Use of an HSPA8 inhibitor in combination with a BRAF inhibitor in the preparation of a medicament for treating colorectal cancer

Abstract

The present invention provides the use of the combination of an HSPA8 inhibitor and a BRAF inhibitor in the preparation of a medicament for treating colorectal cancer. Experimental evidence of the present invention shows that high expression of HSPA8 can serve as a biological marker for poor prognosis of BRAF V600E colorectal cancer; HSPA8 degrades CAV1 through the CMA pathway, releases β-catenin into the nucleus, and activates the Wnt pathway; HSPA8 binds to the KISFN motif of CAV1, and phosphorylation of CAV1 S168 mediated by p38MAPK can promote their interaction; the combined use of an HSPA8 inhibitor and a BRAF V600E-targeted drug can significantly enhance the tumor suppression effect.

Description

Technical Field

[0001] The present invention relates to the use of the combination of an HSPA8 inhibitor and a BRAF inhibitor in the preparation of a medicament for treating colorectal cancer, and belongs to the field of medicaments. Background Art

[0002] According to the latest global cancer statistics, colorectal cancer (CRC) has become the second most lethal malignancy globally. Although the combination of surgery and chemotherapy has a good therapeutic effect on early CRC, there is still a lack of effective clinical treatment methods for patients with advanced CRC, and the 5-year survival rate of patients is less than 14%. CRC is considered a highly heterogeneous disease, and different CRC patients can be classified by specific mutant genotypes. Among them, the BRAF V600E mutation (occurring in about 12% of metastatic CRC patients) is considered an important prognostic marker for metastatic CRC. The median overall survival of CRC patients with the BRAF V600E mutation is only about 11 months. Although the response rate of BRAF V600E-specific inhibitors (such as dabrafenib, encorafenib, and agorafenib) in melanoma is as high as 80%, the response rate in colorectal cancer is only 5%. There is an urgent need to find ways to improve the drug reactivity.

[0003] BRAF is a serine / threonine kinase. After receiving an activation signal, it can regulate the activation of the downstream MAPK-ERK pathway, thereby affecting physiological activities such as cell division and cell differentiation. When valine at position 600 of BRAF is replaced by glutamate (i.e., the BRAF V600E mutation), BRAF enters a constitutively active state, which is generally considered a driver gene mutation for the occurrence of various tumors. When clinically treating tumors with BRAF V600E-targeted inhibitors, compensatory regulation may occur in tumor cells, resulting in the continued activation of the BRAF downstream pathway, thus leading to poor treatment effects. Research shows that the low response rate of BRAF V600E patients to targeted drugs may be due to the activation of the downstream MAPK pathway by cell bypasses. A representative molecular mechanism is that EGFR can activate the downstream MEK pathway by activating CRAF. In addition, bypass activation may also involve the activation of other signaling pathways (such as the PI3K / Akt pathway, etc.). Based on the above research, clinically, the therapeutic effect is enhanced by combining inhibitors of BRAF downstream molecules or bypass pathways. Currently, the response rate of the combination of BRAF and MEK inhibitors can reach 12%, the combination of BRAF, EGFR, and MEK inhibitors can reach 21%, the combination of BRAF and PI3K inhibitors can reach 23%, and the combination of BRAF, PI3K, and EGFR inhibitors can reach 32%, indicating that there may be other molecular mechanisms leading to drug resistance in cells.

[0004] Although the development of BRAF V600E-targeted drugs has progressed smoothly, and several BRAF V600E-targeted inhibitors (such as dabrafenib, encorafenib, agorafenib, etc.) have been marketed or are undergoing clinical trials, the phenomenon of drug resistance often occurs in the actual clinical application of BRAF V600E-targeted small molecule inhibitors. Recent studies have explored the potential mechanisms of adaptive drug resistance in BRAF V600E melanoma. It has been found that during the treatment with BRAF V600E-targeted drugs, the activation of the MAPK cascade pathway downstream of BRAF may occur in tumor cells, leading to the reactivation of the survival pathway of tumor cells and drug resistance. The above studies have elucidated part of the mechanism of BRAF V600E tumor drug resistance and have significantly improved the therapeutic effect of melanoma. Currently, the response rate of BRAF V600E-targeted drugs for melanoma can reach up to 80%, while the remission rate of colorectal cancer is only 5%. Therefore, it is urgent to study the mechanism of poor prognosis in BRAF V600E colorectal cancer patients and find new diagnostic and therapeutic targets for BRAF V600E.

[0005] Liu Yanfei, et al., Research progress of HSP70 inhibitors in the treatment of colorectal cancer, Chinese Journal of Cancer Biotherapy, 2022, 29(6), disclosed that the HSP70 inhibitor VER-155008 can exert anti-tumor effects by inhibiting the ATPase activity of HSP70 or interacting with different domains of HSP70.

[0006] Regarding the clinical phenomenon of poor prognosis in BRAF V600E colorectal cancer patients, there is currently no relevant report on the combined use of HSPA8 inhibitors and BRAF inhibitors. Summary of the Invention

[0007] In order to overcome the above technical problems, the present invention provides the use of the combination of HSPA8 inhibitor and BRAF inhibitor in the preparation of a drug for treating colorectal cancer.

[0008] The present invention provides the use of the combination of HSPA8 inhibitor and BRAF inhibitor in the preparation of a drug for treating colorectal cancer.

[0009] Among them, the molar ratio of the HSPA8 inhibitor to the BRAF inhibitor is: (2.5 - 20):(5 - 20).

[0010] Among them, the BRAF inhibitor is a BRAF V600E-targeted drug; the HSPA8 inhibitor is VER155008.

[0011] Among them, the BRAF inhibitors are: dabrafenib, encorafenib, agorafenib.

[0012] Among them, the dosage ratios of VER155008 to dabrafenib, encorafenib, and agorafenib are respectively:

[0013] VER155008 and dabrafenib: 2.5 μM: 10 μM or 7.5 μM: 20 μM;

[0014] VER155008 and encorafenib: 10 μM: 5 μM or 5 μM: 10 μM;

[0015] VER155008 and agorafenib: 20 μM: 20 μM or 7.5 μM: 20 μM.

[0016] The present invention also provides a pharmaceutical composition for treating colorectal cancer, which is composed of an HSPA8 inhibitor and a BRAF inhibitor, and their molar ratio is: (2.5 - 20):(5 - 20).

