Fateful music from a talented orchestra with a wicked conductor: Connection between oncogenic BRAF, ER stress, and autophagy in human melanoma.
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ABSTRACT:
SUBMITTER: Giglio P
PROVIDER: S-EPMC4905314 | biostudies-other | 2015 Jul-Sep
REPOSITORIES: biostudies-other
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Project description:The notorious unresponsiveness of metastatic cutaneous melanoma to current treatment strategies coupled with its increasing incidence constitutes a serious worldwide clinical problem. Moreover, despite recent advances in targeted therapies for patients with BRAF(V600E) mutant melanomas, acquired resistance remains a limiting factor and hence emphasises the acute need for comprehensive pre-clinical studies to increase the biological understanding of such tumours in order to develop novel effective and longlasting therapeutic strategies. Autophagy and ER stress both have a role in melanoma development/progression and chemoresistance although their real impact is still unclear. Here, we show that BRAF(V600E) induces a chronic ER stress status directly increasing basal cell autophagy. BRAF(V600E)-mediated p38 activation stimulates both the IRE1/ASK1/JNK and TRB3 pathways. Bcl-XL/Bcl-2 phosphorylation by active JNK releases Beclin1 whereas TRB3 inhibits the Akt/mTor axes, together resulting in an increase in basal autophagy. Furthermore, we demonstrate chemical chaperones relieve the BRAF(V600E)-mediated chronic ER stress status, consequently reducing basal autophagic activity and increasing the sensitivity of melanoma cells to apoptosis. Taken together, these results suggest enhanced basal autophagy, typically observed in BRAF(V600E) melanomas, is a consequence of a chronic ER stress status, which ultimately results in the chemoresistance of such tumours. Targeted therapies that attenuate ER stress may therefore represent a novel and more effective therapeutic strategy for BRAF mutant melanoma.
| S-EPMC4423179 | biostudies-literature
Project description:Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.
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Project description:Melanoma patients with oncogenic BRAF(V600E) mutation have poor prognoses. While the role of BRAF(V600E) in tumorigenesis is well established, its involvement in metastasis that is clinically observed in melanoma patients remains a topic of debate. Here, we show that BRAF(V600E) melanoma cells have extensive invasion activity as assayed by the generation of F-actin and cortactin foci that mediate membrane protrusion, and degradation of the extracellular matrix (ECM). Inhibition of BRAF(V600E) blocks melanoma cell invasion. In a BRAF(V600E)-driven murine melanoma model or in patients' tumor biopsies, cortactin foci decrease upon inhibitor treatment. In addition, genome-wide expression analysis shows that a number of invadopodia-related genes are downregulated after BRAF(V600E) inhibition. Mechanistically, BRAF(V600E) induces phosphorylation of cortactin and the exocyst subunit Exo70 through ERK, which regulates actin dynamics and matrix metalloprotease secretion, respectively. Our results provide support for the role of BRAF(V600E) in metastasis and suggest that inhibiting invasion is a potential therapeutic strategy against melanoma.
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Project description:Autophagy is a dynamic cell survival mechanism by which a double-membrane vesicle, or autophagosome, sequesters portions of the cytosol for delivery to the lysosome for recycling. This process can be inhibited using the antimalarial agent chloroquine (CQ), which impairs lysosomal function and prevents autophagosome turnover. Despite its activity, CQ is a relatively inadequate inhibitor that requires high concentrations to disrupt autophagy, highlighting the need for improved small molecules. To address this, we screened a panel of antimalarial agents for autophagy inhibition and chemically synthesized a novel series of acridine and tetrahydroacridine derivatives. Structure-activity relationship studies of the acridine ring led to the discovery of VATG-027 as a potent autophagy inhibitor with a high cytotoxicity profile. In contrast, the tetrahydroacridine VATG-032 showed remarkably little cytotoxicity while still maintaining autophagy inhibition activity, suggesting that both compounds act as autophagy inhibitors with differential effects on cell viability. Further, knockdown of autophagy-related genes showed no effect on cell viability, demonstrating that the ability to inhibit autophagy is separate from the compound cytotoxicity profiles. Next, we determined that both inhibitors function through lysosomal deacidification mechanisms and ultimately disrupt autophagosome turnover. To evaluate the genetic context in which these lysosomotropic inhibitors may be effective, they were tested in patient-derived melanoma cell lines driven by oncogenic BRAF (v-raf murine sarcoma viral oncogene homolog B). We discovered that both inhibitors sensitized melanoma cells to the BRAF V600E inhibitor vemurafenib. Overall, these autophagy inhibitors provide a means to effectively block autophagy and have the potential to sensitize mutant BRAF melanomas to first-line therapies.
