reversing drug resistance. This invention not only reveals a new mechanism of chemotherapy resistance in TNBC, but also establishes CD44v5 as a key therapeutic target for overcoming clinical drug resistance.

[0093] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Application of CD44v5 inhibitors in promoting immunotherapy for triple-negative breast cancer and overcoming cisplatin-resistant chemotherapy.

2. The application according to claim 1, characterized in that, The inhibitors include substances that reduce CD44v5 protein expression or CD44v5 protein content, or substances that inhibit CD44v5 gene expression. These substances include one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses.

3. Application of CD44v5 inhibitors combined with active ingredients in the preparation of drugs for the treatment of triple-negative breast cancer.

4. The application according to claim 3, characterized in that, The CD44v5 exerts a dual regulatory effect through the IL-4R / STAT3 axis, and the dual regulatory effect includes: (1) Regulates multiple immune checkpoint molecules to promote immune escape in triple-negative breast cancer and enhances resistance to anti-PD-1 / PD-L1 therapy; (2) Upregulate SLC7A11 expression and stabilize its membrane localization, inhibit tumor cell ferroptosis, and thus enhance the chemotherapy resistance of triple-negative breast cancer to cisplatin.

5. The application according to claim 3, characterized in that, The application uses CD44v5 expression level as an evaluation index, and then uses CD44v5 inhibitors in combination with PD-1 / PD-L1 inhibitors and cisplatin chemotherapy drugs to enhance the sensitization of triple-negative breast cancer treatment.

6. The application according to claim 3, characterized in that, The drug is used to promote tumor cell apoptosis and ferroptosis, and to restore T cell function.

7. The application according to claim 3, characterized in that, The drug includes a CD44v5 inhibitor and medically approved excipients.

8. The application according to claim 3, characterized in that, The drug includes various acceptable dosage forms, such as injections, pills, capsules, granules, tablets, or oral liquids.

9. A diagnostic model for triple-negative breast cancer, characterized in that, The model uses CD44v5 gene expression level or CD44v5 protein expression level as one of the effective indicators for diagnosis.

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