Proteasomal downregulation of the pro-apoptotic MST2 pathway contributes to BRAF inhibitor resistance in melanoma

Abstract

The RAS-RAF-MEK-ERK pathway is hyperactivated in most malignant melanomas, and mutations in BRAF or NRAS account for most of these cases. BRAF inhibitors (BRAFi) are highly efficient for treating patients with BRAFV600E mutations, but tumours frequently acquire resistance within a few months. Multiple resistance mechanisms have been identified, due to mutations or network adaptations that revive ERK signalling. We have previously shown that RAF proteins inhibit the MST2 proapoptotic pathway in a kinase independent fashion. Here, we have investigated the role of the MST2 pathway in mediating resistance to BRAFi. We show that the BRAFV600E mutant protein, but not the wildtype BRAF protein, strongly binds to MST2 and inhibits MST2 pro-apoptotic signalling. Downregulation of MST2 reduces BRAFi induced apoptosis. In BRAFi resistant cell lines MST2 pathway proteins are downregulated by ubiquitination and subsequent proteasomal degradation rendering cells refractory to MST2 pathway induced apoptosis. Restoration of apoptosis is achieved by increasing MST2 pathway protein expression using proteasome inhibitors. In summary, we show that the MST2 pathway is involved in the acquisition of BRAFi resistance in melanoma.

Interaction of LATS1 with SMAC links the MST2/Hippo pathway with apoptosis in an IAP-dependent manner

“…The MST2 pathway role in melanoma is poorly understood and most studies have solely focussed on the role of YAP [38]. In a parallel study, we have seen that loss of expression of MST2 and LATS1 is common in melanoma cell lines that have acquired resistance to BRAF inhibitors [19]. We have demonstrated that oncogenic BRAF can inhibit MST1/2 in these cell lines and prevent the activation of its proapoptotic signal.…”

“…2A). Since RASSF1A expression is lost in these cell lines [19,28,29], we postulated that the lack of expression of RASSF1A could impair LATS1 proapoptotic signalling in melanoma, explaining the resistance to LATS1 overexpression observed in these cell lines. Hence, we studied the effect that RASSF1A re-expression had in the regulation of MST2-LATS1 interaction in A375 cells.…”

“…BRAF inhibition activates the LATS1 proapoptotic signalling, and it is enhanced by RASSF1A Since it was previously reported that MST1/2 kinases can be bound and inhibited by mutant BRAF [18,19], we tested whether BRAF inhibition could trigger LATS1 proapoptotic signalling alone or in combination with RASSF1A restoration. Using LATS1 phosphorylation state as readout of LATS1 activation, we found that increasing amounts of RASSF1A slightly promoted LATS1 activity in growing conditions and that the BRAF inhibitor Vemurafenib enhanced LATS1 activation (Fig.…”

“…Initial proteomics datasets used in this project have been published before [19,23,27] and are available in PRIDE repository. The original data sets described in this manuscript are deposited in PRIDE repository [48], access number PXD032781.…”

“…Thus, silencing of the proapoptotic MST2 pathway seems to be a common event in aggressive melanoma and restoration of RASSF1A expression is sufficient to induce cell death in melanoma cell lines [13]. Importantly, several lines of evidence indicate that the ERK pathway, which is commonly deregulated in melanomas due BRAF or NRAS mutations, can contribute to silencing the MST2 pathway proapoptotic signal through different mechanisms: (i) RAF1 and mutant BRAF V600E can block LATS1 activity by direct binding and inhibition of MST kinases [18,19]; (ii) activated AKT inhibits MST2 by direct phosphorylation [20]; (iii) mutant NRAS can inhibit MST2 activation, potentially through AKT activation [21]. Also, there is growing evidence supporting the importance of LATS1 in the regulation of apoptosis, including the regulation of p53, PUMA and mediation of death receptor-dependent apoptosis [15,22,23].…”