[0017] Among them, the BRAF inhibitor is a BRAF V600E - targeted drug; the HSPA8 inhibitor is VER155008.

[0018] Among them, the BRAF inhibitors are: dabrafenib, encorafenib, and agorafenib.

[0019] The dosage ratios of the said VER155008 to dabrafenib, encorafenib, and agorafenib are respectively:

[0020] VER155008 and dabrafenib: 2.5 μM: 10 μM or 7.5 μM: 20 μM;

[0021] VER155008 and encorafenib: 10 μM: 5 μM or 5 μM: 10 μM;

[0022] VER155008 and agorafenib: 20 μM: 20 μM or 7.5 μM: 20 μM.

[0023] This invention elucidates the role of HSPA8 in regulating BRAF V600E CRC progression through CMA-dependent CAV1 degradation. The upregulation of HSPA8 is a potential cause of poor prognosis in BRAF V600E CRC patients, which promotes tumor progression by activating the Wnt / β-catenin signaling pathway through a CAV1-dependent mechanism; CAV1 sequesters β-catenin in the cytoplasm by directly binding to β-catenin, preventing it from entering the nucleus and activating downstream pathways. HSPA8 interacts with CAV1 through the KIFSN motif, promoting CMA-mediated degradation of CAV1, thereby releasing β-catenin into the nucleus and activating the Wnt / β-catenin pathway. In addition, the small molecule inhibitor VER155008 of HSPA8 can increase the sensitivity of BRAF V600E CRC cells to BRAF inhibitors. HSPA8 can serve as a predictive and prognostic biomarker for CRC patients, as well as a therapeutic target for alleviating drug resistance in patients with BRAF V600E mutations. This invention combines HSPA8 inhibitors with BRAF inhibitors, which is expected to enhance the sensitivity of BRAF V600E colorectal cancer to BRAF inhibitors, thereby reducing the dosage of BRAF inhibitors clinically, improving the safety of clinical medication, and effectively slowing down the process of drug resistance generation. Brief Description of the Drawings

[0024] Figure 1 . The BRAF V600E mutation significantly affects the survival prognosis of colorectal cancer patients. (A) Overall survival of CRC patients with BRAF wild-type, V600E mutation, or other mutations according to the metastatic colorectal cancer database (MSKCC, Cancer cell, 2018). (B) Overall survival of CRC patients with BRAF wild-type or V600E mutation according to the CTPAC-2 database.

[0025] Figure 2 . Omics data screening shows that HSPA8 is a potential drug target for BRAF V600E colorectal cancer. (A) GO enrichment scatter plot. The horizontal axis represents the normalized enrichment score (NES), and the vertical axis represents the gene set size (the top 10 enriched pathways statistically). (B) Venn diagram showing 16 genes screened by cross-referencing the GOCC_AUTOPHAGOSOME gene set, TCGA BRAF co-expressed genes, and chEMBL drug target gene sets in the GSE98314 dataset. (C) Evaluation of candidate genes involved in BRAF V600E-targeted drug therapy based on the GSE98314 dataset.

[0026] Figure 3. HSPA8 is highly expressed in BRAF V600E colorectal cancer and is associated with poor prognosis in CRC patients. (A-C) According to the TCGA, CCLE, or CTPAC-2 databases, the mRNA levels of HSPA8 were examined in colorectal cancer patients with wild-type BRAF and BRAF V600E mutations. (D-E) According to the TCGA or CTPAC-2 databases, the mRNA levels of HSPA8 were examined in colorectal cancer patients with wild-type BRAF and BRAF missense mutations. (F) According to the TCGA database, the overall survival of patients with high or low expression of HSPA8 was plotted (P = 0.0047).

[0027] Figure 4 . HSPA8 is highly expressed in colorectal cancer tissues. (A) Representative fields of immunohistochemical staining of HSPA8 in normal colorectal tissues or colorectal cancer tissues, scale bar: 50 μm. (B) Statistical quantification of immunohistochemical staining of HSPA8 in normal colorectal tissues or colorectal cancer tissues (P < 0.0001). (C-D) According to the TCGA and GSE20916 databases, the mRNA levels of HSPA8 in normal or CRC patients (P = 0.0028 and P < 0.0001). (E-F) Immunoblotting experiments and statistics on the expression levels of HSPA8 in normal colorectal tissues and CRC patient tissues (P = 0.0011).

[0028] Figure 5 . The expression level of HSPA8 is linearly correlated with the migration ability of colorectal cancer. (A-B) Immunoblot analysis and statistical quantification of the expression levels of HSPA8 in multiple colorectal cancer cell lines. (C) Representative images of colorectal cells undergoing migration. Scale bar: 100 μm. (D) The expression level of HSPA8 is positively correlated with the number of migrating cells (P = 0.0250).

[0029] Figure 6 . HSPA8 is highly expressed in colorectal cancer tissues with a high-metastasis phenotype. (A) Representative fields of immunohistochemical staining of HSPA8 in normal colon, transition zone, and colorectal cancer tissues. Scale bar: 100 μm. (B) Representative images of immunohistochemical staining of HSPA8 in in situ colorectal cancer tissues (n = 25) or metastatic tumor tissues (n = 15), scale bar: 100 μm (upper), 25 μm (lower). (C) Statistical chart of immunohistochemical staining of HSPA8 in primary or metastatic sites of CRC (P = 0.0458). (D) According to the Oncomine database Ramaswamy Multi-cancer, the mRNA levels of HSPA8 in in situ tissues or tumor metastases of CRC patients (P = 0.0156).

[0030] Figure 7. Schematic diagram of mouse model construction. (A - B) Steps for constructing orthotopic colorectal cancer or tail vein injection mouse models.

[0031] Figure 8 . Figures demonstrating that HSPA8 promotes tumor metastasis in orthotopic models and tail vein metastasis models. (A) Representative images of liver metastases and H&E staining of mouse tissues; Scale bars: 50 mm (left), 50 μm (middle), 10 μm (right). (B) Statistical quantification of the number of liver metastasis nodules of the indicated CRC cells (P = 0.0199). (C) Representative images of lung metastases and H&E staining of mouse tissues; Scale bars: 50 mm (left), 50 μm (middle), 10 μm (right). (D) Statistical quantification of the number of lung metastasis nodules of the indicated CRC cells (P = 0.0087).

