Oncogenic BRAF Signaling Research Articles

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, albeit short term, clinical responses. Re-activation 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, promoting cell motility but 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 plays a critical role in promoting the metastasis of B-RAF mutant melanomas.

Abstract 4294: Copper is required for oncogenic BRAF signaling and tumorigenesis.

Abstract The BRAF serine/threonine kinase is mutated, typically at V600, to remain in the active oncogenic state in a large fraction of melanoma, thyroid cancers, and hairy cell leukemia, and to a lesser extent in a wide spectrum of other cancers, thereby activating the kinases MEK1 and MEK2 to stimulate the MAPK pathway and promote cancer. Excitingly, ATP inhibitors of oncogenic BRAF and MEK provide a survival advantage in metastatic melanoma and early clinical studies suggest that coupling BRAF and MEK kinase inhibitors may be even more effective. Thus, the combination of multiple approaches to inhibit MAPK signaling holds great promise for the treatment of BRAF mutation-positive cancers, especially in terms of overcoming resistance. In this regard, we previously found that copper (Cu) influx enhanced MEK1 phosphorylation of its substrates ERK1/2 through a Cu-MEK1 interaction. We show here that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced MAPK signaling and oncogenic BRAFV600E-mediated tumorigenesis, which could be rescued by expressing activated ERK2. Importantly, a Cu chelator used in the treatment of Wilson's disease reduced tumor growth of not only BRAFV600E-transformed cells, but also cells resistant to a BRAF inhibitor. Taken together, these results suggest that Cu-chelation therapy could be repurposed for the treatment of BRAFV600E mutation-positive cancers. Citation Format: Donita C. Brady, Matthew S. Crowe, Michelle L. Turski, Dennis J. Thiele, Christopher M. Counter. Copper is required for oncogenic BRAF signaling and tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4294. doi:10.1158/1538-7445.AM2013-4294

Pharmacodynamic Effects and Mechanisms of Resistance to Vemurafenib in Patients With Metastatic Melanoma

PURPOSE To assess pharmacodynamic effects and intrinsic and acquired resistance mechanisms of the BRAF inhibitor vemurafenib in BRAF(V600)-mutant melanoma, leading to an understanding of the mechanism of action of vemurafenib and ultimately to optimization of metastatic melanoma therapy. METHODS In the phase II clinical study NP22657 (BRIM-2), patients received oral doses of vemurafenib (960 mg twice per day). Serial biopsies were collected to study changes in mitogen-activated protein kinase (MAPK) signaling, cell-cycle progression, and factors causing intrinsic or acquired resistance by immunohistochemistry, DNA sequencing, or somatic mutation profiling. Results Vemurafenib inhibited MAPK signaling and cell-cycle progression. An association between the decrease in extracellular signal-related kinase (ERK) phosphorylation and objective response was observed in paired biopsies (n = 22; P = .013). Low expression of phosphatase and tensin homolog showed a modest association with lower response. Baseline mutations in MEK1(P124) coexisting with BRAF(V600) were noted in seven of 92 samples; their presence did not preclude objective tumor responses. Acquired resistance to vemurafenib associated with reactivation of MAPK signaling as observed by elevated ERK1/2 phosphorylation levels in progressive lesions and the appearance of secondary NRAS(Q61) mutations or MEK1(Q56P) or MEK1(E203K) mutations. These two activating MEK1 mutations had not previously been observed in vivo in biopsies of progressive melanoma tumors. CONCLUSION Vemurafenib inhibits tumor proliferation and oncogenic BRAF signaling through the MAPK pathway. Acquired resistance results primarily from MAPK reactivation driven by the appearance of secondary mutations in NRAS and MEK1 in subsets of patients. The data suggest that inhibition downstream of BRAF should help to overcome acquired resistance.

