BRAF Silencing by Short Hairpin RNA or Chemical Blockade by PLX4032 Leads to Different Responses in Melanoma and Thyroid Carcinoma Cells

BRAF-activating mutations have been reported in several types of cancer, including melanoma (∼70% of cases), thyroid (30-70%), ovarian (15-30%), and colorectal cancer (5-20%). Mutant BRAF has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase pathway. BRAF silencing induces regression of melanoma xenografts, indicating the essential role of BRAF for cell survival. We set up an inducible short hairpin RNA system to compare the role of oncogenic BRAF in thyroid carcinoma versus melanoma cells. Although BRAF knockdown led to apoptosis in the melanoma cell line A375, the anaplastic thyroid carcinoma cell ARO underwent growth arrest upon silencing, with little or no cell death. Reexpression of the thyroid differentiation marker, sodium iodide symporter, was induced after long-term silencing. The different outcome of BRAF down-regulation in the two cell lines was associated with an opposite regulation of p21CIP1/WAF1 expression levels in response to the block of the BRAF mitogenic signal. These results were confirmed using a specific BRAF small-molecule inhibitor, PLX4032. Restoration of p21CIP1/WAF1 expression rescued melanoma cells from death. Altogether, our data indicate that oncogenic BRAF inhibition can have a different effect on cell fate depending on the cellular type. Furthermore, we suggest that a BRAF-independent mechanism of cell survival exists in anaplastic thyroid cancer cells. (Mol Cancer Res 2008;6(5):751–9)

[1]  V. Trovisco,et al.  BRAF mutations are associated with some histological types of papillary thyroid carcinoma , 2004, The Journal of pathology.

[2]  A. Pinchera,et al.  Correlation between B-RAFV600E mutation and clinico-pathologic parameters in papillary thyroid carcinoma: data from a multicentric Italian study and review of the literature. , 2006, Endocrine-related cancer.

[3]  F. Holstege,et al.  Specific inhibition of gene expression using a stably integrated, inducible small‐interfering‐RNA vector , 2003, EMBO reports.

[4]  A. Nicholson,et al.  Mutations of the BRAF gene in human cancer , 2002, Nature.

[5]  J. Fagin,et al.  High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas. , 1993, The Journal of clinical investigation.

[6]  R. Marais,et al.  Is BRAF the Achilles' Heel of Thyroid Cancer? , 2006, Clinical Cancer Research.

[7]  M. Nikiforova,et al.  Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer. , 2005, The Journal of clinical investigation.

[8]  D. Barford,et al.  Mechanism of Activation of the RAF-ERK Signaling Pathway by Oncogenic Mutations of B-RAF , 2004, Cell.

[9]  M. Nikiforova,et al.  BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. , 2003, The Journal of clinical endocrinology and metabolism.

[10]  David P. Davis,et al.  Oncogenic BRAF is required for tumor growth and maintenance in melanoma models. , 2006, Cancer research.

[11]  N. Lydon,et al.  Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis. , 1997, Blood cells, molecules & diseases.

[12]  D. Sidransky,et al.  Exon 15 BRAF Mutations Are Uncommon in Melanomas Arising in Nonsun-Exposed Sites , 2004, Clinical Cancer Research.

[13]  M. Santoro,et al.  BRAF Is a Therapeutic Target in Aggressive Thyroid Carcinoma , 2006, Clinical Cancer Research.

[14]  A. Ross,et al.  The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells , 1996, Molecular and cellular biology.

[15]  M. Gorospe,et al.  Protective role of p21(Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells , 1996, Molecular and cellular biology.

[16]  S. Filetti,et al.  p53 re-expression inhibits proliferation and restores differentiation of human thyroid anaplastic carcinoma cells , 1997, Oncogene.

[17]  K. Smalley,et al.  A pivotal role for ERK in the oncogenic behaviour of malignant melanoma? , 2003, International journal of cancer.

[18]  P. Meltzer,et al.  High frequency of BRAF mutations in nevi , 2003, Nature Genetics.

[19]  C. Marshall,et al.  B-RAF is a therapeutic target in melanoma , 2004, Oncogene.

[20]  J. Fagin,et al.  Conditional BRAFV600E expression induces DNA synthesis, apoptosis, dedifferentiation, and chromosomal instability in thyroid PCCL3 cells. , 2005, Cancer research.

[21]  M. Gorospe,et al.  p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells. , 1997, Oncogene.

[22]  M. Laiho,et al.  Human melanoma cell line UV responses show independency of p53 function. , 1999, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[23]  J. Pouysségur,et al.  Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Fagin,et al.  Inhibitors of Raf Kinase Activity Block Growth of Thyroid Cancer Cells with RET/PTC or BRAF Mutations In vitro and In vivo , 2006, Clinical Cancer Research.

[25]  P. Ladenson,et al.  BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. , 2005, The Journal of clinical endocrinology and metabolism.

[26]  Hans Clevers,et al.  The β-Catenin/TCF-4 Complex Imposes a Crypt Progenitor Phenotype on Colorectal Cancer Cells , 2002, Cell.

[27]  T. Giordano,et al.  Correlation Between Genetic Alterations and Microscopic Features, Clinical Manifestations, and Prognostic Characteristics of Thyroid Papillary Carcinomas , 2006, The American journal of surgical pathology.

[28]  T. Sugimura,et al.  cDNA cloning and characterization of ret activated in a human papillary thyroid carcinoma cell line. , 1990, Biochemical and biophysical research communications.

[29]  B. Druker,et al.  Oncogenes and Tumor Suppressors (795 articles) , 2004 .

[30]  D. Tuveson,et al.  Suppression of BRAF(V599E) in human melanoma abrogates transformation. , 2003, Cancer research.

[31]  J. Castro,et al.  Suppression of oncogenic NRAS by RNA interference induces apoptosis of human melanoma cells , 2005, International journal of cancer.

[32]  G. Babcock,et al.  Oncogenic RAS Induces Accelerated Transition through G2/M and Promotes Defects in the G2 DNA Damage and Mitotic Spindle Checkpoints* , 2006, Journal of Biological Chemistry.

[33]  R. Testi,et al.  p21(Waf1/Cip1/Sdi1) mediates shear stress-dependent antiapoptotic function. , 2004, Cardiovascular research.