[0032] Figure 9 . Figure showing a positive correlation between HSPA8 expression level and BRAF - targeted drug IC50 in BRAFV600E - mutant cell lines. (A) Scatter plot and trend line of IC50 values and HSPA8 mRNA expression levels of BRAF wild - type cells and BRAFV600E - mutant cells treated with AZ628 in the CCLE database. (B) Scatter plot and trend line of IC50 values and HSPA8 mRNA expression levels of BRAF wild - type cells and BRAFV600E - mutant cells treated with SB590885 in the CCLE database. (C) Cell viability graph of RKO wild - type or HSPA8 - knockout cell lines treated or untreated with dabrafenib. **P < 0.01.

[0033] Figure 10 . Figure showing a negative correlation between HSPA8 expression and drug responsiveness in colorectal cancer and melanoma. (A) Statistical analysis of HSPA8 protein expression levels in tissue samples of colorectal cancer and melanoma patients in the HumanProtein Atlas database. (B) IC50 data of BRAF inhibitors in different colorectal cancer and melanoma cell lines in the CCLE database.

[0034] Figure 11. HSPA8 is upregulated and activates the MAPK pathway after BRAFV600E and BRAF targeted drug treatment. (A) Western blot assay was used to examine the expression level of HSPA8 in SW480 cells transfected with siNC, siBRAF, siBRAF + BRAFWT or siBRAF + BRAFV600E plasmids. (B) RT-qPCR assay was used to examine the expression level of HSPA8 mRNA in SW480 cells transfected with siNC, siBRAF, siBRAF + BRAFWT or siBRAF + BRAFV600E plasmids. (C) Western blot assay was used to examine the change level of HSPA8 expression in BRAFV600E colorectal cancer cell lines HT29 and RKO after treatment with dabrafenib. (D) GSEA enrichment analysis was performed to enrich the KEGG MAPK pathway in RKO and RKOshHSPA8 cell lines.

[0035] Figure 12 . Knockout of HSPA8 significantly enhances the therapeutic effect of BRAF inhibitors. (A) Colony formation assay was used to examine the effect of dabrafenib or encorafenib treatment and whether HSPA8 was knocked out on the colony formation ability of RKO cell line. (B) Transwell assay was used to examine the effect of dabrafenib or encorafenib treatment and whether HSPA8 was knocked out on the migration and invasion abilities of RKO cell line. Scale bar: 100 μm. ***P < 0.001, **P < 0.01, *P < 0.05.

[0036] Figure 13 . HSPA8 inhibitor sensitizes BRAF inhibitor in RKO cells. (A-D) Cell viability assay was used to examine the inhibitory effect of VER155008 (5 μM) combined with dabrafenib, encorafenib and agrafenib on the viability of RKO. (E) Summary table shows the IC50 values of each group in treating RKO. ***P < 0.001, *P < 0.05.

[0037] Figure 14 HSPA8 inhibitor sensitizes BRAF inhibitor in HT29 cells. (A-D) Cell viability assay was used to examine the inhibitory effect of VER155008 (5 μM) combined with dabrafenib, encorafenib and agrafenib on the viability of HT29. (E) Summary table shows the IC50 values of each group in treating HT29. ***P < 0.001, **P < 0.01.

[0038] Figure 15 . HSPA8 inhibitor and BRAF inhibitor have a drug synergy effect in RKO cells. (A-F) Heat map shows the cell activity inhibition rate and drug synergy coefficient of RKO cells treated with different concentrations of VER155008 and BRAF inhibitors dabrafenib, encorafenib or agrafenib.

[0039] Figure 16 .The figure showing the drug synergy between HSPA8 inhibitor and BRAF inhibitor in HT29 cells. (A-F) Heatmap showing the inhibition rate of cell viability and the drug synergy coefficient after treatment of HT29 cells with different concentrations of VER155008 and BRAF inhibitors dabrafenib, encorafenib or agorafenib.

[0040] Figure 17 .The figure showing the regulation of EMT marker protein expression by the combination of HSPA8 inhibitor and BRAF inhibitor. Immunoblotting was used to detect the expression levels of EMT markers in cells treated with VER155008, encorafenib or the combination of the two drugs.

[0041] Figure 18 .The figure showing the regulation of invasion and migration abilities of BRAF V600E colorectal cancer cells by the combination of HSPA8 inhibitor and BRAF inhibitor. (A-H) Transwell assay was used to detect the migration and invasion levels of RKO cells or HT29 cells treated with VER155008, dabrafenib, encorafenib or the combination of these drugs. Scale bar: 50 μm. ***P < 0.001, *P < 0.05.

[0042] Figure 19 .The figure showing the regulation of scratch wound healing ability of BRAF V600E colorectal cancer cells by the combination of HSPA8 inhibitor and BRAF inhibitor. (A-D) Scratch assay was used to detect the scratch wound healing ability of RKO cells or HT29 cells treated with VER155008, dabrafenib, encorafenib or the combination of these drugs. Scale bar: 200 μm. **P < 0.01, *P < 0.05.

[0043] Figure 20 .The figure showing the enhanced tumor killing effect of the combination of HSPA8 inhibitor and BRAF inhibitor in a mouse model. (A) Tumor pictures of mouse subcutaneous tumors treated with different treatment groups. Scale bar: 1 cm. (B) Tumor volumes at different time points after treatment with different treatment groups. (C) Tumor masses of different treatment groups. (D) Body weight statistics of mice in different treatment groups at different time points. ***P < 0.001.

[0044] Figure 21 .The figure showing the regulation of EMT marker protein expression by the combination of HSPA8 inhibitor and BRAF inhibitor in mouse tumor tissues. (A-E) Immunohistochemistry was used to detect the expression levels of Ki67, CAV1, claudin-1 and slug in mouse tumor tissues after treatment with VER155008, encorafenib alone or in combination. Scale bar: 50 μm. ***P < 0.001, **P < 0.01.