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Project description:Activating mutations in NRAS and BRAF are found frequently in cutaneous melanomas. Because concurrent mutations of both BRAF and RAS are extremely rare, it is thought that transformation by RAS and BRAF occurs through a common mechanism. Also, there is evidence for a relationship of synthetic lethality between NRAS and BRAF oncogenes that leads to selection against cells with a hyperactive mitogen-activated protein kinase (MAPK) pathway. However, it is not known whether the hyperactivation of the MAPK pathway by overexpression of either oncogene alone could also inhibit melanoma tumorigenesis. Here, we show that in melanoma cells with oncogenic BRAF (mBRAF), high levels of mBRAF induce hyperactivation of ERK and senescence-like phenotype and trigger autophagy by inhibiting the mammalian target of rapamycin complex signaling. Growth inhibition and cell death caused by high mBRAF levels are partially rescued by downregulation of BRAF protein or inhibition of autophagy, but not by inhibition of the MAPK or apoptotic pathways. In nude mice, growth of mBRAF-overexpressing tumors is inhibited. Quantitative immunohistochemical analysis of human melanomas and cell lines showed a significant positive correlation between the levels of BRAF protein and autophagy marker light chain 3. Our data suggest that high oncogenic BRAF levels trigger autophagy, which may have a role in melanoma tumor progression.
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Project description:Malignant melanoma is among the most aggressive cancers and its incidence is increasing worldwide. Targeted therapies and immunotherapy have improved the survival of patients with metastatic melanoma in the last few years; however, available treatments are still unsatisfactory. While the role of the BRAF-MEK1/2-ERK1/2 pathway in melanoma is well established, the involvement of mitogen-activated protein kinases MEK5-ERK5 remains poorly explored. Here we investigated the function of ERK5 signaling in melanoma. We show that ERK5 is consistently expressed in human melanoma tissues and is active in melanoma cells. Genetic silencing and pharmacological inhibition of ERK5 pathway drastically reduce the growth of melanoma cells and xenografts harboring wild-type (wt) or mutated BRAF (V600E). We also found that oncogenic BRAF positively regulates expression, phosphorylation, and nuclear localization of ERK5. Importantly, ERK5 kinase and transcriptional transactivator activities are enhanced by BRAF. Nevertheless, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Accordingly, combination of MEK5 or ERK5 inhibitors with BRAFV600E inhibitor vemurafenib is more effective than single treatments in reducing colony formation and growth of BRAFV600E melanoma cells and xenografts. Overall, these data support a key role of the ERK5 pathway for melanoma growth in vitro and in vivo and suggest that targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, might represent a novel approach for melanoma treatment.
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Project description:Purpose: NRAS mutations in malignant melanoma are associated with aggressive disease requiring rapid antitumor intervention, but there is no approved targeted therapy for this subset of patients. In clinical trials, the MEK inhibitor (MEKi) binimetinib displayed modest antitumor activity, making combinations a requisite. In a previous study, the BRAF inhibitor (BRAFi) vemurafenib was shown to induce endoplasmic reticulum (ER) stress that together with inhibition of the RAF-MEK-ERK (MAPK) pathway amplified its proapoptotic activity in BRAF-mutant melanoma. The present study investigated whether this effect might extent to NRAS-mutant melanoma, in which MAPK activation would be expected.Experimental Design and Results: BRAFi increased pERK, but also significantly increased growth inhibition and apoptosis induced by the MEKi in monolayer, spheroids, organotypic, and patient-derived tissue slice cultures of NRAS-mutant melanoma. BRAFi such as encorafenib induced an ER stress response via the PERK pathway, as detected by phosphorylation of eIF2α and upregulation of the ER stress-related factors ATF4, CHOP, and NUPR1 and the proapoptotic protein PUMA. MEKi such as binimetinib induced the expression of the proapoptotic protein BIM and activation of the mitochondrial pathway of apoptosis, the latter of which was enhanced by combination with encorafenib. The increased apoptotic rates caused by the combination treatment were significantly reduced through siRNA knockdown of ATF4 and BIM, confirming its critical roles in this process.Conclusions: The data presented herein encourage further advanced in vivo and clinical studies to evaluate MEKi in combination with ER stress inducing BRAFi as a strategy to treat rapidly progressing NRAS-mutant melanoma. Clin Cancer Res; 23(20); 6203-14. ©2017 AACR.
