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The RET tyrosine kinase is a functional receptor for neurotrophic ligands of the glial cell line-derived neurotrophic factor (GDNF) family. Loss of function of RET is associated with congenital megacolon or Hirschsprung's disease, whereas germ-line point mutations causing RET activation are responsible for multiple endocrine neoplasia type 2 (MEN2A, MEN2B, and familial medullary thyroid carcinoma) syndromes. Here we show that the expression of a constitutively active RET-MEN2A oncogene promotes survival of rat pheochromocytoma PC12 cells upon growth factor withdrawal. Moreover, we show that the RET-MEN2A-mediated survival depends on signals transduced by the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) cascades. Thus, in PC12 cells, RET-MEN2A associates with the PI3K regulatory subunit p85 and promotes activation of Akt (also referred to as protein kinase B) in a PI3K-dependent fashion; in addition, RET-MEN2A promotes MAPK activation. PI3K recruitment and Akt activation as well as MAPK activation depend on RET-MEN2A tyrosine residue 1062. As a result, tyrosine 1062 of RET-MEN2A is essential for RET-MEN2A-mediated survival of PC12 cells cultured in growth factor-depleted media.

The Akt/protein kinase B serine/threonine kinase is a downstream effector of phosphoinositide 3-kinase (PI3K). Akt is an important component of mitogenic and antiapoptotic signaling pathways and is implicated in neoplastic transformation. Thyroid cells in culture retain a differentiated phenotype consisting of epithelial cell morphology and the expression of several tissue-specific genes. The survival and proliferation of these cells depend on thyrotropin and a mixture of five additional hormones that includes insulin. The regulation of proliferation and the expression of the thyroid differentiation program are intimately connected processes. As a result, oncogenes that induce hormone-independent proliferation invariably impair the expression of the thyroid-specific differentiation markers. Given that thyrotropin and insulin stimulate Akt activation in thyroid cells, we set out to determine the effects of Akt on thyroid cell proliferation, survival, and differentiation. To this end, we expressed constitutively active myristylated Akt (myrAkt) in PC Cl 3 thyroid cells. The myrAkt-expressing cells continued to proliferate, even in the absence of hormones, and they were resistant to programmed cell death induced by starvation. These effects were paralleled by the induction of the G1 cyclins D3 and E and by the inhibition of induction of the proapoptotic Fas, Fas ligand, and BAD genes in starved cells. However, in marked contrast with several other oncogenes, myrAkt did not interfere with the expression of thyroid differentiation functions. These results unveil the existence of an Akt-triggered thyroid cell pathway that modulates proliferation and survival without affecting the expression of the thyroid cell differentiated phenotype.

// Serena Bonin 1, * , Alessia Parascandolo 2, * , Cinzia Aversa 1 , Renzo Barbazza 1 , Nobuo Tsuchida 3 , Maria Domenica Castellone 4 , Giorgio Stanta 1 and Giancarlo Vecchio 5, 6, 7 1 Dipartimento di Scienze Mediche, Universita di Trieste-Cattinara, Trieste, Italy 2 IRCSS SDN, Naples, Italy 3 Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan 4 Istituto di Endocrinologia e Oncologia Sperimentale G.Salvatore, CNR, Naples, Italy 5 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita di Napoli Federico II, Naples, Italy 6 Istituto Superiore di Oncologia, Naples, Italy 7 Istituto Superiore di Oncologia, Genoa, Italy * Co-first authors Correspondence to: Giancarlo Vecchio, email: vecchio@unina.it Keywords: breast cancer; luminal B; lymph node metastasis; α-L-Fucosidase-1; immunohistochemistry Received: October 06, 2017 Accepted: February 01, 2018 Epub: February 07, 2018 Published: March 16, 2018 ABSTRACT Background: The lysosomal enzyme α-L-Fucosidase-1 (FUCA-1) catalyzes the hydrolytic cleavage of terminal fucose residues. FUCA-1 gene is down-regulated in highly aggressive and metastatic human tumors as its inactivation perturbs the fucosylation of proteins involved in cell adhesion, migration and metastases. Results: Negativity to FUCA-1 was significantly related to the development of later recurrences in breast cancer patients with lymph node involvement at diagnosis. Cancer specific survival of luminal B LN+ patients was influenced by FUCA-1 expression as luminal B LN+ patients with positive expression had a longer cancer specific survival. FUCA-1 mRNA expression was inversely related to cancer stage and lymph node involvement. WB and qPCR analysis of FUCA-1 expression in breast cancer-derived cell lines confirmed an inverse relationship with tumor aggressiveness. Conclusions: This study shows that, within LN+ breast cancer patients, FUCA-1 is able to identify a sub-set of non recurrent patients characterized by the positive expression of FUCA-1 and that, within luminal B LN+ patients, the expression of FUCA-1 predicts longer cancer specific survival. Methods: We have analyzed FUCA-1 in 305 breast cancer patients by Immunohistochemistry (IHC), and by qPCR in breast cancer patients and in breast cancer cell lines.

