Oncogenic B-RAFV600E Signaling Induces the T-Box3 Transcriptional Repressor to Repress E-Cadherin and Enhance Melanoma Cell Invasion

Approximately 50% of melanomas require oncogenic B-RAFV600E signaling for proliferation, survival, and metastasis, and the use of highly selective B-RAF inhibitors has yielded remarkable, although short-term, clinical responses. Reactivation of signaling downstream of B-RAF is frequently associated with acquired resistance to B-RAF inhibitors, and the identification of B-RAF targets may therefore provide new strategies for managing melanoma. In this report, we applied whole-genome expression analyses to reveal that oncogenic B-RAFV600E regulates genes associated with epithelial–mesenchymal transition in normal cutaneous human melanocytes. Most prominent was the B-RAF-mediated transcriptional repression of E-cadherin, a keratinocyte–melanoma adhesion molecule whose loss is intimately associated with melanoma invasion and metastasis. Here we identify a link between oncogenic B-RAF, the transcriptional repressor Tbx3, and E-cadherin. We show that B-RAFV600E induces the expression of Tbx3, which potently represses E-cadherin expression in melanocytes and melanoma cells. Tbx3 expression is normally restricted to developmental embryonic tissues and promoting cell motility, but it is also aberrantly increased in various cancers and has been linked to tumor cell invasion and metastasis. We propose that this B-RAF/Tbx3/E-cadherin pathway has a critical role in promoting the metastasis of B-RAF-mutant melanomas

Oncogenic activation of MEK/ERK primes melanoma cells for adaptation to endoplasmic reticulum stress

Cancer cells commonly undergo chronic endoplasmic reticulum (ER) stress, to which the cells have to adapt for survival and proliferation. We report here that in melanoma cells intrinsic activation of the ER stress response/unfolded protein response (UPR) is, at least in part, caused by increased outputs of protein synthesis driven by oncogenic activation of mitogen-activated protein kinase kinase/extracellular signal–regulated kinase (MEK/ERK) and promotes proliferation and protects against apoptosis induced by acute ER stress. Inhibition of oncogenic BRAFV600E or MEK-attenuated activation of inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) signaling of the UPR in melanoma cells. This was associated with decreased phosphorylation of eukaryotic initiation factor 4E (eIF4E) and nascent protein synthesis and was recapitulated by knockdown of eIF4E. In line with this, introduction of BRAFV600E into melanocytes led to increases in eIF4E phosphorylation and protein production and triggered activation of the UPR. Similar to knockdown of glucose-regulated protein 78 (GRP78), inhibition of XBP1 decelerated melanoma cell proliferation and enhanced apoptosis induced by the pharmacological ER stress inducers tunicamycin and thapasigargin. Collectively, these results reveal that potentiation of adaptation to chronic ER stress is another mechanism by which oncogenic activation of the MEK/ERK pathway promotes the pathogenesis of melanoma

Preexisting MEK1(P124) mutations diminish response to BRAF inhibitors in metastatic melanoma patients

Background: MEK1 mutations in melanoma can confer resistance to BRAF inhibitors, although preexisting MEK1P124 mutations do not preclude clinical responses. We sought to determine whether recurrent, preexisting MEK1P124 mutations affected clinical outcome in BRAF inhibitor–treated patients with melanoma. Methods: Data from four published datasets were analyzed to determine whether preexisting MEK1P124 mutations affect radiologic response or progression-free survival (PFS) in patients with BRAFV600-mutant metastatic melanoma treated with vemurafenib or dabrafenib. The effects of MEK1P124 mutations on MAPK pathway activity and response to BRAF inhibition were also investigated in a series of cell models. Results: In a pooled analysis of 123 patients, the presence of a pretreatment MEK1P124 mutation (N = 12, 10%) was associated with a poorer RECIST response (33% vs. 72% in MEK1P124Q/S vs. MEK1P124 wild-type, P = 0.018), and a shorter PFS (median 3.1 vs. 4.8 months, P = 0.004). Furthermore, MEK1P124Q/S mutations were shown to have independent kinase activity and introduction of these mutations into a BRAF-mutant melanoma cell line diminished inhibition of ERK phosphorylation by dabrafenib and enhanced clonogenic survival in the presence of dabrafenib compared with cells ectopically expressing wild-type MEK1. Consistent with these data, two BRAF-mutant cell lines with endogenous MEK1P124 mutations showed intermediate sensitivity to dabrafenib, but were highly sensitive to downstream inhibition of MEK or ERK. Conclusion: Taken together, our data indicate that preexisting MEK1P124 mutations are associated with a reduced response to BRAF inhibitor therapy and identify a subset of patients with BRAF-mutant melanoma likely to benefit from combination therapies involving MEK or ERK inhibitors.8 page(s

Clinical significance of intronic variants in BRAF inhibitor resistant melanomas with altered BRAF transcript splicing

Abstract Alternate BRAF splicing is the most common mechanism of acquired resistance to BRAF inhibitor treatment in melanoma. Recently, alternate BRAF exon 4–8 splicing was shown to involve an intronic mutation, located 51 nucleotides upstream of BRAF exon 9 within a predicted splicing branch point. This intronic mutation was identified in a single cell line but has not been examined in vivo. Herein we demonstrate that in three melanomas biopsied from patients with acquired resistance to BRAF inhibitors, alternate BRAF exon 4–8 splicing is not associated with this intronic branch point mutation. We also confirm that melanoma cells expressing BRAF splicing variants retain exquisite sensitivity to existing FDA-approved MEK inhibitors

BRAF inhibitor resistance mechanisms in metastatic melanoma : spectrum and clinical impact

Purpose: Multiple BRAF inhibitor resistance mechanisms have been described, however, their relative frequency, clinical correlates, and effect on subsequent therapy have not been assessed in patients with metastatic melanoma. Experimental Design: Fifty-nine BRAFV600-mutant melanoma metastases from patients treated with dabrafenib or vemurafenib were analyzed. The genetic profile of resistance mechanisms and tumor signaling pathway activity was correlated with clinicopathologic features and therapeutic outcomes. Results: Resistance mechanisms were identified in 58% progressing tumors and BRAF alterations were common. Gene expression analysis revealed that mitogen-activated protein kinase (MAPK) activity remained inhibited in 21% of resistant tumors, and the outcomes of patients with these tumors were poor. Resistance mechanisms also occurred in pretreatment biopsies and heterogeneity of resistance mechanisms occurred within patients and within tumors. There were no responses to subsequent targeted therapy, even when a progressing tumor had a resistance mechanism predicted to be responsive. Conclusions: Selecting sequential drugs based on the molecular characteristics of a single progressing biopsy is unlikely to provide improved responses, and first-line therapies targeting multiple pathways will be required.13 page(s