c-CBL regulates melanoma proliferation, migration, invasion and the FAK-SRC-GRB2 nexus

Abstract

Melanoma is one of the most aggressive and lethal forms of skin cancer. Despite recent improvements in targeted therapies, many patients with advanced disease fail to achieve lasting tumor regression. Therefore, it is important to develop novel druggable targets that can be exploited to improve clinical outcome. Here, we studied the role of Casitas B-lineage lymphoma (c-CBL), an E3 ubiquitin ligase, in human melanoma. Employing quantitative real-time PCR and Western blot analysis in a panel of human melanoma cell lines (A375, G361, Hs-294T, SK-Mel-2, SK-Mel-28 and 451Lu), we found that c-CBL is strongly expressed in human melanoma cells at the mRNA and protein levels. Further, we determined c-CBL levels in clinical samples of melanomas and benign melanocytic nevi, using quantitative Nuance multispectral imaging. Compared to benign nevi, melanomas showed an overlapping range of c-CBL immunoreactivity. Small interfering RNA (siRNA)-mediated knockdown of c-CBL resulted in decreased proliferation, clonogenic survival and migration of melanoma cells. Furthermore, it also resulted in decreased cellular invasion in a 3D spheroid assay system. C-CBL and FAK are regulated by SRC, and FAK binds SRC and GRB2. C-CBL E3 ligase domain regulates receptor tyrosine kinase internalization through ubiquitination and its ring finger domain stabilizes the FAK-SRC-actin cytoskeleton thereby promoting cellular motility. C-CBL knockdown was associated with decreased protein and/or mRNA levels of SRC, FAK and GRB2. Taken together, we have provided evidence that c-CBL plays a role in melanoma cell proliferation, migration and invasion as well as inhibition of the FAK-GRB2-SRC nexus. Our findings indicate that additional studies are warranted to further dissect the role of c-CBL in melanoma and determine the therapeutic potential of its inhibition.