// Carminia Maria Della Corte 1 , Vincenza Ciaramella 1 , Concetta Di Mauro 2 , Maria Domenica Castellone 3 , Federica Papaccio 1 , Morena Fasano 1 , Ferdinando Carlo Sasso 4 , Erika Martinelli 1 , Teresa Troiani 1 , Ferdinando De Vita 1 , Michele Orditura 1 , Roberto Bianco 2 , Fortunato Ciardiello 1 , Floriana Morgillo 1 1 Oncologia Medica, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale “F. Magrassi e A. Lanzara”, Seconda Universita degli Studi di Napoli, Naples, Italy 2 Oncologia Medica, Dipartimento di Medicina Clinica e Chirurgia, Universita degli studi di Napoli “Federico II”, Naples, Italy 3 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Istituto di Endocrinologia ed Oncologia Sperimentale “G. Salvatore” (IEOS), University of Naples “Federico II”, Naples, Italy 4 Medicina Interna, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale “F. Magrassi e A. Lanzara”, Seconda Universita degli Studi di Napoli, Naples, Italy Correspondence to: Floriana Morgillo, e-mail: florianamorgillo@yahoo.com Keywords: metformin, MEK, selumetinib, pimasertib, NSCLC Received: August 13, 2015 Accepted: November 25, 2015 Published: December 11, 2015 ABSTRACT Purpose: Metformin, widely used as antidiabetic drug, showed antitumoral effects expecially in combination with chemotherapy. Our group recently has demonstrated that metformin and gefitinib are synergistic in LKB1 -wild-type NSCLC cells. In these models, metformin as single agent induced an activation and phosphorylation of mitogen-activated-protein-kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization. Experimental design: Since single agent metformin enhances proliferating signals through the RAS/RAF/MAPK pathway, and several MEK inhibitors (MEK-I) demonstrated clinical efficacy in combination with other agents in NSCLC, we tested the effects of metformin plus MEK-I (selumetinib or pimasertib) on proliferation, invasiveness, migration abilities in vitro and in vivo in LKB1 positive NSCLC models harboring KRAS wild type and mutated gene. Results: The combination of metformin with MEK-I showed a strong anti-proliferative and proapoptotic effect in Calu-3, H1299, H358 and H1975 human NSCLC cell lines, independently from the KRAS mutational status. The combination reduced the metastatic behaviour of NSCLC cells, via a downregulation of GLI1 trascritional activity, thus affecting the transition from an epithelial to a mesenchymal phenotype. Metformin and MEK-Is combinations also decreased the production and activity of MMP-2 and MMP-9 by reducing the NF-jB (p65) binding to MMP-2 and MMP-9 promoters. Conclusions: Metformin potentiates the antitumor activity of MEK-Is in human LKB1 -wild-type NSCLC cell lines, independently from the KRAS mutational status, through GLI1 downregulation and by reducing the NF-jB (p65)-mediated transcription of MMP-2 and MMP-9.

Small cell lung carcinoma (SCLC) often features the upregulation of the Sonic hedgehog (Shh) pathway leading to activation of Gli transcription factors. SCLC cells secrete bombesin (BBS)-like neuropeptides that act as autocrine growth factors. Here, we show that SCLC tumor samples feature co-expression of Shh and BBS-cognate receptor (gastrin-releasing peptide receptor (GRPR)). We also demonstrate that BBS activates Gli in SCLC cells, which is crucial for BBS-mediated SCLC proliferation, because cyclopamine, an inhibitor of the Shh pathway, hampered the BBS-mediated effects. BBS binding to GRPR stimulated Gli through its downstream G?q and G?12/13 GTPases, and consistently, other G?q and G?13 coupled receptors (such as muscarinic receptor, m1, and thrombin receptor, PAR-1) and constitutively active G?qQL and G?12/13QL mutants stimulated Gli. By using cells null for G?q and G?12/13, we demonstrate that these G proteins are strictly necessary for Gli activation by BBS. Moreover, by using constitutively active Rho small G-protein (Rho QL) as well as its inhibitor, C3 toxin, we show that Rho mediates G-protein-coupled receptor (GPCR)-, G?q- and G?12/13-dependent Gli stimulation. At the molecular level, BBS caused a significant increase in Shh gene transcription and protein secretion that was dependent on BBS-induced GPCR/G?q-12/13/Rho mediated activation of nuclear factor ?B (NF?B), which can stimulate a NF-?B response element in the Shh gene promoter. Our data identify a novel molecular network acting in SCLC linking autocrine BBS and Shh circuitries and suggest Shh inhibitors as novel therapeutic strategies against this aggressive cancer type.Oncogene advance online publication, 21 April 2014; doi:10.1038/onc.2014.104.

To identify genes involved in the transformation of thyroid follicular cells, we explored, using DNA oligonucleotide microarrays, the transcriptional response of PC Cl3 rat thyroid epithelial cells to the ectopic expression of the RET/PTC oncogenes. We found that RET/PTC was able to induce the expression of CXCR4, the receptor for the chemokine CXCL12/SDF-1alpha/beta. We observed that CXCR4 expression correlated with the transforming ability of the oncoprotein and depended on the integrity of the RET/PTC-RAS/ERK signaling pathway. We found that CXCR4 was expressed in RET/PTC-positive human thyroid cancer cell lines, but not in normal thyroid cells. Furthermore, we found CXCR4 expression in human thyroid carcinomas, but not in normal thyroid samples by immunohistochemistry. Since CXCR4 has been recently implicated in tumor proliferation, motility and invasiveness, we asked whether treatment with SDF-1alpha was able to induce a biological response in thyroid cells. We observed that SDF-1alpha induced S-phase entry and survival of thyroid cells. Invasion through a reconstituted extracellular matrix was also supported by SDF-1alpha and inhibited by a blocking antibody to CXCR4. Taken together, these results suggest that human thyroid cancers bearing RET/PTC rearrangements may use the CXCR4/SDF-1alpha receptor-ligand pathway to proliferate, survive and migrate.