Oncogenic B-Raf signaling in melanoma cells controls a network of microRNAs with combinatorial functions

Over two-thirds of melanomas have activating mutations in B-Raf, leading to constitutive activation of the B-Raf/MKK/ERK signaling pathway. The most prevalent mutation, B-RafV600E, promotes cancer cell behavior through mechanisms that are still incompletely defined. Here, we used a sensitive microarray profiling platform to compare microRNA (miRNA) expression levels between primary melanocytes and B-RafV600E-positive melanoma cell lines, and between melanoma cells treated in the presence and absence of an MKK1/2 inhibitor. We identified a network of >20 miRNAs deregulated by B-Raf/MKK/ERK in melanoma cells, the majority of which modulate the expression of key cancer regulatory genes and functions. Importantly, miRNAs within the network converge on protein regulation and cancer phenotypes, suggesting that these miRNAs might function combinatorially. We show that miRNAs augment effects on protein repression and cell invasion when co-expressed, and gene-specific latency and interference effects between miRNAs were also observed. Thus, B-Raf/MKK/ERK controls key aspects of cancer cell behavior and gene expression by modulating a network of miRNAs with cross-regulatory functions. The findings highlight the potential for complex interactions between coordinately regulated miRNAs within a network.

Abstract 4215: Integrin-dependent mechanisms for sensitizing melanomas to chemotherapy

Abstract BRAF is mutated in 50-70% of human melanomas, resulting constitutive activation of the MEK-ERK1/2 signal transduction pathway and resistance to apoptosis. Agents that target the oncogenic BRAF signaling pathway are revolutionizing the treatment of metastatic melanoma. However, it is already evident that there is both intrinsic and acquired resistance to this class of compounds suggesting that combinatorial therapies may be needed to combat melanoma resistance. We recently described a novel compound, denoted vinculin activating peptide (VAP) that engages defective integrins with the extracellular matrix and consequently sensitizes melanomas to chemotherapy. VAP is relatively large and the minimal sequences required for the sensitization of melanomas to chemotherapy are not known. In this study, we investigated the smallest domains of VAP that are required for its chemosensitizing effects. Here, we report that VAP contains three small domains, termed vinculin binding sites (VBSs) which are sufficient to bind the integrin-associated protein, vinculin and sensitize melanoma cells to chemotherapeutics. Out of the three VBSs within VAP, VBS3 exhibits stronger binding to vinculin and increases cell-matrix adhesion and focal adhesion number to a greater extent. Furthermore, VBS3 enhances melanoma sensitivity to chemotherapy more than VBS1 or VBS2. The residues of VBS3 required for the sensitization of melanomas have been investigated and suggest that divergent residues are important for chemosensitization. Collectively these data suggest that small molecule mimetics of VBS3 would be an ideal therapy to overcome the resistance of metastatic melanomas to chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4215. doi:1538-7445.AM2012-4215

Early [18F]Fluorodeoxyglucose–Positron Emission Tomography Responses in Metastatic Melanoma: What Do They Mean?

Inhibition of oncogenic BRAF signaling has shown substantial benefit in the approximately 50% of patients with metastatic melanoma whose tumors harbor an activating mutation. Vemurafenib, a selective and potent inhibitor of mutated BRAF kinase, has rapidly moved from early clinical trials to status as an approved drug on the basis of significant improvements in progression-free survival (PFS), overall survival, and response rate compared with standard dacarba

Mutationally Activated BRAFV600E Elicits Papillary Thyroid Cancer in the Adult Mouse

Mutated BRAF is detected in approximately 45% of papillary thyroid carcinomas (PTC). To model PTC, we bred mice with adult-onset, thyrocyte-specific expression of BRAF(V600E). One month following BRAF(V600E) expression, mice displayed increased thyroid size, widespread alterations in thyroid architecture, and dramatic hypothyroidism. Over 1 year, without any deliberate manipulation of tumor suppressor genes, all mice developed PTC displaying nuclear atypia and marker expression characteristic of the human disease. Pharmacologic inhibition of MEK1/2 led to decreased thyroid size, restoration of thyroid form and function, and inhibition of tumorigenesis. Mice with BRAF(V600E)-induced PTC will provide an excellent system to study thyroid tumor initiation and progression and the evaluation of inhibitors of oncogenic BRAF signaling.

