Abstract
BRAF and MEK inhibitors have improved outcomes for patients with BRAF-mutant melanoma, but their efficacy is limited by both intrinsic and acquired resistances. Activation of the PI3K pathway can mediate resistance to these agents, providing a strong rationale for combination therapy in melanoma. Here, a panel of nine low-passage human metastatic melanoma cell lines with BRAF mutations was tested in cell proliferation and protein expression assays for sensitivity to inhibitors of MEK (selumetinib) and BRAF (vemurafenib) as single agents and in combination with inhibitors of pan-PI3K (ZSTK474), pan-PI3K/mTOR (BEZ235), individual PI3K isoforms (p110α, A66; p110β, TGX-221; p110γ, AS-252424; p110δ, idelalisib), or mTORC1/2 (KU-0063794). Selumetinib and vemurafenib potently inhibited cell proliferation in all cell lines, especially in those that expressed low levels of phosphorylated AKT (pAKT). ZSTK474 and BEZ235 also inhibited cell proliferation in all cell lines and enhanced the antitumor activity of selumetinib and vemurafenib in the majority of lines by either interacting synergistically or additively to increase potency or by inducing cytotoxicity by significantly increasing the magnitude of cell growth inhibition. Furthermore, ZSTK474 or BEZ235 combined with selumetinib to produce robust inhibition of pERK, pAKT, and pS6 expression and synergistic inhibition of NZM20 tumor growth. The inhibitors of individual PI3K isoforms or mTORC1/2 were less effective at inhibiting cell proliferation either as single agents or in combination with selumetinib or vemurafenib, although KU-0063794 synergistically interacted with vemurafenib and increased the magnitude of cell growth inhibition with selumetinib or vemurafenib in certain cell lines. Overall, these results suggest that the sensitivity of BRAF-mutant melanoma cells to BRAF or MEK inhibitors is at least partly mediated by activation of the PI3K pathway and can be enhanced by combined inhibition of the BRAF/MEK and PI3K/mTOR signaling pathways.
Highlights
BRAF mutations leading to constitutive activation of the RAS/ RAF/MEK/ERK pathway and increased cell cycle progression, differentiation, survival, migration, and angiogenesis are reported in 40–50% of melanoma cases [1]
Since the antiproliferative activity of selumetinib and vemurafenib was less pronounced in BRAF-mutant melanoma cell lines that expressed phosphorylated AKT (pAKT), we investigated if inhibition of PI3K activity could promote growth inhibition in BRAF-mutant melanoma cell lines
Since pS6 expression can predict responsiveness to BRAF and MEK inhibitors in BRAF-mutant melanoma cells [25, 26], we investigated pS6 expression after 1 or 24 h treatment with selumetinib, ZSTK474 and BEZ235 alone and in combination with three cell lines that were highly sensitive to selumetinib and vemurafenib (NZM3, NZM11, and NZM20) and three cell lines that were less sensitive (NZM6, NZM7, and NZM12)
Summary
BRAF mutations leading to constitutive activation of the RAS/ RAF/MEK/ERK pathway and increased cell cycle progression, differentiation, survival, migration, and angiogenesis are reported in 40–50% of melanoma cases [1]. Among the multiple potential mechanisms of intrinsic and acquired resistance to BRAF and MEK inhibition that have been identified [8,9,10,11,12,13,14], the PI3K signaling pathway has been frequently implicated. Persistent activity of mTORC1, which operates downstream of both the PI3K and RAS/RAF/MEK/ERK signaling pathways, can lead to resistance following BRAF or MEK inhibition [19, 25, 26]. Compensatory signaling through the RAS/RAF/MEK/ ERK pathway following receptor tyrosine kinase (RTK) upregulation may promote resistance to PI3K pathway inhibition [27,28,29,30]
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