The Evaluation of FAK-FAT Domain Inhibition in Melanoma Subtypes

Abstract

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is an essential component of the focal adhesion complex, which are hubs for signal transduction and mediation of important cellular processes. FAK activity is dependent on integrin-regulated signaling and includes both enzymatic and scaffolding functions due to its multi-domain structure. Through its scaffolding focal adhesion targeting (FAT)-domain, FAK associates with other complex proteins to maintain focal adhesion function. Specifically, the FAK-FAT domain’s interaction with the adapter protein, paxillin, is a critical interaction which mediates focal adhesion turnover, cell motility, and assembly of these adhesion complexes. Additionally, through its scaffolding function, the FAT-domain of FAK regulates cell proliferation, survival, tumorigenesis, adhesion, invasion, and migration. Importantly, FAK is reported to be highly overexpressed in a wide range of tumor types. Within the context of melanoma, numerous studies show that the proper functioning of the FAK-FAT domain is necessary for proliferation, survival, invasion, and metastasis of tumor cells. Presently, FAK-inhibitors in current clinical trials target the kinase domain and have shown limited efficacy in patient outcomes. Given these poor outcomes, in part due to resistance mechanisms to kinase inhibition, targeting the FAT-domain is further suggested as an efficacious therapeutic approach in cancer. Protein analysis studies between a variety of melanoma subtypes showed that NRAS-mutant melanoma cell lines exhibit an increase in tyrosine phosphorylation at Y925, a marker for the FAK-FAT domain. This contrasts with the absence of pY925 in BRAF-mutant melanoma cell lines, suggesting a potential FAT-domain sensitivity in NRAS-mutant melanoma but not BRAF-mutants. To investigate this potential dependence, NRAS- and BRAF-mutant melanoma subtypes were evaluated for sensitivity to FAK-FAT domain inhibition. A novel stapled peptide UA-2012 was modeled after the LD2 motif of paxillin, as this protein is important to FAK-FAT domain function. An endogenous dominant-negative inhibitor, adFRNK, was utilized as the positive control for FAT-domain inhibition. The results showed that NRAS-mutant melanoma cells were highly sensitive to either mode of FAT-domain inhibition, by UA-2012 or adFRNK, showing anti-viability, anti-invasion, anti-adhesion, and pro-apoptotic effects. However, the BRAF-mutant melanoma cells did not display these effects, suggesting that BRAF-mutant melanoma cells are not sensitive to FAT-domain inhibition. Disruption of focal adhesion complexes and the FAK-paxillin interaction by the FAT inhibitors were observed in NRAS-mutant melanoma cells, corroborating that the FAT-domain is critical component of cancer cell phenotype in this subtype of melanoma. This work holds promise for bridging current gaps in melanoma therapy by targeting the FAK-FAT in NRAS-mutant melanoma.