title: C-Raf is associated with disease progression and cell proliferation in a subset of melanomas.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Yale Cancer Center and Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors.forename: Lucia B
authors.initials: LB
authors.lastname: Jilaveanu
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Zito
authors.forename: Saadia A
authors.initials: SA
authors.lastname: Aziz
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Conrad
authors.forename: John C
authors.initials: JC
authors.lastname: Schmitz
authors.forename: Mario
authors.initials: M
authors.lastname: Sznol
authors.forename: Robert L
authors.initials: RL
authors.lastname: Camp
authors.forename: David L
authors.initials: DL
authors.lastname: Rimm
authors.forename: Harriet M
authors.initials: HM
authors.lastname: Kluger
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001557:Benzenesulfonates / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D015331:Cohort Studies; D018450:Disease Progression; D005260:Female; D020868:Gene Silencing; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D008875:Middle Aged; D009506:Nevus / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009536:Niacinamide / Q000031:analogs & derivatives; D010636:Phenols / Q000494:pharmacology; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000077157:Sorafenib; D055815:Young Adult
chemical_list: D001557:Benzenesulfonates; D007211:Indoles; D010636:Phenols; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D034741:RNA, Small Interfering; D009536:Niacinamide; D000077157:Sorafenib; D019908:Proto-Oncogene Proteins c-raf; C489251:5-iodo-3-((3,5-dibromo-4-hydroxyphenyl)methylene)-2-indolinone