[0045] Figure 22 . H&E staining images of major organs in mice after the combination of HSPA8 inhibitor and BRAF inhibitor. The H&E experiment examined the pathological changes of major organs such as the heart, liver, spleen, lung, and kidney in mice after treatment with VER155008, encorafenib alone or in combination. Detailed implementation methods

[0046] The sources of the drugs used in the present invention are as follows:

[0047] VER155008 was purchased from Selleck (Catalog No.: S7751); dabrafenib was purchased from Beyotime (Catalog No.: SD5919); encorafenib was purchased from Tauto (Catalog No.: T6487); agorafenib was purchased from MCE (Catalog No.: HY-15200)

[0048] Test Example 1: Test on the combination of HSPA8 inhibitor and BRAF inhibitor of the present invention in the treatment of colorectal cancer

[0049] 1. Materials and methods

[0050] 1.1 Experimental materials and equipment

[0051] 1.1.1 Reagents and materials

[0052] a) Parental cell line

[0053]

[0054] b) Small molecule drugs

[0055]

[0056] c) Antibodies

[0057]

[0058]

[0059] d) Other reagents

[0060]

[0061]

[0062] 1.1.2 Experimental instruments and equipment

[0063]

[0064] 1.2 Experimental procedures

[0065] 1.2.1 Cell culture

[0066] 1.2.1.1 Cell resuscitation

[0067] a) Preheat the culture medium in a 37°C constant temperature water bath.

[0068] b) Quickly take out the target cells frozen in liquid nitrogen and thaw them in a 37°C constant temperature water bath.

[0069] c) Centrifuge the thawed cryotube (800 rpm, 3 min), resuspend the cell pellet with the preheated culture medium, and add the cell suspension to the prepared culture dish.

[0070] d) Gently shake well and then place the culture dish in a cell culture incubator (5% CO2, 37°C) for culturing. Observe the growth condition under a microscope after overnight attachment, and change the culture medium.

[0071] 1.2.1.2 Cell passage

[0072] a) Place the cell culture dish under a microscope. When the cell density reaches 80%-90%, passage is required.

[0073] b) Aspirate the culture medium, add PBS to rinse the residual culture medium, add 1-2 ml of trypsin (0.25%) and shake evenly, and place it in the incubator for digestion. The digestion time is determined according to operation experience.

[0074] c) Observe under a microscope. When the cells become round and cells float up when gently tapping the bottom of the culture dish, it indicates that the cell digestion is complete. Add 1-2 ml of complete culture medium to the culture dish to neutralize the trypsin and resuspend the cells, transfer them to a 15 ml centrifuge tube, and centrifuge (800 rpm, 3 min).

[0075] d) After centrifugation, discard the supernatant, add complete culture medium, resuspend the cells, inoculate a part of the cell suspension into the culture dish for continued culturing, and the other part of the cells can be used for experimental operations (such as plating, cryopreservation, etc.).

[0076] 1.2.1.3 Cell counting

[0077] a) Digest adherent cells into a cell suspension according to the method in 2.2.1.2, and pipette to mix evenly.

[0078] b) Dilute the original cell solution by a certain multiple, aspirate 10 μl of the cell suspension, and add it between the coverslip and the hemocytometer, avoiding the generation of bubbles.

[0079] c) Use a counter to calculate the total number of cells in the grid. The cell concentration is [(total number of cells in the 4×4 grid

[0080] / 4)×10 4 × dilution multiple] cells / ml.

[0081] 1.2.1.4 Cell cryopreservation

[0082] a) Prepare a programmable cooling box and cryotubes, and make marks on the cryotubes in advance;

[0083] b) Digest adherent cells according to the method in 2.2.1.2 and resuspend them in a centrifuge tube for centrifugation (800 rpm, 3 min);

[0084] c) Discard the supernatant, add cryopreservation solution according to the cell quantity and the number of cryopreserved samples, resuspend the cells in the cryotubes with the cryopreservation solution, place them in the programmable cooling box, store overnight at -80 °C, and then transfer them to a liquid nitrogen tank for cryopreservation.

[0085] 1.2.3 Western Blot

[0086] 1.2.3.1 Solution Preparation

[0087] a) Prepare RIPA lysis buffer according to the formula, adjust the pH to 7.4 with hydrochloric acid solution, store at 4 °C, and add Cocktail protease inhibitor and phosphatase inhibitor before use;

[0088]

[0089]

[0090] b) Preparation of 12% SDS-PAGE gel;

[0091]

[0092] c) SDS-PAGE electrophoresis buffer formula:

[0093]

[0094] d) Transfer buffer formula:

[0095]

[0096] e) SDS-PAGE loading buffer (5×loading buffer) formula, aliquot and store at -20 °C;

[0097]

[0098]

[0099] f) TBS (20×), pH = 7.6, store at room temperature;

[0100]

[0101] g) TBST formula:

[0102] 1.2.3.2 Protein sample processing

[0103] a) Discard the culture medium, wash twice with PBS buffer, scrape the cells with a cell scraper and collect them in a 1.5 ml centrifuge tube. Centrifuge at 4000 rpm for 3 min at 4°C and discard the supernatant; if it is a tissue sample, cut it into small pieces with scissors, add an appropriate amount of liquid nitrogen for treatment, grind it and collect it in a 1.5 ml centrifuge tube;

[0104] b) Prepare an appropriate amount of RIPA lysis buffer pre-added with protease inhibitor (Cocktail), vortex and mix well, and place on ice;

[0105] c) Add an appropriate amount of RIPA lysis buffer to resuspend the cells according to the amount of cell precipitate, pipette and mix well, and place on ice for lysis for 30 min;

[0106] d) Ultrasonically treat the cells with an ultrasonic crusher, 2 s each time, repeat 3 times;

[0107] e) Centrifuge the lysed sample at 4°C (13000 rpm, 15 min), aspirate the supernatant and transfer it to a new 1.5 ml EP tube for use;

[0108] f) Quantify the protein, add an appropriate amount of 5×SDS-PAGE loading buffer, vortex and mix well, heat at 100°C

[0109] After 10 min, prepare the WB sample, store it in a -20°C refrigerator or perform the subsequent WB experiment.

[0110] 1.2.3.3 SDS-PAGE electrophoresis

[0111] a) Install the prepared SDS-PAGE gel into the electrophoresis tank, add electrophoresis buffer, and pull out the comb; b) Take an appropriate amount of the prepared protein sample and evenly add it to the sample wells;

[0112] c) Keep the voltage at 80 V when the protein sample is in the stacking gel. When the protein sample is in the separating gel (the protein Marker starts to separate), adjust the voltage to 120 V. Pause the voltage when the protein sample is separated to the appropriate position and prepare for membrane transfer.

