SASH1 Suppresses Triple Negative Breast Cancer Cell Invasion Through Actin-mediated Regulation Of Hippo/YAP Pathway

Purpose:Breast cancer is a serious threat to women's health.The incidence of breast cancer is still the highest in the incidence of female malignant tumors and the second highest in mortality of the world.Triple-negative breast cancer(TNBC)is devoid of the expression of progesterone receptor,estrogen receptor and human epidermal growth factor receptor 2 and there is no effective targeted therapeutics against it.Therefore,there is an urgent need to identify the novel targets for the treatment of TNBC.SASH1(SAM and SH3 domain 1)is a member of the SLY signal adaptor family of proteins that inhibit migration and invasion of a variety of tumor cells.It is down-regulated in a variety of tumors such as invasive breast cancer and liver metastatic colon cancer.Low expression of SASH1 is closely related to poor prognosis.SNP screening of tumor tissues from 30 cases of TNBC patients indicated that SASH1 is an important genetic risk factor for TNBC,but the mechanisms by which SASH1 elicits tumor-supressive effects in TNBC are unclear.At present,there are few reports dissecting the mechanisms of SASH1 in cancer.Liu et al found that SASH1 inhibited the proliferation and invasion of cervical cancer cells by inhibiting FAK expression in cervical cancer cells.It has been reported that in colon cancer,SASH1 interacted with F-actin to induce actin polymerization.In addition,SASH1 binded and colocalized with cortactin,which is involved in cell migration and adhesion,revealing the possible mechanism by which the down-regulated expression of SASH1 promoted colon cancer metastasis and progression.There is another literature demonstrating that SASH1 regulates melanoma cell migration through the SASH1-IQGAP1-E-cadherin-dependent pathway.Additionally,SASH1 may inhibit the invasion and metastasis of hepatoma cells by down-regulating the Shh-Gli1 and PI3K-AKT pathways.Yes associated protein(YAP)and WW domain–containing transcription regulator 1(WWTR1 or TAZ)are transcriptional coactivators downstream of the Hippo signaling pathway and its activation and expression levels are regulated by a variety of factors.Upon Hippo pathway activation,the Hippo core kinases MST1/2-LATS1/2 phosphorylates YAP/TAZ and promotes the degradation of YAP/TAZ by the proteasome pathway,thereby playing a tumor-suppressive role.Actin polymerization and depolymerization regulate the nuclear localization of YAP in tumor cells;as such the cross-talk between F-actin and Hippo/YAP pathways is an important way to regulate the metastasis of breast cancer.Our study provides a new direction for the clinical targeted therapy of TNBC by investigating the molecular mechanism of SASH1 regulating TNBC cell invasion.Method:1.The expression of SASH1 protein in 68 cases of non-TNBC and 75 cases of TNBC tissues was analyzed by immunohistochemistry.Statistical analysis was performed to confirm the correlation between SASH1 expression and clinicopathological features of TNBC patients.Immunohistochemical analysis of 12 cases of TNBC tissue sections and 8 cases of non-TNBC tissue sections showed the expreesion of SASH1.2.BT549 and MDA-MB-468 cells stably depletion of SASH1 were constructed and the efficiency of SASH1 knock-down was checked by immunoblotting.3.Transwell and anoikis assays were used to detect the effects of stable knockdown or adenovirus-mediated expression of SASH1 on MDA-MB-231 and MDA-MB-468 cell invasion.4.The in vivo effects of stable knockdown of SASH1 on TNBC cell invasion were detected in a chicken embryo-based tumor invasion model as well as in nude mice of pulmonary metastasis induced by intravenous injection of tail vein.5.Whole-genome sequencing of MDA-MB-468 cell lines with stably depletion of SASH1 and control cells were performed by RNAseq.The differences of signal pathway and gene expression were