Preliminary Study Of Mechanism Of SASH1in Breast Cells

OBJECTIVE: To investigate the expression and clinical significanceof the novel tumor suppressor SAM-and SH3-domain containing1(SASH1)protein in invasive ductal carcinoma (IDC) and the potential molecularmechanism of SASH1on the regulation of proliferation, apoptosis andmetastasis of cells.METHODS:(1) The expressions of SASH1were detected in31para-carcinoma tissues and41carcinoma tissues from patients with IDCby immunohistochemistry and western blot respectively. The results ofSASH1were analyzed by combination with clinical and pathological dataof IDC.(2) Retrovirus vector of SBP-Flag-SASH1-pBABE-puro wastransfected into HEK-293T cells for the construction of stable HEK-293Tcells expressing exogenous SASH1after puromycin screening. Exogenousfusion protein expression of Flag-SASH1was identified by western blot.Pull-down assay， LC-MS/MS analysis and immunoprecipitation wereperformed to explore the key SASH1binding proteins which mightregulate the proliferation，apoptosis and metastasis of tumor cells. (3) SASH1-siRNA1and SASH1-siRNA2was transfected intoMDA-MB-231cell lines, compared with blank group and negative-siRNA.Western blot detection the interference effect of SASH1protein and theexpression of Phospho-ERK1/2after72hours.RESULTS:(1)The expression of SASH1protein was significantlylower in cancer tissues than that in para-carcinoma tissues（F=11.162，p=0.01）. Immunohistochemistry assay indicated that the positiveexpression rates of SASH1in the para-carcinoma tissues were77.4%（24/31）, which was higher than that (31.7%，13/41）in the cancer tissues,and there was a significant difference between the positive expression rateof SASH1in para-carcinoma tissues and tumor tissues (p<0.01). Thepositive expression of SASH1was correlated with histological grade andthe metastasis of axillary lymph node, but not with patients’age and tumorsize.(2) Recombinant plasmid of SBP-Flag-SASH1-pBABE-puro wassuccessfully stably expressed in HEK-293T cells. Pull-down assay，LC-MS/MS analyses and immunoprecipitation indicated that SASH1hadno intereaction with MAP2K2, but there was a close intereaction betweenSASH1and MAP4K4.(3) The SASH1-siRNA can efficiently block the expression of SASH1,and the expression of Phospho-ERK1/2was increased in group ofSASH1-siRNA. CONCLUSION:（1）The loss or inhibition of SASH1expressionmight play an important role in tumorigenesis and development of IDC.(2) There is interactions between SASH1and MAP4K4, MAP4K4isan important candidate binding protein of SASH1and may mediatemultiple cellular functions of SASH1.(3) The expression of Phospho-ERK1/2was increased when theexpression of SASH1was suppressed, SASH1may cross-talk withERK1/2signaling pathways probably through the mediation of MAP4K4to promote many cell processes including cell proliferation and migration.