Despite the remarkable clinical response of melanoma harboring BRAF mutations to

Despite the remarkable clinical response of melanoma harboring BRAF mutations to BRAF inhibitors (BRAFi), most tumors become resistant. tumors (Davies et al., 2002). Tumors harboring constitutively active BRAFV600E exhibit highly active mitogen-activated protein kinase (MAPK) signaling, which is implicated in their transformation (Lopez-Bergami, 2011). Success in targeting oncogenic kinase activity has encouraged the development of therapies targeting the BRAF mutation, an approach that has produced a growing number of BRAF inhibitors (BRAFi), including vemurafenib and dabrafenib. These reagents represent significant advances in the clinical management of melanoma relative to the previous first-line therapy, dacarbazine (Chapman et al., 2011; Flaherty et al., 2010; Hauschild et al., 2012; Sosman et al., 2012). Nonetheless, some tumors treated with BRAFi exhibit intrinsic drug resistance, while others develop adaptive resistance over time. This remains a major obstacle in the long-term effectiveness of BRAFi-based therapy (Ribas and Flaherty, 2011) and thus is the subject of intense study. Numerous pathways reportedly underlie BRAFi resistance, including reactivation of MAPK signaling through NRAS or MEK1 mutations, BRAF splicing or gene amplification, and upregulation of receptor tyrosine kinases (RTKs) or growth factors (Abel et al., 2013; Nazarian et al., 2010; Poulikakos et al., 2011; Shi et al., 2012; Wagle et al., 2011; Wilson et al., 2012). In addition, altered signaling pathways, such as PI3K/AKT/mTOR and MITF/PGC1alpha, are implicated in BRAFi resistance (Haq et al., 2013; Shi et al., 2011; Villanueva et al., 2010). However, it is currently not possible to predict which tumors will exhibit chemoresistance. These hurdles have stimulated interest in novel combination therapies, including 123583-37-9 supplier BRAFi, but it remains challenging to identify which patients should undergo such regimens (Sullivan and Flaherty, 2013). Defining the mechanisms that underlie intrinsic/primary resistance or adaptive resistance and detecting them prior to initiating treatment could accelerate the development of rational combination therapies aimed at overcoming BRAFi resistance. Given the importance 123583-37-9 supplier of ubiquitin proteasome system (UPS) components in tumor development, progression, and resistance mechanisms (Hoeller and Dikic, 2009; Qi et al., 2008, 2010, 2013), we sought to determine whether UPS components may also contribute to BRAFi resistance of melanoma. To identify components of the UPS that potentially drive BRAFi resistance, we performed functional screening of a small interfering RNA (siRNA) library against UPS-related genes. We then assessed positive hits for differentially expressed genes in data 123583-37-9 supplier sets of BRAFi-resistant melanomas. The 123583-37-9 supplier combined analyses led us to identify the E3 ubiquitin ligase RNF125, which is downregulated in resistant melanomas, as a component of intrinsic resistance to BRAFi. We demonstrate the role of RNF125 in regulating JAK1 and EGFR expression, and establish the importance of this regulation for chemoresistance of melanoma to BRAFi. RESULTS Identification of RNF125 in BRAFi-Resistant Melanomas To define mechanisms underlying melanoma cell resistance to BRAFi, we evaluated the potential deregulation of UPS factors in BRAFi-resistant melanoma. To this end, we performed an unbiased screen of a siRNA library, including 123583-37-9 supplier 1,173 genes encoding most of the UPS-associated proteins. We performed the screen using melanoma cell lines (Lu1205 parental, sensitive [Lu1205S]), which became resistant in the presence of increasing concentrations (up to 5 M) of the BRAFi PLX4032 (Lu1205 resistant [Lu1205R]; Figures 1A and S1A). As previously reported, resistant cultures exhibited a high ERK activation correlated with BRAFi resistance, with an overall IC50 increase of 20- to 400-fold (Greger et al., 2012; Su et al., 2012; Figure S1A). Potential changes in viability of the parental and BRAFi-resistant Lu1205 cultures were monitored following transfection of cells with three siRNAs targeting each of the 1,173 Mouse monoclonal to PRAK UPS-related genes (Figure 1A). An initial screen of the parental line identified 18 genes for which inhibition conferred a growth advantage in the presence of BRAFi (1 M; Figures 1B and 1C). Among these genes, inhibition of CUL3, RBX1, or WDR24 conferred the most potent net growth advantage (Figures 1B and 1C). In agreement with this finding, an independent study reported that downregulation of Cul3 and Rbx1 confers a growth advantage in melanoma cells treated with BRAFi (Shalem et al., 2014). None of the UPS-related genes identified in the Lu1205S cells were able to alter the growth of.

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