HSPA8 Activates Wnt/β‐Catenin Signaling to Facilitate BRAF V600E Colorectal Cancer Progression by CMA‐Mediated CAV1 Degradation

Abstract BRAF V600E attracts wide attention in the treatment of colorectal cancer (CRC) as stratifying and predicting a refractory classification of CRC. Recent evidence indicates that Wnt/β‐catenin signaling is broadly activated and participates in the refractoriness of BRAF V600E CRC, but the underlying molecular mechanism needs to be elucidated. Here, heat shock 70 kDa protein 8 (HSPA8), an essential regulator in chaperone‐mediated autophagy (CMA), is identified as a potential therapeutic target for advanced BRAF V600E CRC. These results show that HSPA8 is transcriptionally upregulated in BRAF V600E CRC, which promotes CMA‐dependent degradation of caveolin‐1 (CAV1) to release β‐catenin into the nucleus and thus activates the Wnt/β‐catenin pathway, contributing to metastasis and progression of BRAF V600E CRC. Of note, HSPA8 directly interacts with the KIFSN motif on CAV1, the interaction can be enhanced by p38 MAPK‐mediated CAV1 S168 phosphorylation. Furthermore, pharmacological targeting HSPA8 by VER155008 exhibits synergistic effects with BRAF inhibitors on CRC mouse models. In summary, these findings discover the important role of the HSPA8/CAV1/β‐catenin axis in the development of refractory BRAF V600E CRC and highlight HSPA8 as a predictive biomarker and therapeutic target in clinical practice.


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Figure S1.BRAF V600E mutation induces elevated HSPA8 expression in human colorectal cancer.

Figure S1 .
Figure S1.BRAF V600E mutation induces elevated HSPA8 expression in human colorectal cancer.(A-B) HSPA8 mRNA levels in CRC patients with or without BRAF missense mutation according to TCGA and CTPAC-2 perspective dataset (Student's t test).(C) Immunoblotting assays of SW480 cells transfected with siNC, siBRAF, siBRAF + BRAF WT or siBRAF + BRAF V600E.(D) Real-time qPCR analysis was performed to examine the mRNA expression levels of HSPA8 (mean ± SEM) in SW480 cells transfected with siNC or siBRAF, followed by overexpression of BRAF WT or BRAF V600E.(E-G) The predicted sequence of the interaction between BRAF downstream transcription factors (e.g., c-Myc, c-FOS, c-JUN) and the HSPA8 promoter according to JASPAR.(H) Real-time qPCR analysis was performed to examine the mRNA expression levels of MYC, FOS, or JUN (mean ± SEM) in SW480 cells transfected with BRAF WT or BRAF V600E plasmid.(I) ChIP-qPCR analysis.Chloroplasts fixed with formaldehyde (FA) were extracted from SW480 cells transfected with BRAF or BRAF V600E plasmid.Then, the DNA was sonicated into short fragments under specific conditions, immunoprecipitated with the indicated antibodies, and analyzed by quantitative real-time RT-PCR.Error bars indicate standard deviations for triplicates.(J) Immunoblotting assays of HSPA8 expression in HEK293T cells transfected with Vector or Flag-c-Myc plasmid.(K) Real-time qPCR analysis was performed to examine the HSPA8 mRNA expression levels (mean ± SEM) in HEK293T cells transfected with Vector or Flag-c-Myc plasmid.(L-M) HSPA8 expression levels showed a positive correlation with MYC expression levels according to the TCGA and cBioportal dataset Sidra-LUMC AC-ICAM (Pearson correlation test).***P < 0.001, **P < 0.01, *P < 0.05, and data are the mean ± SEM from at least three independent experiments.

Figure S2 .
Figure S2.HSPA8 expression is positively correlated with metastasis in human colorectal cancer.

Figure S2 .
Figure S2.HSPA8 expression is positively correlated with metastasis in human colorectal cancer.(A) Representative images of HSPA8 immunohistochemical staining in normal colorectal tissue or CRC tissue.Scale bar: 200 μm, 50 μm (enlarged).(B) Statistical quantification of HSPA8 immunohistochemical staining in normal colorectal tissue or CRC tissue (P < 0.0001, paired Student's t test).(C-D) HSPA8 mRNA levels in normal or CRC patients according to TCGA and GSE20916 dataset (P = 0.0028 and P < 0.0001, Student t test).(E-F) Immunoblotting analysis of HSPA8 expression levels in normal colorectal tissue or CRC tissue (P = 0.0011, Student's t test).(G) Immunoblot analysis of HSPA8 expression levels in several CRC cell lines.(H) Statistical quantification of HSPA8 expression levels in (G).(I) Representative images of migrated colorectal cells.Scale bar: 100 μm.(J) HSPA8 expression levels showed a positive correlation with migrated cell numbers (P = 0.0250, Pearson correlation test).(K) The genetic background of selected colorectal cancer cell lines.(L-M) The steps of constructing an orthotropic or tail vein injection mice model of CRC.