compared and verified by Realtime-PCR.6.The expression of SASH1,F-actin and Hippo/YAP pathway-associated proteins were determined by western blot assay.7.The binding of SASH1 with Hippo/YAP pathway-related proteins was detected by immunoprecipitation.8.The co-localization between SASH1 and F-actin,the formation of F-actin and the localization of YAP were examined by immunofluorescence analysis.9.Phosphorylation of SASH1 protein by LATS1/2 was detected using phospho-taggel.10.Construction of SASH1 wild-type and mutant plasmids was achieved by regular molecular cloning.Results:1.Correlation analysis between SASH1 and TNBC.1.1 Bioinformatics results showed that the expression of SASH1 in breast cancer was low,and the expression of SASH1 in TNBC patients is significantly lower than that in non-TNBC patients.Immunohistochemical results of 12 cases of TNBC and 8 cases non-TNBC indicated that the expression of SASH1 is lower in TNBC patients.1.2 Immunohistochemistry analysis was performed on tumor tissues from 68 cases of non-TNBC and 75 cases of TNBC patients.The results showed that the percentage of low expression of SASH1 in the cytoplasm was 43.1%(28/43)in non-TNBC patients,while that in TNBC patients was 60.8(45/74),the difference was significant(p=0.037),suggesting that the expression of SASH1 in TNBC patients was significantly lower than that in non-TNBC patients.The results of clinical correlation analysis showed that the expression of SASH1 was significantly correlated with molecular subtypes in breast cancer.But it not correlated with age,primary tumor infiltration(T),and tumor stage(TNM),lymphocyte node metastasis(N),the expression of p53,E-Cadherin and Ki67.2.SASH1 suppresses TNBC cell invasion.2.1 Knocking down SASH1 promoted the invasion of TNBC cells(MDA-MB-231 and MD1-MB-468).2.2 Overexpression of SASH1 significantly inhibited cell invasion in TNBC cells.2.3 Knocking down SASH1 enhanced the invasive ability of TNBC cells in vivo.3.The molecular mechanism of SASH1 inhibiting cell invasion in TNBC cells.3.1 Data from RNAseq showed that SASH1 was closely related to extracellular matrix signaling.Knocking down SASH1 significantly increased the expression of ARHGAP42,which was verified by real-time quantitative PCR and Western blot assays.3.2 Down-regulation of SASH1 inhibited the formation of F-actin in TNBC cells and promoted the transition from F-actin to G-actin.3.3 Down-regulation of SASH1 inhibited the phosphorylation of YAP in TNBC cells and promoted the nucleation of YAP,lead to increased transcriptional regulation of CYR61 expression.3.4 Overexpression of SASH1 by adenovirus could rescue SASH1depletion-induced the down-regulated phosphorylation of YAP along with the upregulated nuclear localization of YAP in TNBC cells.3.5 The F-actin polymerization inhibitor Jasplakinolide inhibited stably depletion of SASH1-induced increase of the nuclear localization of YAP in TNBC cells.3.6 SASH1 regulates the expression of ARHGAP42,and SASH1 inhibits cell invasion depending on ARHGAP42 in TNBC cells.3.7 Both MST1/2 and LATS1/2 were binding to SASH1,and SASH1 regulateed cell invasion depending on LATS1 in TNBC cells.3.8 Upon irradiation,LATS1/2 phosphorylated SASH1 at S407.3.9 SASH1 inhibited cell invasion in TNBC cells depending on the site at S407.Conclusion:1.SASH1 expression was relatively low in breast cancer patients and low SASH1 expression was correlated with poor prognosis and molecular subtypes in breast cancer.In addition,significantly lower in TNBC patients than in non-TNBC patients.2.SASH1 inhibited cell invasion in TNBC through F-actin/YAP axis.3.SASH1 regulated cell invasion in TNBC cells depending on ARHGAP42.4.SASH1 regulated cell invasion in TNBC cells depending on LATS1.5.SASH1 interacted with MST1/2 and LAST1/2.Upon irradiation,SASH1 was phosphorylated by LATS1/2 at S407.6.SASH1 inhibited cell invasion in TNBC cells depending on the site at S407.