Vemurafenib Potently Induces Endoplasmic Reticulum Stress–Mediated Apoptosis in BRAFV600E Melanoma Cells

Abstract

The V600E mutation in the kinase BRAF is frequently detected in melanomas and results in constitutive activation of BRAF, which then promotes cell proliferation by the mitogen-activated protein kinase (MAPK) signaling pathway. Although the BRAFV600E kinase inhibitor vemurafenib has remarkable antitumor activity in patients with BRAFV600E-mutated melanoma, its effects are limited by the onset of drug resistance. We found that exposure of melanoma cell lines with the BRAFV600E mutation to vemurafenib decreased the abundance of anti-apoptotic proteins and induced intrinsic mitochondrial apoptosis. Vemurafenib-treated melanoma cells showed increased cytosolic concentration of calcium, a potential trigger for endoplasmic reticulum (ER) stress, which can lead to apoptosis. Consistent with an ER stress-induced response, vemurafenib decreased the abundance of the ER chaperone protein GRP78, increased the abundance of the spliced isoform of the transcription factor X-box protein 1 (XBP1) (which transcriptionally activates genes involved in ER stress responses), increased the phosphorylation of the translation initiation factor eIF2α (which would be expected to inhibit protein synthesis), and induced the expression of ER stress-related genes. Knockdown of the ER stress response protein ATF4 significantly reduced vemurafenib-induced apoptosis. Moreover, the ER stress inducer thapsigargin prevented invasive growth of tumors formed from vemurafenib-sensitive melanoma cells in vivo. In melanoma cells with low sensitivity or resistance to vemurafenib, combination treatment with thapsigargin augmented or induced apoptosis. Thus, thapsigargin or other inducers of ER stress may be useful in combination therapies to overcome vemurafenib resistance.

Targeting ER stress–induced autophagy overcomes BRAF inhibitor resistance in melanoma

“…A previous report found that the ER stress response that ensues after BRAFi in some BRAF mutant cell lines may contribute to BRAFi-associated apoptosis (48). In our present work, chemical els, as expected, due to simultaneous autophagy induction and distal blockade.…”

Dangerous liaisons: flirtations between oncogenic BRAF and GRP78 in drug-resistant melanomas

“…In BRAF V600E melanoma cells, knockdown of ATF4, an ER stressinduced transcription factor acting in the PERK pathway, markedly reduced vemurafenib-associated cell death. Conversely, vemurafenib combined with either thapsigargin or tunicamycin, both of which are known inducers of ER stress, enhanced apoptosis in vemurafenibresistant or -insensitive melanoma cells (6). These results suggest that pharmacological enhancers of ER stress, particularly those that stimulate the PERK pathway, may be useful adjuncts to vemurafenib treatment and may overcome the rapidly developing drug resistance seen in melanoma patients.…”

“…The ER luminal domains of the three ER stress sensors, PERK, ATF6, and IRE1, are highly homologous, and all three proteins bind GRP78; therefore, it would be expected that the sequestration of GRP78 by BRAF V600E might activate all three ER stress-signaling pathways. On the other hand, earlier studies have shown that GRP78, which is induced by ATF6, is decreased in melanoma cells after vemurafenib exposure (6). At this stage, not only is it unclear how BRAF inhibitors promote BRAF entry into the ER, but it also remains to be determined how and why the sequestration of GRP78 by BRAF preferentially activates PERK.…”

“…Dynamins assemble as rings around membrane tubules where they are thought to actively "pinch off" membranes (5). Complete loss of Dnm2 results in embryonic lethality in mice (6). In humans, CNM is caused by dominant DNM2 mutations (7), and overexpression of a CNM-linked DNM2 mutation (R465W) in mouse muscle results in myopathic features, including centralized nuclei, muscle atrophy, and deformed T-tubules (8).…”

Novel BRAF mutation in melanoma: A case report

“…This mutation modifies the spatial structure of activation P-loop, causing a 500-fold increase in BRAF kinase activity and facilitating the acquisition of secondary genetic events in cancer progression (3). Target therapy for cutaneous melanomas is focused on this mutation: small molecules (vemurafenib and dabrafenib) act as Tyrosine Kinase Inhibitors (TKIs), and interrupt the BRAF/MEK step in RAS/MEK/ERK pathway, inducing apoptosis in mutated cells (4). Less frequent BRAF mutations (5,6) with alterations in the same crucial site, such as Threonine599 and Serine601 (7), have been described.…”