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Project description:It has been well documented that miRNAs can modulate the effectiveness of cancer-associated signaling pathways. Mitogen-activated protein kinase (MAPK/ERK) signaling plays an essential role in the progression of many cancers, including melanoma and colon cancers. However, no single miRNA is reported to directly target multiple components of the MAPK/ERK pathway. We performed a miRNA PCR array screening with various MAPK/ERK signaling activities. The miRNA array data revealed that the expression of miR-524-5p was decreased in cells with an active MAPK/ERK pathway and confirmed that the expression of miR-524-5p is inversely associated with the activity of the MAPK/ERK pathway. We demonstrated that miR-524-5p directly binds to the 3'-untranslated regions of both BRAFandERK2 and suppresses the expression of these proteins. Because BRAF and ERK2 are the main components of MAPK signaling, the overexpression of miR-524-5p effectively inhibits MAPK/ERK signaling, tumor proliferation, and melanoma cell migration. Moreover, tumors overexpressing miR-524-5p were significantly smaller than those of the negative control mice. Our findings provide new insight into the role of miR-524-5p as an important miRNA that negatively regulates the MAPK/ERK signaling pathway, suggesting that miR-524-5p could be a potent therapeutic candidate for melanoma treatment.
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Project description:BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors elicit a transient anti-tumor response in ∼ 80% of BRAF(V600)-mutant melanoma patients that almost uniformly precedes the emergence of resistance. Here we used a mouse model of melanoma in which melanocyte-specific expression of Braf(V618E) (analogous to the human BRAF(V600E) mutation) led to the development of skin hyperpigmentation and nevi, as well as melanoma formation with incomplete penetrance. Sleeping Beauty insertional mutagenesis in this model led to accelerated and fully penetrant melanomagenesis and synchronous tumor formation. Treatment of Braf(V618E) transposon mice with the BRAF inhibitor PLX4720 resulted in tumor regression followed by relapse. Analysis of transposon insertions identified eight genes including Braf, Mitf, and ERas (ES-cell expressed Ras) as candidate resistance genes. Expression of ERAS in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thymoma viral oncogene homolog 1), a phenotype reverted by combinatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associated with phosphorylation/inactivation of BAD, and that the resistance of hepatocyte growth factor-treated human melanoma cells to PLX4720 can be reverted by treatment with the BAD-like BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD pathway in resistance to BRAF inhibition and illustrate an in vivo approach for finding drug resistance genes.
| S-EPMC4330752 | biostudies-literature
Project description:Autophagy is a conserved process in eukaryotes that contributes to cell survival in response to stress. Previously, we found that ER stress induces autophagy in a manner dependent upon IRE1b, an ER membrane-associated factor involved in the splicing of bZIP60 mRNA. IRE1 is a dual protein kinase and ribonuclease, and here we studied the involvement of the protein kinase catalytic domain, nucleotide binding and RNase domains of IRE1b in activating autophagy. Autophagy was assessed by quantifying the numbers of autophagosomes in transgenic Arabidopsis seedlings bearing mutations in the various IRE1b domains. The results showed that nucleotide binding and RNase activity of IRE1b are required for ER stress-mediated autophagy. The RNase activity is involved in IRE1b’s mRNA splicing function, but its principal splicing target, bZIP60, is not involved in IRE1b’s activation of autophagy. We therefore considered other roles for IRE1b in the activation of autophagy. Clustering of ER localized IRE1b-YFP was observed when seedlings were subjected to ER stress, and so we investigated whether IRE1b clustering induced autophagy. However, the RNase knockout mutation in IRE1b still undergoes clustering, suggesting that IRE1b clustering does not induce autophagy. In response to ER stress, the RNase of IRE1 has been found to engage in another activity called Regulated Ire1-Dependent Decay of Messenger RNA (RIDD), which is the promiscuous degradation of other mRNA in response to ER stress. By analyzing the RNA-seq data, 12 RIDD target genes were picked up for testing their role in inhibiting autophagy, and glucosidase 21 and peroxidase 14 are proved to be degraded to support the induction of autophagy by ER stress.
2018-03-31 | GSE99576 | GEO