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C-Raf is associated with disease progression and cell proliferation in a subset of melanomas.

ABSTRACT: Raf-kinases include three major isoforms. Although the role of B-Raf in melanoma is well established, little is known about C-Raf. We studied effects of C-Raf knockdown in vitro and assessed expression of C-Raf in a large cohort of melanomas and nevi.Using specific siRNAs, we knocked down C-Raf expression, and determined the effect on viability, MAP extracellular signal-regulated kinase (ERK)/ERK kinase signaling, and apoptosis in seven melanoma cell lines. We determined the IC(50) of the C-Raf inhibitors sorafenib and GW5074, and studied the effects of GW5074 on cell signaling. Using an automated method to measure in situ protein expression, we quantified C-Raf expression in 263 nevi and 523 melanomas.C-Raf was knocked down in three cell lines with detectable phospho-C-Raf, resulting in decreased viability in two of the three (YULAC and YUROB). This resulted in decreased Bcl-2 expression and phospho-Bad cleavage, without affecting phospho-MEK and phospho-ERK. Sensitivity to sorafenib and GW5074 varied. GW5074 inhibited mitogen-activated protein kinase signaling without Bcl-2 and phospho-Bad down-regulation. C-Raf was highly expressed in melanomas compared with nevi (P < 0.0001), and no nevi had high C-Raf expression. C-Raf expression was higher in metastatic than primary specimens (P = 0.0225).C-Raf siRNA knock-down results in decreased viability of YULAC (B-Raf(V600K)) and YUROB (B-Raf(WT)) melanoma cells, likely mediated by Bcl-2 inhibition rather than mitogen-activated protein kinase inhibition. Cotargeting C-Raf and parallel pathways might be an effective therapeutic approach for melanoma. C-Raf expression is up-regulated in a subset of melanomas but not in nevi, suggesting that it might be a valuable diagnostic marker and therapeutic target.

SUBMITTER: Jilaveanu LB

PROVIDER: S-EPMC2763114 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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C-Raf is associated with disease progression and cell proliferation in a subset of melanomas.

Jilaveanu Lucia B LB Zito Christopher R CR Aziz Saadia A SA Conrad Patricia J PJ Schmitz John C JC Sznol Mario M Camp Robert L RL Rimm David L DL Kluger Harriet M HM

Clinical cancer research : an official journal of the American Association for Cancer Research 20090908 18

<h4>Purpose</h4>Raf-kinases include three major isoforms. Although the role of B-Raf in melanoma is well established, little is known about C-Raf. We studied effects of C-Raf knockdown in vitro and assessed expression of C-Raf in a large cohort of melanomas and nevi.<h4>Experimental design</h4>Using specific siRNAs, we knocked down C-Raf expression, and determined the effect on viability, MAP extracellular signal-regulated kinase (ERK)/ERK kinase signaling, and apoptosis in seven melanoma cell l ...[more]

PMID: 19737955

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Project description:Simple Summary NRAS-mutant melanoma is a highly aggressive subtype with few treatment options. Although both BRAF-mutant and NRAS-mutant melanomas have activation of the MEK/ERK pathway, MEK inhibitors (MEKi) are only effective for the BRAF-mutant subtype. The aim of this study was to understand why MEKi are ineffective in NRAS-mutant melanomas with the long-term goal of identifying new treatment regimens. Here, we show that ABL and DDR kinases are critically important for MEKi resistance because they cooperate to promote the stability of key proteins involved in driving melanoma growth and survival. FDA-approved drugs that inhibit ABL1/2 and DDR1 have been used for decades to treat leukemia. We showed that one such inhibitor prevents MEKi resistance from developing in a NRAS-mutant melanoma animal model. Thus, the data in this study provide the rationale for testing the use of drugs targeting ABL1/2 and DDR1 in combination with MEKi for patients with NRAS-mutant melanomas who have failed to respond to immunotherapy. Abstract Melanomas harboring NRAS mutations are a particularly aggressive and deadly subtype. If patients cannot tolerate or the melanomas are insensitive to immune checkpoint blockade, there are no effective 2nd-line treatment options. Drugs targeting the RAF/MEK/ERK pathway, which are used for BRAF-mutant melanomas, do little to increase progression-free survival (PFS). Here, using both loss-of-function and gain-of-function approaches, we show that ABL1/2 and DDR1 are critical nodes during NRAS-mutant melanoma intrinsic and acquired MEK inhibitor (MEKi) resistance. In some acquired resistance cells, ABL1/2 and DDR1 cooperate to stabilize RAF proteins, activate ERK cytoplasmic and nuclear signaling, repress p27/KIP1 expression, and drive RAF homodimerization. In contrast, other acquired resistance cells depend solely on ABL1/2 for their survival, and are sensitive to highly specific allosteric ABL1/2 inhibitors, which prevent β-catenin nuclear localization and destabilize MYC and ETS1 in an ERK-independent manner. Significantly, targeting ABL1/2 and DDR1 with an FDA-approved anti-leukemic drug, reverses intrinsic MEKi resistance, delays acquisition of acquired resistance, and doubles the survival time in a NRAS-mutant mouse model. These data indicate that repurposing FDA-approved drugs targeting ABL1/2 and DDR1 may be a novel and effective strategy for treating patients with treatment-refractory NRAS-driven melanomas.

Project description:Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ?7% of human malignancies and ?60% of melanomas. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFR? (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFR? RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFR?-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFR? or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFR? or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.

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C-Raf is associated with disease progression and cell proliferation in a subset of melanomas.

Publications

C-Raf is associated with disease progression and cell proliferation in a subset of melanomas.

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