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The epithelial-mesenchymal transition (EMT) is a crucial process in tumour progression, by which epithelial cells acquire a mesenchymal phenotype, increasing its motility and the ability to invade distant sites. Here, we describe the molecular mechanisms by which BRAF, TGFβ and the Src/FAK complex cooperatively regulate EMT induction and cell motility of anaplastic thyroid cancer cells. Analysis of EMT marker levels reveals a positive correlation between TGFβ and Snail expression, with a concomitant downregulation of E-cadherin, accompanied by an increase of cell migration and invasion. Furthermore, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with its inhibitor PLX4720 reverses the TGFβ-mediated effects on Snail, E-cadherin, migration and invasion. Moreover, BRAF induces TGFβ secretion through a MEK/ERK-dependent mechanism. In addition, TGFβ activates the Src/FAK complex, which in turn regulates the expression of Snail and E-cadherin as well as cell migration. The inhibition of Src with the inhibitor SU6656 or abrogation of FAK expression with a specific siRNA reverses the TGFβ-induced effects. Interestingly, we demonstrate that activation of the Src/FAK complex by TGFβ is independent of BRAF signalling, since inhibition of this oncogene does not affect its phosphorylation. Our data strongly suggest that TGFβ induces EMT and aggressiveness of thyroid cancer cells by parallel mechanisms involving both the BRAF/MEK/ERK and Src/FAK pathways independently. Thus, we describe novel functions for Src/FAK in mediating the EMT program and aggressiveness regulated by TGFβ, establishing the inhibition of these proteins as a possible effective approach in preventing tumour progression of BRAF-expressing thyroid tumours. © 2015 Wiley Periodicals, Inc.

The dysregulation of autophagy is important in the development of many cancers, including thyroid cancer, where V600EBRAF is a main oncogene. Here, we analyse the effect of V600EBRAF inhibition on autophagy, the mechanisms involved in this regulation and the role of autophagy in cell survival of thyroid cancer cells. We reveal that the inhibition of V600EBRAF activity with its specific inhibitor PLX4720 or the depletion of its expression by siRNA induces autophagy in thyroid tumour cells. We show that V600EBRAF downregulation increases LKB1-AMPK signalling and decreases mTOR activity through a MEK/ERK-dependent mechanism. Moreover, we demonstrate that PLX4720 activates ULK1 and increases autophagy through the activation of the AMPK-ULK1 pathway, but not by the inhibition of mTOR. In addition, we find that autophagy blockade decreases cell viability and sensitize thyroid cancer cells to V600EBRAF inhibition by PLX4720 treatment. Finally, we generate a thyroid xenograft model to demonstrate that autophagy inhibition synergistically enhances the anti-proliferative and pro-apoptotic effects of V600EBRAF inhibition in vivo. Collectively, we uncover a new role of AMPK in mediating the induction of cytoprotective autophagy by V600EBRAF inhibition. In addition, these data establish a rationale for designing an integrated therapy targeting V600EBRAF and the LKB1-AMPK-ULK1-autophagy axis for the treatment of V600EBRAF-positive thyroid tumours.

Somatostatin (SST) is one of the main regulators of thyroid function. It acts by binding to its receptors, which lead to the dissociation of G proteins into Gαi and Gβγ subunits. However, much less is known about the function of Gβγ in thyroid cells. Here, we studied the role of SST and Gβγ dimers released upon SST stimulation on the Ras-ERK1/2 pathway in FTRL-5 thyroid cells. We demonstrate that SST activates Ras through Gi proteins, since SST-induced Ras activation is inhibited by pertussis toxin. Moreover, the specific sequestration of Gβγ dimers decreases Ras-GTP and phosphorylated ERK1/2 levels, and overexpression of Gβγ increases ERK1/2 phosphorylation induced by SST, indicating that Gβγ dimers released after SST treatment mediate activation of Ras and ERK1/2. On the other hand, SST treatment does not modify the expression of the thyroid differentiation marker sodium/iodide symporter (NIS) through ERK1/2 activation. However, SST increases AKT activation and the inhibition of the Src/PI3K/AKT pathway increases NIS levels in SST-treated cells. Thus, we conclude that, in thyroid cells, signalling from SST receptors to ERK1/2 involves a Gβγ-mediated signal acting on a Ras-dependent pathway. Moreover, we demonstrate that SST might regulates NIS expression through a Src/PI3K/AKT-dependent mechanism, but not through ERK1/2 signalling, showing the main role of this hormone in thyroid function.

Introduction Large scale sequencing studies have identified that 93% of the colorectal cancer (CRC) patients carry at least one mutation in genes implicated in Wnt signalling pathway. Notably, majority of the CRC patients (88%) carry either APC or b-catenin mutations that can activate the Wnt signalling pathway. Recent evidences suggest that Wnt/b-catenin signalling activity is regulated by CDH17 in hepatocellular carcinoma. As CDH17 is exclusively expressed in the intestine and overexpressed in CRC, we hypothesised that CDH17 could be utilised as a therapeutic target to treat CRC patients. Material and methods RNA interference-based stable knockdowns were established in a panel of CRC cells with varying mutations in APC and b-catenin. Wnt signalling activity of the cells were measured by TOPflash assay. Apoptosis studies were performed using fluorescence activated cell sorting. Cells were further subjected to immunoprecipitations with anti-CDH17 and anti-b-catenin antibodies followed by label-free quantitative proteomics analysis. A monoclonal antibody was developed to block CDH17 and sensitise CRC cells to chemotherapeutic drugs. Results and discussions Knockdown of CDH17 in CRC cells downregulated b-catenin and attenuated Wnt signalling activity irrespective of APC and/or b-catenin mutations. Furthermore, CDH17 silencing induced apoptosis and sensitised CRC cells to the chemotherapeutic drugs 5-Fluorouracil. Immunoprecipitations using anti-CDH17 and anti-b-catenin antibodies followed by label-free quantitative proteomics analysis highlighted no direct interaction between CDH17 and b-catenin hence implying an indirect regulation of b-catenin expression and Wnt signalling pathway by CDH17. The analysis revealed Ecadherin and FAT1 as common interactors of CDH17 and bcatenin. Quantitative proteomic analysis of cell lysates revealed the upregulation of FAT1, a negative regulator of Wnt signalling pathway, upon knockdown of CDH17. Monoclonal antibodies developed against CDH17 were able to increase apoptosis and sensitivity of CRC cells to 5-Fluorouracil. Conclusion Overall, these findings suggest that CDH17 can attenuate Wnt signalling pathway and induce apoptosis irrespective of the APC and b-catenin mutational status. As Wnt signalling pathway is aberrated in 93% of CRC patients, the membrane protein CDH17 can be exploited as therapeutic target to treat CRC.

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