The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells
Rosa Marina Melillo, Maria Domenica Castellone, Valentina Guarino, Valentina De Falco, Anna Maria Cirafici, Giuliana Salvatore, Fiorina Caiazzo, Fulvio Basolo, Riccardo Giannini, Mogens Kruhoffer, Torben Orntoft, Alfredo Fusco, Massimo Santoro
Rosa Marina Melillo, Maria Domenica Castellone, Valentina Guarino, Valentina De Falco, Anna Maria Cirafici, Giuliana Salvatore, Fiorina Caiazzo, Fulvio Basolo, Riccardo Giannini, Mogens Kruhoffer, Torben Orntoft, Alfredo Fusco, Massimo Santoro
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Article Oncology

The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells

Abstract

In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade.

Authors

Rosa Marina Melillo, Maria Domenica Castellone, Valentina Guarino, Valentina De Falco, Anna Maria Cirafici, Giuliana Salvatore, Fiorina Caiazzo, Fulvio Basolo, Riccardo Giannini, Mogens Kruhoffer, Torben Orntoft, Alfredo Fusco, Massimo Santoro

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Figure 1

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RET/PTC-mediated ERK activation is dependent on RAS and BRAF. (A) Schema...
RET/PTC-mediated ERK activation is dependent on RAS and BRAF. (A) Schematic representation of the RET, HRAS, and BRAF constructs. The RET/PTC breakpoint and RET tyrosines 1015 and 1062 are indicated. Residues V600 and K483 are mutated to E and M, respectively, in the BRAF(V600E) and BRAF(K–) plasmids. Residues G12 and S17 are mutated to V and N in the HRAS(V12) and HRAS(N17) plasmids. C, CAAX tail; CR1–3, conserved BRAF regions 1–3; Cys, cysteine-rich; EC, extracellular domain; ED, HRAS effector domain; H, heterogeneous region; JX, juxtamembrane; SP, RET signal peptide; TK, tyrosine kinase; TM, transmembrane. (B) Protein lysates (500 μg) extracted from HEK293 cells transfected with the indicated plasmids underwent immunoprecipitation with anti-tag (myc) antibody. Kinase assay was performed with GST-MEK as a substrate. BRAF and RET/PTC3 were detected by Western blotting (W.B.) with anti-myc and anti-RET antibodies, respectively. RAS expression was detected with an anti-RAS monoclonal antibody that also recognizes the endogenous protein. (C) HEK293 cells transfected with the indicated plasmids were harvested, and protein extracts were subjected to immunoblotting with anti–phospho-p44/p42 ERK (pERK) antibodies. The blot was reprobed with anti-p44/p42 antibodies for normalization. RET/PTC3 and RAS were detected by Western blotting with specific antibodies. These experiments are representative of at least 3 independent assays. (D) Transient BRAF suppression was achieved by RNA interference. Whole cell lysates were prepared 48 hours after transfection and analyzed for protein expression by Western blotting with the indicated antibodies. siRNA(SCR), scrambled siRNA.