In this study, we report that NRG1/ERBB3 signaling is dramatically enhanced in V600 BRAF harboring melanoma cells treated with RAF and MEK inhibitors and diminishes inhibitor effects on cell viability and tumor growth. Central to the enhanced ERBB3 signaling by PLX4032/AZD6244 is FOXD3, a transcription factor that is induced by RAF/MEK inhibition and can protect cells from PLX4032-mediated death. ERBB3 partners with ERBB2 and the enhanced signaling from ERBB3/ERBB2 complexes can be overcome by combining BRAF inhibitors with the ERBB2/EGFR inhibitor lapatinib. These data suggest that this combination, as well as others that target ERBB3/ERBB2 signaling, may have therapeutic value in the clinic to improve the efficacy of BRAF inhibitors and prolong duration of response.
Our data provide evidence that upregulation of ERBB3 through FOXD3 is a form of adaptive resistance to RAF/MEK inhibitors in mutant BRAF melanoma. We previously showed that FOXD3 was induced upon disruption of mutant BRAF signaling in melanoma and was capable of promoting survival of cells treated with PLX4032 (vemurafenib)/PLX4720 (22
). Here, we identify ERBB3
as a direct transcriptional target of FOXD3. This links the regulation of ERBB3 to the mutant BRAF/MEK/ERK pathway for what we believe is the first time. Regulation of ERBB3
by other forkhead box transcription factors has been previously reported. FOXO3a and FOXO1 promote the upregulation of ERBB3
in breast cancer cells treated with lapatinib via effective inhibition of PI3K/AKT signaling (26
). While we did not observe upregulation of ERBB3 by lapatinib or PI3K inhibitors in melanoma cells (data not shown), this compensatory feedback mechanism has a number of parallels to the model that we propose. Additionally, FOXA1 was shown to bind to the ERBB3
intronic enhancer region in androgen receptor–driven (AR-driven) breast cancer. In response to androgen stimulation, FOXA1 and AR were recruited to intron 1, where they promoted ERBB3
). We found that FOXD3 strongly enriched the intronic enhancer region of ERBB3
. While it is unclear whether FOXD3 occupies the same binding sites as FOXA1, FOXD3 is a pioneering factor for FOXA1 at certain loci during development (41
). It would be interesting to know whether FOXD3 target genes in melanoma are also known targets of FOXA1.
RAF/MEK inhibitors sensitize V600 mutant BRAF melanoma cells to NRG1β, resulting in a dramatic increase in AKT phosphorylation. Increased PI3K/AKT signaling is one previously identified mechanism of resistance to BRAF inhibition (15
). In our experiments, activation of AKT was seen regardless of PTEN status, which has been shown to be one determinant of responsiveness to BRAF inhibition (17
). Consistent with the importance of AKT signaling in response to RAF inhibitors, we found that directly inhibiting AKT with MK2206 was able to enhance the efficacy of PLX4032 and ablate the protective effects of NRG1β on 1205Lu and WM115 cells (Supplemental Figure 7, A–C). These data also indicate that AKT is one of the main effectors of ERBB3-mediated resistance to PLX4032. Interestingly, inhibition of either BRAF or MEK1/2 led to the decreased phosphorylation of S6 ribosomal protein. but treatment with NRG1β restored S6 ribosomal protein phosphorylation, indicating a shift of translational control from ERK1/2 to AKT signaling. This restoration of protein translation as well as the actions of AKT on apoptotic and cell-cycle proteins may contribute to the enhanced cell viability.