[0113] 1.2.3.4 Membrane transfer

[0114] a) Add an appropriate amount of methanol (200 mL) to the transfer buffer (800 mL) and mix well. Prepare the PVDF membrane and filter paper, and prepare the membrane transfer items such as the membrane transfer tank, membrane transfer clip, and ice bag;

[0115] b) Activate the PVDF membrane with methanol and then place it in the transfer buffer for later use. Assemble the transfer cassette according to the "sandwich" structure sequence of cathode / sponge / 4 layers of filter paper / gel / PVDF membrane / 4 layers of filter paper / sponge / anode. Pay attention to avoiding the generation of air bubbles between the PVDF membrane and the gel;

[0116] c) Ensure that the transfer cassette is placed in the transfer tank, add the transfer solution and an ice pack, and set a constant current of 300 mA for transfer;

[0117] Determine the transfer time according to the molecular weight of the target protein and the pore size of the PVDF membrane, about 1.5 - 2.5 h; d) After the transfer is completed, take out the PVDF membrane from the transfer cassette and put it into the milk blocking solution containing 5% skim milk powder that has been completely dissolved in TBST buffer, and place it on a shaker for blocking for 1 - 2 h. 1.2.3.5 Incubate antibodies and chemiluminescence

[0118] a) After the blocking is completed, use TBST buffer to wash the remaining milk on the PVDF membrane clean;

[0119] b) Compare the position of the protein Marker, cut the PVDF membrane into appropriate-sized strips according to the molecular weight of the target protein, put them into a hybridization incubation bag and add the diluted primary antibody. Avoid generating air bubbles during the operation, and incubate overnight on a shaker at 4°C;

[0120] c) Recover the primary antibody, take out the strips, wash them 3 times with TBST buffer, 10 min each time;

[0121] d) Put the washed strips into a new hybridization incubation bag, add an appropriate amount of diluted secondary antibody according to the resistance of the primary antibody, and incubate at room temperature for 2 h;

[0122] e) Take out the strips, wash them 6 times with TBST buffer, 10 min each time. Pay attention to preventing the strips from sticking to each other or sticking to the membrane washing box during the washing process;

[0123] f) Prepare the developing working solution according to the instructions of the chemiluminescence developing kit; Place the strips in the developing working solution for 30 s, and shake back and forth to fully soak the strips in the developing working solution;

[0124] g) Use an integrated chemiluminescence imaging system for development.

[0125] g) Use an integrated chemiluminescence imaging system for development.

[0126] 1.2.6 Transwell migration and invasion assays

[0127] a) Add 600 μl of complete medium to a 24-well plate, and place the sterilized and dried 8-μm special 24-well plate

[0128] Gently place the Transwell chamber with the pore size into the well, avoiding the formation of air bubbles between the chamber membrane and the liquid surface;

[0129] b) After digesting the cells, resuspend the cells in serum-free medium. Calculate the volumes of the required serum-free medium and cell suspension respectively according to the counting results, so that the number of cells inoculated in each chamber is 4×10 4 cells / 200 μl;

[0130] c) If performing a migration experiment, directly add 200 μl of the cell suspension dropwise in a spiral manner to the chamber. If performing an invasion experiment, the chamber needs to be pre-coated with Matrigel and then add

[0131] 200 μl of the cell suspension, and place it in an incubator for 1 - 2 days;

[0132] d) Take out the chamber, discard the medium in the chamber, wipe off the cells on the inner side of the chamber, and add 4% paraformaldehyde for fixation (1 h);

[0133] e) Discard the fixing solution, stain with crystal violet solution (30 min). Gently wipe off the residual crystal violet solution with a cotton ball, dry it, and then take a photo and count under a microscope.

[0134] 1.2.7 Scratch assay

[0135] a) Use a marker pen to draw 3 parallel lines on the back of a 12-well plate for each well as positioning auxiliary lines;

[0136] b) Inoculate 3×10 5 RKO cells or 5×10 5 HT29 cells into a 12-well plate respectively, gently shake well, and place it in a 37°C cell incubator overnight;

[0137] c) When the cell growth confluence rate reaches 90 - 100%, use the well plate lid as a "ruler", and use a yellow pipette tip to vertically scratch along the positioning auxiliary line at the bottom of each well;

[0138] d) After scratching, replace it with double-free medium and immediately take a photo of the scratch using an optical microscope to observe the width of the scratch in each field of view;

[0139] e) Place the cells in a 37°C cell incubator and continue to culture. Take a photo of the scratch field of view again at 24 h - 48 h, and observe the width of the scratch in each field of view at this time;

[0140] f) Use Image J software to compare and analyze the scratch widths at different time periods to obtain the migration distance of the cells during this period.

[0141] 1.2.8 Detection of cell proliferation by MTT method

[0142] a) Count the cells at 1000 cells / 100 μl and seed them in a 96-well plate. Add physiological saline to all the wells in the outer circle of the 96-well plate to prevent evaporation of the culture medium.

[0143] b) Incubate in a cell culture incubator for 1, 3, 5, and 7 days respectively. Add 10 μl of filtered MTT solution to each well and incubate in the cell culture incubator for 2 - 4 h.

[0144] c) Discard the supernatant and add 150 μl of DMSO to each well to dissolve the crystals.

[0145] d) After the crystals are fully dissolved, measure the absorbance at 570 nm using a multifunctional microplate reader and plot the cell growth curve.

[0146] 1.2.9 Immunohistochemical staining

[0147] 1.2.9.1 Prepare the following solutions:

[0148] a) PBS (phosphate buffer solution), pH 7.4, stored at room temperature.

[0149]

[0150] b) Antigen retrieval solution

[0151]

[0152]

[0153] 1.2.9.2 Tissue fixation, embedding, and sectioning

[0154] a) Collect fresh tissue and fix it with 4% paraformaldehyde for 1 - 2 days.

[0155] b) Put the fixed tissue into a tissue embedding cassette and rinse it slowly with running water overnight.

[0156] c) Dehydrate the tissue sequentially in gradient ethanol: soak in 75%, 85%, and 95% ethanol for 1 h each, and soak in 100%

[0157] ethanol 3 times, 30 min each time.