Parsing out the complexity of RAF inhibitor resistance

The discovery in 2002 of frequent mutation of BRAF in cancer was a milestone event in our understanding of the genetics of melanoma (1). This observation, amongst others, led to the current understanding that melanoma is a collection of genetically divergent neoplasms united by common histopathologic criteria (2). Moreover, the implicit promise of this discovery was that inhibition of oncogenic BRAF signaling would improve the treatment of patients with metastatic disease, which had largely defied the best efforts of medical oncology. However, the clinical failure of initial RAF or MEK inhibitor trials, combined with evidence that BRAF was mutated in benign nevi, led to skepticism that BRAF→MEK→ERK signaling was important for melanoma maintenance (3-6). In 2010, this early promise was finally realized when Plexxicon and Roche scientists in collaboration with a world-wide consortium of medical oncologists, described the properties of PLX-4032, a pan-RAF inhibitor that elicited striking tumor regressions in Phase I clinical trials (7,8). Indeed, the remarkable (~80%) response rate of patients to PLX-4302 garnered considerable attention from the press and excitement in the melanoma research community (9). One of the remarkable, but unsung, aspects of PLX-4032′s success was the critical role that drug formulation played in obtaining sufficiently sustained inhibition of BRAF→MEK→ERK signaling in patients (7).

NFAT signalling is a novel target of oncogenic BRAF in metastatic melanoma

Background:Metastatic melanoma is the most deadly form of skin cancer and with an overall 5-year survival rate of <11%, there is an acute need for novel therapeutic strategies. Activating mutations in the BRAF oncogene are present in 50–70% of cases and contribute to tumourigenesis, thus, defining downstream targets of oncogenic BRAF may help define novel targets for therapeutic intervention. The Ca2+/calcineurin-regulated transcription factor, Nuclear factor of activated T-cells (NFAT), is important in the pathogenesis of several human cancers, target genes of which are also known to contribute to melanoma progression. One such NFAT target gene is COX-2, increased expression of which correlates with poor prognosis; however, upstream regulators of COX-2 in melanoma remain undefined. Therefore, the aim of this study was to evaluate NFAT expression and activity in metastatic melanoma and establish whether or not oncogenic BRAF signalling modulates NFAT activity and determine if NFAT is a key upstream regulator of COX-2 in melanoma.Methods:Nuclear factor of activated T-cells transcriptional activity and protein expression were determined in three human metastatic melanoma cell lines with differing B-RAF mutational status. NFAT activation by oncogenic BRAFV600E was explored by BRAFV600E overexpression and application of the specific MEK inhibitor PD98059. Regulation of COX-2 expression by NFAT was investigated using NFAT-targeted siRNA, calcineurin inhibitors cyclosporin A and FK506, in addition to COX-2 luciferase reporter vectors that selectively lacked NFAT binding sites.Results:NFAT transcriptional activity was increased in BRAF-mutated melanoma cells compared with wild-type cells. Furthermore, in wild-type cells, overexpression of BRAFV600E increased NFAT activity, which was blocked by the MEK inhibitor PD98059. Using calcineurin inhibitors and siRNA-mediated knockdown of NFAT2 and 4, we show NFAT is required for COX-2 promoter activation and protein induction in metastatic melanoma cells.Conclusion:NFAT2 and 4 are expressed in human metastatic melanoma cell lines and are activated by oncogenic BRAFV600E via MEK/ERK signalling. NFAT is an important upstream regulator of COX-2 in metastatic melanoma. Furthermore, as the BRAF/MEK/ERK pathway is hyperactive in other malignancies and MEK/ERK are also activated by oncogenic RAS in 30% of all human cancers, the potential to exploit NFAT signalling for therapeutic benefit warrants further investigation.

