Colorectal cancer cells with the BRAFV600E mutation are addicted to the ERK1/2 pathway for growth factor-independent survival and repression of BIM

Abstract

The RAF-mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated kinase 1/2 (RAF-MEK1/2-ERK1/2) pathway is activated in many human tumours and can protect cells against growth factor deprivation; however, most such studies have relied upon overexpression of RAF or MEK constructs that are not found in tumours. Here we show that expression of the endogenous BRAFV600E allele in mouse embryonic fibroblasts from conditional knock-in transgenic mice activates ERK1/2, represses the BH3-only protein BIM and protects cells from growth factor withdrawal. Human colorectal cancer (CRC) cell lines harbouring BRAFV600E are growth factor independent for the activation of ERK1/2 and survival. However, treatment with the MEK1/2 inhibitors U0126, PD184352 or the novel clinical candidate AZD6244 (ARRY-142886) overcomes growth factor independence, causing CRC cell death. BIM is de-phosphorylated and upregulated following MEK1/2 inhibition in all CRC cell lines studied and knockdown of BIM reduces cell death, indicating that repression of BIM is a major part of the ability of BRAFV600E to confer growth factor-independent survival. We conclude that a single endogenous BRAFV600E allele is sufficient to repress BIM and prevent death arising from growth factor withdrawal, and CRC cells with BRAFV600E mutations are addicted to the ERK1/2 pathway for repression of BIM and growth factor-independent survival.

Tumour cell survival signalling by the ERK1/2 pathway

“…12 Both of these studies reveal that BIM EL is the major form of BIM that is subject to regulation by MEK inhibitors and demonstrate that tumour cells with BRAF 600E exhibit a strong, constitutive signal for BIM EL degradation through the proteasome. 57,58 They also reveal some important details: first, inhibition of MEK alone in complete medium can cause an increase in BIM expression but relatively little cell death; 57 second, even complete ablation of BIM by siRNA affords only 60% protection against death arising from serum withdrawal and MEK inhibition. 58 Both these observations suggest that either BIM expression must reach a certain threshold to induce death or that BIM acts in concert with another BH3-only protein.…”

“…57,58 They also reveal some important details: first, inhibition of MEK alone in complete medium can cause an increase in BIM expression but relatively little cell death; 57 second, even complete ablation of BIM by siRNA affords only 60% protection against death arising from serum withdrawal and MEK inhibition. 58 Both these observations suggest that either BIM expression must reach a certain threshold to induce death or that BIM acts in concert with another BH3-only protein. Indeed, studies have suggested that ERK1/2 signalling protects melanoma cells from anoikis by inhibiting both BAD and BIM.…”

“…57 Similarly, serum withdrawal and MEK inhibition by U0126 or AZD6244 gives rise to a strong increase in BIM expression in BRAF 600E -positive colorectal cancer cell lines and death under these conditions is significantly reduced by siRNAmediated ablation of BIM. 58 Indeed, the clinical candidate AZD6244 caused a striking increase in BIM EL expression in COLO205 cells 58 and promoted regression of COLO205 xenografts in vivo. 12 Both of these studies reveal that BIM EL is the major form of BIM that is subject to regulation by MEK inhibitors and demonstrate that tumour cells with BRAF 600E exhibit a strong, constitutive signal for BIM EL degradation through the proteasome.…”

“…This is particularly evident in the case of serum withdrawal; for example, BRAF 600E -positive melanoma and colorectal cancer cells appear to be addicted to the ERK1/2 pathway for growth factor-independent survival as they undergo cell death when subjected to serum withdrawal combined with MEK inhibition. 57,58 Inhibition of MEK in invasive melanoma cells sensitizes them to anoikis 59 whereas papillary thyroid cancer cell lines may or may not undergo apoptosis upon inhibition of the ERK1/2 pathway. 60,61 It is now clear from several studies that BIM is a major target of ERK1/2-dependent survival signalling in BRAF 600E -positive tumour cells.…”

Intrinsic resistance to the MEK1/2 inhibitor AZD6244 (ARRY‐142886) is associated with weak ERK1/2 signalling and/or strong PI3K signalling in colorectal cancer cell lines

“…4,5 The BRAF 600E mutant can replicate many of the effects of mutant RAS; indeed, expression of endogenous BRAF 600E promotes anchorage-independent growth, morphologic transformation, cell proliferation and cell survival in primary mouse embryo fibroblasts. [6][7][8][9][10][11] As the only physiologically defined substrate of RAF, MEK1/2 plays a key role in mediating the effects of RAS and RAF. 12,13 Since genetic inhibition of the ERK1/2 pathway reverses RAS and RAF transformation, and pharmacological inhibition of MEK1/2 is feasible, 14 this pathway has attracted much attention in the search for new chemotherapeutics and selective small molecule MEK1/2 inhibitors have been identified, including PD184352 (CI-1040), PD0325901 and AZD6244 (ARRY-142886).…”