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.

Figure 1

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.

Figure 2

Generation of thyroid cell cultures. (A) Protein lysates (50 μg) extracted from the indicated cell lines underwent Western blotting with anti-RET, -tag (myc), or -RAS antibodies. Equal protein loading was ascertained by anti-tubulin immunoblot. (B) Mass populations of PC cells transfected with the indicated plasmids were photographed using a phase-contrast light microscope (magnification, ×150). RET/PTC3-, RET/PTC3(Y1015F)-, BRAF(V600E)-, and HRAS(V12)-expressing cells displayed a transformed morphology, whereas RET/PTC3(Y1062F)-expressing cells were virtually indistinguishable from parental cells. (C) Protein extracts from the indicated cell lines were subjected to immunoblotting with anti–phospho-p44/42 ERK antibodies. The blot was reprobed with anti-p44/42 antibodies for normalization. (D) BRAF targeting by siRNA but not by scrambled siRNA induced BRAF downregulation and ERK inhibition, as shown by Western blotting with specific antibodies.

Figure 3

The transformed phenotype of RET/PTC3 thyroid cells requires the integrity of the Y1062-RAS-BRAF-ERK pathway. (A) PC cells were transfected with the indicated plasmids or the empty vector and either selected in neomycin-containing medium or left in the absence of 6H. Three weeks later, colonies were stained with crystal violet and counted. The ratio of neomycin-resistant clones to 6H-independent colonies was calculated. The results of 3 independent experiments performed in duplicate ± SD are reported [the number of 6H-independent colonies induced by HRAS(V12) was set at 100]. Inset: Protein lysates (50 μg) underwent Western blotting with the indicated antibodies. cyc D1, cyclin D1. (B) Matrigel invasion of parental and transformed PC cells. Where indicated, in PC RET/PTC3 cells, suppression of endogenous BRAF was obtained by transfection of RNA interference against BRAF, and ERK inhibition was achieved by U0126 treatment. Cells were seeded in the upper chamber of 8-μM-pore transwells and incubated for 24 hours. Thereafter, filters were fixed and stained. The upper surface was wiped clean and cells on the lower surface photographed (top) and then solubilized. Absorbance at 570 nm was measured with a microplate reader. Cell migration is expressed as percentage with respect to RET/PTC3 cells, whose migration was arbitrarily set at 100. Each column represents the average ± SD of 3 independent experiments (bottom). (C) Suppression of endogenous BRAF in PC RET/PTC3 cells was obtained by transfection of RNA interference against BRAF. Cells were counted at different time points, and the average results of 3 independent experiments are reported.

Figure 4

Graphic representation of the gene expression changes (fold change of at least 4) in RET/PTC3, BRAF(V600E), and HRAS(V12) cells versus baseline (PC): upregulated genes (A) and downregulated genes (B). The numbers of upregulated and downregulated genes are represented on the y axis. Thick arrows indicate the Y1062-dependent genes; thin arrows indicate the targets common to RET/PTC3, BRAF(V599E), and HRAS(V12). The different groups of genes are highlighted. Average SLRs (ASLR) are indicated.

Figure 5

Expression levels of selected genes (A) and chemokines (B) in human PTC samples versus 5 normal thyroid tissues as determined by Q-RT-PCR. The PTC samples were characterized for the presence of either a RET/PTC rearrangement or a BRAF(V600E) mutation. For each target (indicated on the x axis), the expression levels values of tumors (y axis) were calculated relative to the average expression level in normal tissues (TN). All the experiments were performed in triplicate; SDs were smaller than 25% in all cases (data not shown). P values were calculated by the Mann-Whitney U test.

Figure 6

Chemokines and chemokine receptors are expressed in human PTC-derived cell lines. (A) CXCL1 and CXCL10 secretion in human PTC cells was evaluated by ELISA. Experiments were performed in triplicate, and the average value of the results ± SD was plotted. Normal thyroid cells (P5) were used as a negative control. (B) Expression levels of CXCR2 and CXCR3 in PTC cell lines were evaluated by Q-RT-PCR. Expression values were calculated relative to the expression level in normal P5 cells. Experiments were performed in triplicate, and the average value of the results ± SD was plotted. (C) Flow cytometric analysis of surface expression of CXCR2 and CXCR3 in TPC1 cells. (D) TPC1 cells were transfected by BRAF or scrambled siRNA and harvested 72 or 96 hours later. Protein lysates were subjected to immunoblotting with anti-BRAF and anti–phospho-p44/p42 ERK antibodies. (E) BRAF siRNA interference in TPC1 cells affected chemokine production as determined by ELISA.

Figure 7

Functional activities of CXCL1 and CXCL10 in human PTC cell lines. (A) Stimulation with CXCL1 and CXCL10 (100 ng/ml) induced time-dependent ERK and AKT activation in TPC1 cells. Cell lysates were harvested at the indicated time points; Western blots were probed with the indicated antibodies. (B) BrdU incorporation was measured to evaluate S-phase entry upon treatment with CXCL1 or CXCL10 or the indicated inhibitors. The average of the results of 3 independent experiments ± SD is indicated. (C) TPC1 cells were allowed to migrate for 24 hours toward serum-free medium or, where indicated, a gradient of CXCL1 or CXCL10. Where indicated, cells were preincubated with blocking antibodies, control antibodies, chemical inhibitors, or PTX. Cells were treated with either BRAF siRNA or U0126. Representative micrographs (left) and absorbance at 570 nm (average ± SD of 3 experiments; right) are shown.