[0158] d) Immerse in xylene 2 times, 1 h each time.

[0159] e) Immerse in paraffin 3 times, 1 h each time.

[0160] f) Embed the tissue in paraffin using a tissue embedding machine and store it at -20 °C overnight.

[0161] g) Cut the tissue wax block into 4 - 6 μm sections

[0162] h) Place the sections on a floating table at 45 °C until they are stretched, then place them on a spreading table at 65 °C and store them in a section box after spreading. 1.2.9.3 Dewaxing, hydration and staining of sections

[0163] a) Place the tissue on an iron rack and bake the sections at 65 °C for 2 h;

[0164] b) Immerse the tissue in xylene twice, 10 min each time;

[0165] c) Hydrate the tissue in gradient ethanol in sequence: soak in 100%, 95%, 85%, and 75% ethanol for 2 min each; d) Place it on a shaker and rinse it slowly with deionized water twice, 5 min each time;

[0166] e) Block with hydrogen peroxide: soak the tissue in 3% H2O2, protect from light for 10 min, then place it on a shaker and rinse it slowly with ddH2O twice, 5 min each time;

[0167] f) Put the sections into an antigen retrieval box, perform antigen retrieval with antigen retrieval solution and a pressure cooker, and turn off the power after 3 - 5 min after the steam comes up;

[0168] 3 - 5 min later;

[0169] g) After the pressure cooker depressurizes, take out the antigen retrieval box and cool it to room temperature, discard the antigen retrieval solution, and wash it twice with PBS buffer, 5 min each time;

[0170] h) Take out the tissue sections, wipe off the water stains, circle the tissue with a wax pen, add an appropriate amount of goat serum (diluted 1:20) blocking solution, and block at 37 °C in a humidified box for 1 h;

[0171] i) Absorb the serum with filter paper, add an appropriate amount of primary antibody (diluted 1:200) and evenly cover the tissue surface, and incubate at 4 °C in a humidified box overnight;

[0172] j) Recover the primary antibody, put the sections back into the antigen retrieval box, wash them 3 times with PBS buffer, 5 min each time; k) Add an appropriate amount of secondary antibody and evenly cover the tissue surface, and incubate at 37 °C in a humidified box for 1 h;

[0173] l) Discard the secondary antibody, wash it 3 times with PBS buffer, 5 min each time;

[0174] m) Develop color with DAB chromogenic solution, observe the tissue staining under the microscope until it reaches an appropriate degree, and quickly place the sections in tap water to terminate the color development;

[0175] n) Stain the nucleus with hematoxylin solution, treat for 20 s, and then rinse it with tap water to turn blue for about 30 min;

[0176] o) Dehydrate the tissue in gradient ethanol in sequence, soak in 75%, 85%, 95% ethanol for 2 min each, 100%

[0177] Soak in alcohol twice, 2 minutes each time, and soak in xylene twice, 5 minutes each time;

[0178] p) Drop an appropriate amount of neutral gum for sealing the slides, avoid generating air bubbles during the process, and after completion, place the slides in a fume hood to air dry.

[0179] 1.2.10 Experimental animal models

[0180] The breeding of BALB / c nude mice was carried out according to the standards of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The use of experimental animals was reviewed and approved by the Institutional Ethics Committee of Sichuan University.

[0181] 1.2.10.1 Nude mouse tail vein model

[0182] a) House male BALB / c nude mice (6 weeks old) in a SPF-class animal room for 1 week to adapt to the environment; b) After digesting adherent cells, count them at 1×10 6 cells / 100 μl, and dilute to an appropriate volume with serum-free medium

[0183] and place on ice;

[0184] c) Use an auxiliary device for tail vein injection for intravenous injection, 100 μl / mouse, 6 mice / group;

[0185] d) If drug administration is required, administer the drug by gavage 3 days after tail vein injection;

[0186] e) Sacrifice the mice after one month, count and photograph the lung tumor metastases, fix them with 4% paraformaldehyde, and perform subsequent immunohistochemistry operations.

[0187] 1.2.10.2 Nude mouse colorectal orthotopic tumor model

[0188] a) House male BALB / c nude mice (6 weeks old) in a SPF-class animal room for 1 week to adapt to the environment; b) After digesting adherent cells, count them at 2.5×10 5 cells / 25 μl, and dilute to an appropriate volume with serum-free medium

[0189] and place on ice;

[0190] c) Inject 25 μl / mouse into the left lobe of the liver along the liver margin, 6 mice / group;

[0191] d) Two weeks later, the mice were sacrificed, the liver tumor metastases were counted and photographed, and then fixed in 4% paraformaldehyde solution for subsequent embedding, immunohistochemistry, and HE staining.

[0192] 1.2.11 Statistical analysis

[0193] a) The data differences between the two groups were compared using a two-tailed Student’s t-test.

[0194] b) The data correlation was analyzed using Pearson correlation test and linear regression analysis.

[0195] c) The survival analysis was performed using the log-rank (Mantel-Cox) test.

[0196] d) At least three independent repeated experiments were conducted to obtain data. GraphPad Prism 9.0 software was used for graphing and statistical difference analysis, specifically shown as *, P < 0.05; **, P < 0.01; ***, P < 0.001;

[0197] NS, no significant difference.

[0198] 2. Results

[0199] 2.1 BRAF V600E mutation induces increased HSPA8 expression in human colorectal cancer

[0200] 2.1.1 Screening of relevant targets for BRAF V600E colorectal cancer refractory

[0201] Recent studies have found that the BRAF V600E mutation in colorectal cancer can be used as a marker to predict the prognosis of colorectal cancer patients. The mortality rate of colorectal cancer patients with BRAF V600E increases by more than 2 times after metastasis compared with that of BRAF wild-type patients. Therefore, exploring the related molecular mechanisms of BRAF V600E colorectal cancer refractory has important pathological significance. The impact of the BRAF V600E mutation on the prognosis of colorectal cancer patients was explored in different databases ( Figure 1 ), and it was found that the prognosis of colorectal cancer patients with BRAF V600E was significantly worse than that of wild-type BRAF colorectal cancer patients, and the impact of the V600E mutation was more significant compared with other common mutations.