Prior reports have highlighted the upregulation of RTKs, such as IGF1R or PDGFRβ, in melanoma as possible mechanisms of resistance to RAF inhibitors (14
). We did not detect enhanced signaling from either RTK in response to their respective ligands when cells were pretreated with PLX4032 for 24 hours. This would suggest that these receptors become overexpressed or hyperactivated later in the development of resistance. Indeed, the adaptive mechanism we propose likely allows cells to persist until they acquire a permanent mechanism of resistance. Consistent with this notion, ERBB3 shows enhanced signaling within a few hours of drug treatment. We also observed a marked increase in phospho-ERBB3 in xenografts after 5-day treatment with PLX4720, indicating in vivo relevance. Increased ERBB3 phosphorylation was also detected in 2 out of 3 on-treatment patient samples available to us. Interestingly, vemurafenib-associated increased ERBB3 phosphorylation was also detected in 4 out of 11 progressing patients (counting samples from Figure , B and C), and thus, it may be associated with acquired resistance in some cases. Basal ERBB3 expression was variable across cell lines (Supplemental Figure 8A), and it is therefore likely that the upregulation of ERBB3, as opposed to its basal expression, modulates the response to RAF inhibitor. Additionally, endogenous NRG1 was expressed at very low levels in melanoma cells (Supplemental Figure 8A) and was not enhanced following treatment with RAF inhibitor (Supplemental Figure 8B). The notion that paracrine stimulation of ERBB3 occurs is supported by evidence that production of NRG1 from dermal fibroblasts influences melanocyte biology (46
Despite lacking the strong kinase activity of its ERBB family members, ERBB3 boasts numerous PI3K-recruiting YXXM motifs and thus serves as a powerful signaling partner for its fellow family members. Furthermore, ERBB3 is upregulated in response to targeted therapies in breast cancer and non–small cell lung carcinoma (24
). Unlike melanoma, these cancers are often driven by oncogenic ERBB signaling, either through ERBB2 amplification in the case of breast cancer or EGFR amplification and/or mutation in lung cancer. In acquired resistance to ERBB2 and EGFR inhibitors, signaling through ERBB3 is restored by either ERBB3 upregulation or compensatory phosphorylation by amplified MET (24
). Our findings add what we believe to be a novel twist to ERBB3 and drug resistance in which ERBB3 signaling is augmented to overcome inhibition of the mutant BRAF/MEK/ERK pathway. A recent study attributed resistance to PLX4032 in mutant BRAF colorectal cancer cells to enhanced EGFR phosphorylation (47
). In colorectal cancer cells, inhibition of EGFR in combination with BRAF was able to ablate cell growth and tumorigenesis but melanoma cells did not show this dependence on EGFR. It is possible that EGFR and ERBB3 are governed by similar feedback loops in colorectal cancer and melanoma cells, respectively. Furthermore, we cannot exclude the possibility of RAF-dependent, but FOXD3-independent, mechanisms that contribute to enhanced ERBB3 sensitivity to NRG1 in melanoma.
Targeted therapies are rapidly displacing conventional chemotherapies for cancers with defined driver mutations. For these therapies to show persistent benefits in the clinic, compensatory mechanisms need to be identified and targeted in concert. We demonstrate that treatment of melanoma cells with lapatinib effectively ablated ERBB3 phosphorylation and NRG1β-mediated growth in vitro and enhanced the antitumor activity of PLX4720 in vivo. Although lapatinib does not target ERBB3 directly, it does effectively inhibit all other members of the ERBB family (40
) and therefore may prevent ERBB3 phosphorylation in response to other ERBB family ligands in vivo. As both vemurafenib and lapatinib are FDA approved, combinatorial treatment in the clinic is likely feasible and could potentially enhance the efficacy and duration of response to vemurafenib and other mutant BRAF inhibitors. It is noted that diarrhea and skin rash are common adverse effects associated with lapatinib treatment (49
), and upregulation of ERBB3 may limit the antitumor actions of lapatinib (27
). Monoclonal antibodies targeting ERBB3 have proven efficacious in lung carcinoma and breast and other nonmelanoma tumor models (27
) and are now entering clinical trials (e.g., NCT00994123; ClinicalTrials.gov). Our in vivo depletion experiments provide the basis for directly targeting ERBB3 in combination with vemurafenib in mutant BRAF melanoma. Ongoing efforts are focused on utilizing clinical grade anti-ERBB3 monoclonal antibodies in combination with RAF inhibitors to more specifically target the ERBB3 adaptive response pathway in melanoma preclinical models.