The role of FOXD3 in melanoma and response to RAF/MEK inhibitors

Melanoma is by far the most aggressive and deadliest form of skin cancer. Over half of melanomas are driven by activating mutations in the serine/threonine kinase B-RAF, and as such, B-RAF inhibitors are now used in the clinical treatment of melanoma. Despite the promise of these inhibitors in the clinic, both intrinsic and acquired resistance has been observed, limiting the efficacy of these drugs. While several mechanisms of acquired resistance have been identified, little is known about the factors controlling intrinsic or adaptive resistance. In addition, the role of B-RAF-regulated transcription factors is unknown. Here we identify FOXD3, a transcription factor essential for stem cell self-renewal and normal neural crest development, as a target of mutant B-RAF. We found that inhibition of B-RAF or MEK1/2 results in the upregulation of FOXD3 at the mRNA and protein level. Furthermore, ectopic expression of FOXD3 induces a potent G0/G1 arrest in melanoma cells, which is mediated by p53 and p21 WAF1/CIP1. Based on data from our laboratory indicating that FOXD3 promotes resistance to B-RAF inhibitors PLX4032 and PLX4720, we sought to identify the FOXD3 transcriptome using microarray analysis combined with next-generation chromatin-immunoprecipitation sequencing. We identified the oncogene ERBB3/HER3 as a direct transcriptional target upregulated by FOXD3 and B-RAF/MEK inhibition. Additionally, signaling in response to the ligand of ERBB3, neuregulin-1 (NRG1), was greatly enhanced by B-RAF/MEK inhibition and FOXD3 expression. Furthermore, increased ERBB3 phosphorylation was observed in melanoma xenografts after treatment of mice with PLX4720. Enhanced NRG1-ERBB3 signaling promoted cell growth in the presence of B-RAF/MEK inhibition, and was dependent on ERBB2/HER2. Inhibition of ERBB2 with lapatinib reversed this growth benefit in vitro and reduced tumor burden when combined with PLX4720 in vivo. Taken together, these findings identify FOXD3 as a novel target of mutant B-RAF signaling in melanoma capable of providing adaptive resistance to B-RAF/MEK inhibition at least in part by upregulating ERBB3 and enhancing NRG1-ERBB3 signaling. These findings suggest that combined inhibition of B-RAF and ERBB3 signaling may provide a new therapeutic option for mutant B-RAF melanoma patients