Figure S3 .
Figure S3.HSPA8 modulates the drug response to BRAF inhibitors in BRAF V600E CRC.(A-B) HSPA8 mRNA levels showed a different correlation with AZ628/SB590885 treatment response with or without BRAF V600E mutation, according to the CCLE database.(C) HT29 and RKO cell lines with the BRAF V600E genotype showed upregulated HSPA8 protein levels after dabrafenib treatment.(D) HSPA8 protein expression levels in CRC and melanoma tissues according to the Human Protein Atlas database.(E) MYC mRNA levels in CRC or melanoma patients according to TCGA dataset (Student's t test).(F) The IC50 values of the colorectal and melanoma BRAF V600E cell lines in the CCLE database were calculated.(G) Cell viability assay of RKO shScramble or shHSPA8 cells treated with or without the indicated concentrations of Dabrafenib for 24 h.***P < 0.001, **P < 0.01, *P < 0.05, and data are the mean ± SEM from at least three independent experiments.

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Figure S4.HSPA8 promotes epithelial-mesenchymal transition and metastasis in BRAF wildtype CRC cells.

Figure S4 .
Figure S4.HSPA8 promotes epithelial-mesenchymal transition and metastasis in BRAF wild-type CRC cells.(A) Immunoblotting analysis of SW480 cells stably expressing shScramble or shHSPA8.(B) Immunoblotting analysis of the effects of HSPA8 on the expression of EMT marker proteins in SW480 cells.(C) HSPA8 mRNA levels showed a negative correlation with CDH1 mRNA levels according to the cBioPortal dataset (P = 0.0149, Pearson correlation test).(D) HSPA8 mRNA levels showed a positive correlation with SAI2 mRNA levels according to the cBioPortal dataset (P < 0.0001, Pearson correlation test).(E-F) Effects of HSPA8 on cell migration and invasion evaluated by transwell assays in SW480 cells.Scale bar: 100 μm.(G-H) Wound healing assay showing the migration of SW480 cells stably expressing shScramble or shHSPA8 after 24 hours.Scale bar: 200 μm.***P < 0.001, **P < 0.01, *P < 0.05.Values are the mean ± SEM from at least three independent experiments.

Figure S5 .
Figure S5.HSPA8 is negatively related with CAV1 expression in colorectal cancer.

Figure S5 .
Figure S5.HSPA8 is negatively related with CAV1 expression in colorectal cancer.(A) Representative images of CAV1 immunohistochemical staining in normal colorectal tissue or CRC tissue.Scale bar: 100 μm, 20 μm (enlarged).(B) Statistical quantification of HSPA8 immunohistochemical staining in CRC tissues or normal tissues (P < 0.0001, paired Student's t test).(C-D) CAV1 mRNA in CRC and adjacent tissues according to the Notherman Colon and TCGA databases.(E-F) CAV1 protein levels in primary and metastatic CRC patients according to GSE2109 and GSE28702 datasets (Student's t test).(G) Representative images of HSPA8 and CAV1 immunohistochemical staining in tissue samples.Scale bar: 100 μm.(H) HSPA8 expression score negatively correlated with CAV1 expression score (P = 0.0007, Pearson correlation test).(I-J) CAV1 mRNA levels in CRC patients with or without BRAF V600E mutation according to TCGA and CPTAC-2 Prospective datasets (Student's t test).(K) Immunoblotting analysis of RKO cells stably expressing shScramble or shHSPA8 treated with or without 20 μM MG132 treatment.

Figure S10 .
Figure S10.HSPA8 inhibitor VER155008 exhibits synergic effect with BRAF inhibitors on BRAF V600E HT29 cells.(A-D) Cell viability assay of HT29 cells treated with VER155008 (5 μM), with or without Dabrafenib, Encorafenib, or Agerafenib for 24 h.(E-F) Summary of the IC 50 values of RKO and HT29 cells under the indicated treatments.(G-L) The drug combination dose-response matrices of VER155008 with Dabrafenib, Encorafenib, or Agerafenib in HT29 cells.The drug interaction landscapes were calculated based on the ZIP model.(M) Representative H&E images of the heart, liver, spleen, lung, and kidney of mice treated with or without VER155008, combined with or without Encorafenib.Scale bar: 200 μm.***P < 0.001, **P < 0.01, *P < 0.05, and data are the mean ± SEM from at least three independent experiments.

Figure S11 .
Figure S11.VER155008 exhibits synergetic effect with BRAF inhibitor on inhibition of metastatic potential.

Figure S11 .
Figure S11.VER155008 exhibits synergetic effect with BRAF inhibitor on inhibition of metastatic potential.(A-H) Transwell assays of RKO and HT29 cells treated with Dabrafenib or Encorafenib, combined with or without VER155008.Scale bar: 50 μm.(I-L) Wound healing assay showing the migration of RKO and HT29 cells treated with Dabrafenib or Encorafenib, combined with or without VER155008.Scale bar: 200 μm.***P < 0.001, **P < 0.01, *P < 0.05, and data are the mean ± SEM from at least three independent experiments.