Knockdown of Slingshot 2 (SSH2) serine phosphatase induces Caspase3 activation in human carcinoma cell lines with the loss of the Birt–Hogg–Dubé tumour suppressor gene (FLCN)

Birt–Hogg–Dubé (BHD) syndrome, is a dominantly inherited familial cancer syndrome associated with susceptibility to renal cell carcinoma (RCC) caused by inactivating mutations in the folliculin ( FLCN ) gene. The precise functions of the FLCN gene product are still under investigation but RCC from BHD patients show loss of the wild-type allele consistent with a tumor suppressor gene function. In a search for potential synthetic-lethal targets for FLCN using a phosphatase siRNA library screening approach, we found that knockdown of SSH2 serine phosphatase (one of the three members of Slingshot family and previously implicated in actin reorganization) specifically induced Caspase3/7 activity in a dose-dependent manner (up to six-fold increase, 10 n M , 72 h) in two human FLCN-deficient cell lines (BHD-origin renal cell carcinoma UOK257 and thyroid carcinoma FTC133) but not in their folliculin expressing isogenic cell lines. SSH2 siRNA-induced knockdown was accompanied by increased expression of SSH1 and SSH3 (suggesting a compensatory regulatory mechanism among members of SSH family). FLCN -null cells exhibited evidence of dysregulated cofilin de/phosphorylation pathways. Knockdown of SSH2 in FLCN -null cells was associated with an alteration in cell cycle kinetics (20% increase in G1, 30% and 40% decrease in S and G2M, respectively). Combination treatment of multiple SSH family ( SSH2 plus SSH1 and/or SSH3 ) siRNAs potentiated induction of Caspase3/7 activity and changes in the cell cycle kinetics. These data indicate that: (a) apoptotic cell death in FLCN -null cells can be triggered by SSH2 knockdown through cell cycle arrest; (b) SSH2 represents a potential therapeutic target for the development of agents for the treatment of BHD syndrome and, possibly, related tumors.