Plexin B1 is repressed by oncogenic B-Raf signaling and functions as a tumor suppressor in melanoma cells

Human melanomas show oncogenic B-Raf mutations which activate the B-Raf/MKK/ERK cascade. We screened microarrays to identify cellular targets of this pathway, and found that genes upregulated by B-Raf/MKK/ERK showed highest association with cell cycle regulators, whereas genes downregulated were most highly associated with axon guidance genes, including plexin-semaphorin family members. Plexin B1 was strongly inhibited by MAP kinase signaling in melanoma cells and melanocytes. In primary melanoma cells, plexin B1 blocked tumorigenesis as measured by growth of colonies in soft agar, spheroids in extracellular matrix, and xenograft tumors. Tumor suppression depended on residues in the C-terminal domain of plexin B1 which mediate receptor GAP activity, and also correlated with AKT inhibition. Interestingly, the inhibitory response to plexin B1 was reduced or absent in cells from a matched metastatic tumor, suggesting that changes occur in metastatic cells which bypass the tumor suppressor mechanisms. Plexin B1 also inhibited cell migration, but this was seen in metastatic cells and not in matched primary cells. Thus, plexin B1 has tumor suppressor function in early-stage cells, while suppressing migration in late-stage cells. Our findings suggest that B-Raf/MKK/ERK provides a permissive environment for melanoma genesis by modulating plexin B1.

Novel Potent BRAF Inhibitors: Toward 1 nM Compounds through Optimization of the Central Phenyl Ring

BRAF, a serine/threonine specific protein kinase that is part of the MAPK pathway and acts as a downstream effector of RAS, is a potential therapeutic target in melanoma. We have developed a series of small-molecule BRAF inhibitors based on a 1H-imidazo[4,5-b]pyridine-2(3H)-one scaffold (ring A) as the hinge binding moiety and a number of substituted phenyl rings C that interact with the allosteric binding site. The introduction of various groups on the central phenyl ring B combined with appropriate A- and C-ring modifications afford very potent compounds that inhibit (V600E)BRAF kinase activity in vitro and oncogenic BRAF signaling in melanoma cells. Substitution on the central phenyl ring of a 3-fluoro, a naphthyl, or a 3-thiomethyl group improves activity to yield compounds with an IC(50) of 1 nM for purified (V600E)BRAF and nanomolar activity in cells.

Functional Proteomics Identifies Targets of Phosphorylation by B-Raf Signaling in Melanoma

Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO(3)(-). The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into three-dimensional collagen matrix.

Regulation of ERK3/MAPK6 expression by BRAF

Several forms of cancer are characterized by frequent activating mutations in the serine/threonine kinase, BRAF. Substitution of glutamic acid for valine at codon 600 (V600E) accounts for approximately 90% of all BRAF activating mutations and leads to stimulation of kinase activity, downstream signaling, and cell transformation. To better understand the molecular pathogenesis induced by oncogenic BRAF signaling, we used microarray gene expression profiling to comprehensively analyze the BRAF-directed transcriptional program of cells expressing a conditionally active form of BRAFV600E. Several novel genes that affect proliferation, cell survival, angiogenesis and immune surveillance were identified as possible mediators of BRAF-induced oncogenic signaling. Moreover, we show that a MAPK family member, extracellular signal-regulated kinase-3 (ERK3/MAPK6) is highly expressed in response to BRAF signaling in this system. Cellular ERK3 protein is highly unstable and pharmacological inhibition of BRAF activity resulted in rapid ERK3 degradation. In melanoma cells, RNAi-mediated knockdown of endogenous BRAF or treatment with MEK inhibitors that prevent ERK1/2 activation led to a reduction in ERK3 levels, indicating that elevated ERK3 expression is mediated through MEK1/2 signaling. These results provide strong evidence for another mode by which BRAF can regulate the ERK protein kinase family and suggest ERK3 to be a potential pharmacodynamic marker for targeting BRAF signaling in melanoma.