The BRAF inhibitor vemurafenib is currently used for treating patients with BRAF V600E mutant melanoma. However, the responses to vemurafenib are generally partial and of relatively short duration. Recent evidence suggests that activation of the epidermal growth factor receptor (EGFR)/erbB signaling pathway may be responsible for the development of BRAF inhibitor resistance in melanoma patients. In this study, we characterized the erbB family of receptors and ligands in melanoma cell lines and examined whether targeting both BRAF and erbB provided enhanced antitumor activity in BRAF mutant melanoma. Variable levels of erbB2, erbB3, and truncated erbB4 were expressed in both BRAF wildtype and mutant melanoma cells with no significant differences between wildtype and mutant lines. EGFR was rarely expressed. Neuregulin 3 and neuregulin 4 were the major erbB ligands released by melanoma cells. Multi-erbB targeting with the irreversible tyrosine kinase inhibitor canertinib exerted a more effective growth inhibitory effect in both BRAF wildtype and mutant melanoma cells compared with the single-erbB or dual-erbB targeting inhibitors, gefitinib, erlotinib, and lapatinib. Canertinib inhibited both EGF-induced and neuregulin 1-induced erbB downstream signaling in both mutant and wildtype cell lines. However, canertinib induced apoptosis and sub-G1 arrest only in mutant cells. Canertinib statistically increased the antiproliferative effects of vemurafenib in the BRAF mutant melanoma cell lines while little or no enhanced effect was observed with the combination treatment in the wildtype cell lines. A combined inhibition strategy targeting BRAF together with multiple erbB family kinases is potentially beneficial for treating BRAF V600E mutant melanoma. Wildtype BRAF melanoma may also benefit from a multi-erbB kinase inhibitor.

Dysregulated activation of the epidermal growth factor receptor (ErbB) family (EGFR, ErBB2, ErbB3, and ErbB4) is involved in promoting the cellular growth of human melanoma. EGFR, ErbB3, and ErbB4 are promising targets of malignant melanoma for new molecular drug development. However, the biology of ErbB family signaling pathways indicates that functional redundancy and heterodimerization among ErbB family members can greatly compromise the therapeutic efficacy of single ErbB targeting tyrosine kinase inhibitors (TKIs). In fact, Phase II clinical trials have already shown that EGFR (ErbB1) targeting TKIs provide limited benefits to melanoma patients. Here, this study has compared single vs. multiple ErbB targeting TKIs for inhibiting the growth of melanoma cells with different ErbB family protein expression profiles. We examined the expression of ErbB family members in 14 different human melanoma cell lines (MEL-526, M14, UPCI-MEL 527.1 and 11 SK-MEL cells). EGFR was expressed in 5 of the cell lines examined (35.7 %). ErbB2 was present in 13 lines (92.9 %); ErbB3 was found in 12 lines (85.7 %); and ErbB4 was found in all the cell lines. Furthermore, individual cell lines had a particular dominant form of ErbB protein. This differential expression pattern of ErbB family proteins was also seen in melanoma tissues from patients. Out of 14 melanoma cases examined by immunohistochemistry, 8 samples (61.5 %) were positive for ErbB1 and ErbB4 expression while ErbB2 was detected in 11 samples (84.6 %). We also found that Neuregulin 3 (which ranged from 500 to 76500 pg/ml) and Neuregulin 4 (which ranged from 26-300 pg/ml), which activate ErbB3 and/or ErbB4, are the major ErbB ligands released by the human melanoma cell lines. TGFβ and Amphiregulin, which mainly bind to EGFR, were released in relatively lower levels by the majority of the cell lines, but high levels of these ligands were distinctly found in individual cell lines. Among all the cell lines examined, HB-EGF was barely undetectable. In addition, different sensitivities towards single ErbB targeting tyrosine kinase inhibitors (TKIs) and pan-ErbB targeting TKIs were seen among the melanoma cells. For example, the TKI sensitivities of MEL526 which expressed no EGFR but high ErbB2, 3, and 4 proteins, was found to be: lapatinib (IC50: 6.7 μM) > canertinib (IC50: 12.4 μM) = erlotinib (IC50: 12.9 μM) > gefitinib (IC50: 23 μM). Similarly, M14 and UPCI-MEL527.1, which also have no EGFR expression but express ErbB 2, 3, 4, also responded better to lapatinib and canertinib respectively than to gefitinib or erlotinib. Our study is the first to suggest that the pan-ErbB targeting approach may be more effective in treatment malignant melanoma. Our findings also suggest that future screening of melanoma patients for their dominantly expressed ErbB protein types can potentially serve as a guide for the choice of effective ErbB TKI in treating malignant melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3864. doi:1538-7445.AM2012-3864