[0202] To identify potential drug targets for the treatment of BRAF V600E colorectal cancer, a related screening process was designed. First, after treating BRAF V600E mutant cells with BRAF V600E-targeted drugs, the gene change database (GSE98314) was used to enrich the 10 most significantly changed pathways, among which autophagosome-related genes were significantly enriched ( Figure 2 A); subsequently, genes co-expressed with the BRAF gene were screened out in the TCGA database and intersected with the aforementioned gene set; to screen potential drug targets, the ChEMBL database (https: / / www.ebi.ac.uk / chembl) was used to screen the candidate proteins again, and finally, 16 candidate proteins were found to be currently known drug targets ( Figure 2 B). According to the data of GSE98314, the correlation between candidate proteins and BRAF inhibitor treatment was ranked ( Figure 2 C), and it was found that the HSPA8 protein was the most relevant, suggesting that it might be a key protein for the poor prognosis of BRAF V600E colorectal cancer patients.

[0203] 2.1.2 HSPA8 is highly expressed in BRAF V600E colorectal cancer and is associated with poor prognosis of CRC patients

[0204] It was verified in the database that HSPA8 was highly expressed in BRAF V600E colorectal cancer patients ( Figure 3 A-E), and predicted a poor prognosis for CRC patients ( Figure 3 F), which was consistent with the screening conclusion. In addition, it was also detected in CRC patient tissues that HSPA8 was significantly highly expressed compared with normal tissues and had the potential to be a tumor marker ( Figure 4 ).

[0205] 2.1.3 HSPA8 is highly expressed in highly metastatic colorectal cancer cells and tissues

[0206] It was found that the expression level of HSPA8 in different colorectal cancer cell lines was linearly correlated with the migration ability of the cell lines ( Figure 5 ). In addition, the expression of HSPA8 in metastatic tissues was significantly higher than that in primary tumors ( Figure 6 ), suggesting that HSPA8 is closely related to tumor metastasis.

[0207] 2.1.4 Verification in mouse models that HSPA8 promotes colorectal cancer metastasis

[0208] Furthermore, an animal model was used to verify the effect of HSPA8 on the metastatic ability of colorectal cancer. An orthotopic mouse model of colorectal cancer and a mouse model of pulmonary metastasis via the tail vein were constructed ( Figure 7)。By separately inoculating wild-type RKO cells and RKO cells with HSPA8 knocked out, it was found that knocking out HSPA8 could significantly reduce the colorectal cancer metastasis in mice in both models ( Figure 8 )。

[0209] 2.1.5 Colorectal cancer cells with high expression of HSPA8 showed poor drug responsiveness

[0210] Furthermore, the connection between the expression level of HSPA8 and the drug responsiveness of colorectal cancer was detected in the database. It was found that in the CCLE database, the expression level of HSPA8 was positively correlated with the IC50 of BRAF-targeted drugs in cell lines with BRAFV600E mutations, while it was the opposite in BRAF wild-type cell lines ( Figure 9 A-B). After knocking out HSPA8 in the RKO cell line, it was found that the RKO cell line was more sensitive to the BRAF inhibitor dabrafenib ( Figure 9 C).

[0211] Previously, it was noted that the response rates of BRAFV600E inhibitors in melanoma and colorectal cancer were very significantly different. In melanoma, it could reach more than 50%, while in colorectal cancer, it was only about 5%. The expression levels of HSPA8 in tissue samples of colorectal cancer patients and melanoma patients were examined in the HumanProteinAtlas database, and it was found that the expression level of HSPA8 in the tumor tissues of colorectal cancer patients was significantly higher than that of melanoma patients ( Figure 10 A). Consistent with this, the CCLE database showed that the IC50 values of various BRAF inhibitors (including dabrafenib, SB590885, PLX4720, etc.) for colorectal cancer cell lines were all greater than those of melanoma cell lines ( Figure 10 B), suggesting that the differential expression of HSPA8 in different tumors may be the reason for the differential response of BRAFV600E to specific inhibitors in different tumors.

[0212] 2.1.6 BRAFV600E mutation and the use of BRAF-targeted inhibitors promote the upregulation of HSPA8 expression and the reactivation of downstream pathways

[0213] To further clarify the changes in the expression level of HSPA8 during BRAFV600E mutation and BRAF inhibitor treatment, cell lines with exogenous expression of BRAF and BRAFV600E were constructed ( Figure 11 A-B). It was found that the expression level of HSPA8 was upregulated in the cell line with exogenous expression of BRAFV600E compared with the cell line with exogenous expression of BRAF. At the same time, after treatment with dabrafenib, the expression levels of HSPA8 in the BRAFV600E colorectal cancer cell lines HT29 and RKO cell lines both increased ( Figure 11C), suggesting that HSPA8 may be an important effector molecule in the progression of BRAFV600E colorectal cancer. By performing GSEA analysis on the protein expression data of the RKO cell line with HSPA8 knockout, it was found that after knocking out HSPA8, the proteins related to the downstream of the MAPK pathway, which is the effector pathway of BRAFV600E, were all downregulated, preliminarily verifying this conjecture( Figure 11 D).

[0214] 2.1.7 The expression level of HSPA8 affects the therapeutic effect of BRAFV600E-targeted inhibitors

[0215] To further verify the effect of HSPA8 on the therapeutic effect of BRAFV600E colorectal cancer treated with BRAF-targeted inhibitors, the RKO cell line with or without HSPA8 knockout was treated with a BRAF inhibitor. It was found that under the condition of treatment with the BRAF-targeted drug in the RKO cell line, the clonal proliferation ability, migration ability, and invasion ability were significantly inhibited after HSPA8 knockout( Figure 12 ), suggesting that HSPA8 has clinical therapeutic prospects in enhancing the therapeutic effect of BRAF-targeted drugs.

[0216] The above experiments demonstrated that the BRAF V600E mutation induces an increase in the expression of HSPA8 in human colorectal cancer, which is closely related to the poor prognosis and low drug responsiveness of patients. After knocking out HSPA8, the EMT process of tumors can be significantly inhibited and the therapeutic effect of BRAF inhibitors can be enhanced, suggesting that targeting HSPA8 can be used as a potential clinical treatment strategy.

