ABSTRACT
The aim of the present study was to expound on the interactions between the mitogen-activated protein kinase (MAPK) and Hippo pathway members, and to further elucidate the molecular mechanisms of melanoma tumorigenesis. Four melanoma cell lines (C32, HS695T, SK-MEL-28 and A375) were used in the present study. Western blotting was used to assess the expression levels of the MAPK and Hippo pathway effector proteins: rapidly accelerated fibrosarcoma-1 proto-oncogene, serine/threonine kinase (RAF-1); serine/threonine kinase 3 (STK3; also known as MST-2); yes-associated protein (YAP); and tafazzin (TAZ). Immunoprecipitation was used to identify interactions between the effector proteins of the Hippo and MAPK pathways. RAF-1 was knocked down in melanoma cells using siRNA transfection, and cell proliferation, migration and invasion were determined by the MTT, wound-healing and Transwell invasion assays, respectively. Additionally, the cell cycle and apoptosis were analyzed by flow cytometry 48 h after RAF-1 knockdown. We found that the expression levels of the four proteins were variable, and that the HS695T cells expressed the highest levels of RAF-1. Immunoprecipitation studies revealed that RAF-1 bound to MST-2 in melanoma cells. Knockdown of RAF-1 inhibited the expression of YAP and TAZ, but did not affect MST-2 expression. Additionally, RAF-1 knockdown in melanoma cells significantly inhibited cell proliferation, migration and invasion, and induced apoptosis in these cells. Collectively, our results indicate that the RAF-1/MST-2 interaction may be a novel link between the MAPK and Hippo pathways.