[0217] 2.2 HSPA8 inhibitors and BRAF inhibitors show a synergistic effect in the treatment of BRAF V600E colorectal cancer

[0218] 2.2.1 HSPA8 inhibitors sensitize BRAF inhibitors in BRAF V600E colorectal cancer cells

[0219] According to the results of previous studies, HSPA8 plays an important role in the progression and drug tolerance of BRAF V600E colorectal cancer cells, suggesting the potential application value of HSPA8 as an intervention target for BRAF V600E colorectal cancer. Therefore, the effect of the combination of the HSPA8 inhibitor VER155008 and different BRAF inhibitors (such as dabrafenib, encorafenib, and agorafenib, etc.) on the IC50 value of BRAF inhibitors was examined. The results showed that the HSPA8 inhibitor VER155008 could reduce the IC50 value of BRAF inhibitors in both the RKO and HT29 cell lines, suggesting that the combination of HSPA8 inhibitors and BRAF can sensitize BRAF V600E colorectal cancer cells( Figure 13 、 14 ).

[0220] 2.2.2. HSPA8 inhibitors and BRAF inhibitors have drug synergy in BRAF V600E colorectal cancer cells

[0221] Next, the synergistic effect of HSPA8 inhibitor VER155008 and BRAF inhibitor was quantitatively evaluated. First, a concentration gradient was set to test the cell killing efficiency of different concentrations of VER155008 and BRAF inhibitors, and then the Chou-Talalay method was used to test the drug combination index (CI) of the two drugs at this concentration. The results showed that the combination of VER155008 and BRAF inhibitors in two BRAF V600E colorectal cancer cells, RKO and HT29, could achieve a good combination index at a specific concentration, suggesting that the combination of the two inhibitors has potential clinical significance ( Figure 15 , 16 ). Based on the experimental results of IC50 and drug combination coefficient, Encofenib has a better comprehensive therapeutic effect than dabrafenib and agorafenib. At the same time, Encofenib is a targeted drug in the FDA-approved therapy for the clinical treatment of colorectal cancer BRAF V600E.

[0222] The drug combination coefficient calculated according to the Chou-Talalay method can determine the optimal combination ratio of two drugs, thereby achieving the effect of 1+1>2. Figure 15 and Figure 16 Based on the data of two colorectal cancer cells, it is estimated that the optimal combination range of VER155008 and dabrafenib is VER155008 (2.5-5μM) or (5-10μM) and dabrafenib (7.5-15μM) or (15-20μM); the optimal combination range of VER155008 and encofenib is VER155008 (5-15μM) or (2.5-7.5μM) and encofenib (2.5-7.5μM) or (7.5-12.5μM); the optimal combination range of VER155008 and agrafenib is VER155008 (10-20μM) or (2.5-12.5μM) and agrafenib (15-20μM) or (10-20μM). The optimal concentrations of VER155008 combined with dabrafenib are (2.5, 10 μM) or (7.5, 20 μM); the optimal concentrations of VER155008 combined with encofenib are (10, 5 μM) or (5, 10 μM); the optimal concentrations of VER155008 combined with agorafenib are (20, 20 μM) or (7.5, 20 μM).

[0223] 2.2.3. Combination of HSPA8 inhibitors and BRAF inhibitors to regulate the expression of EMT marker proteins

[0224] The changes in EMT markers after treatment of RKO cells with the HSPA8 inhibitor VER155008 and the BRAF inhibitor encorafenib alone or in combination were examined by Western blotting. It was found that after combined drug treatment, the expression levels of mesenchymal-like markers such as ZEB1 and vimentin were significantly inhibited, suggesting that the HSPA8 inhibitor and the BRAF inhibitor can significantly inhibit the EMT process( Figure 17 ).

[0225] 2.2.4.Regulation of BRAF V600E colorectal cancer cell metastasis by the combination of HSPA8 inhibitor and BRAF inhibitor

[0226] The effect of the combination of the HSPA8 inhibitor and the BRAF inhibitor on the metastatic phenotype of the BRAF V60E colorectal cancer cell line was further examined. The experimental results showed that the combination of the HSPA8 inhibitor VER155008 and the BRAF inhibitor exhibited an inhibitory effect on cell migration and invasion in both the BRAF V600E colorectal cancer cell lines RKO and HT29( Figure 18 ), and slowed down the scratch healing of tumor cells( Figure 19 ), suggesting that the combined use of the HSPA8 inhibitor and the BRAF inhibitor can effectively inhibit the metastasis of BRAFV600E colorectal cancer cells.

[0227] 2.2.5.The combination of HSPA8 inhibitor and BRAF inhibitor enhances the tumor killing effect in a mouse model

[0228] After verification in vitro, a mouse subcutaneous tumor model was used to examine the combined effect of the HSPA8 inhibitor and the BRAF inhibitor in vivo. It was found that the inhibitory effect on tumors of the HSPA8 inhibitor VER155008 or the BRAF inhibitor encorafenib alone was limited, while after combining VER155008 and encorafenib, the inhibitory effect on tumor volume and mass was significant, and there was no significant effect on the body weight of mice, suggesting that the combination of the HSPA8 inhibitor and the BRAF inhibitor has a good combined effect in mice( Figure 20 ).

[0229] 2.2.6.Regulation of EMT marker protein expression by the combination of HSPA8 inhibitor and BRAF inhibitor in mouse tumor tissues

[0230] Next, immunohistochemistry experiments were used to examine the expression of proliferation- and metastasis-related markers in mouse tumors after treatment with VER155008, encorafenib alone, or in combination. It was found that after the combined treatment of tumor tissues with VER155008 and encorafenib, the expression level of Ki67 decreased significantly, suggesting that tumor proliferation was significantly inhibited; the expression level of the EMT epithelial marker claudin-1 increased significantly after combined treatment, while the expression level of the mesenchymal marker slug decreased significantly, suggesting that the combination of drugs could inhibit the EMT process of tumor cells( Figure 21 ). And this inhibitor had no obvious toxicity to the main organs of mice( Figure 22 ).

Claims

1. Use of the combination of an HSPA8 inhibitor and a BRAF inhibitor in the preparation of a medicament for treating colorectal cancer; The BRAF inhibitor is a BRAF V600E-targeted drug; the HSPA8 inhibitor is VER155008; The BRAF inhibitors are: dabrafenib, encorafenib, and agorafenib.

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