Dottorato in Medicina Molecolare

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Tesi di dottorato

Role of Semaphorin 4C and Plexin-B2 in tumor development and metastasis.

Metastasis is an exceedingly complex process, which occurs through a series of sequential steps that include the invasion of adjacent tissues, intravasation, transport through the circulatory system, arrest at a secondary site, extravasation and growth in a secondary organ. Metastases are the cause of 90% of human cancer deaths. Work over the past decade has revealed crucial functions of the semaphorin–plexin system in mammalian physiology. Plexin B subfamily consists of three members which include Plexin-B1, -B2, -B3. Although Plexin-B2 is well expressed in various cancers and its knock out in mice is embryonic lethal, its relevance in cancer is poorly understood. Moreover, the expression of Sema4C, the best known ligand for PlexinB2, is dramatically up regulated during neurogenesis and down regulated upon differentiation. We hypothesized that Semaphorin 4C could have a role in the context of tumor growth and metastasis, and focused to study its functions by both in vitro and in vivo experiments.

 We analyzed multiple cancer cell lines and found that Sema4C exists as membrane-bound and extracellular shedded form. Over-expression of Sema4C in various cancer lines has a profound impact on the cell phenotype, accompanied by cytoskeletal rearrangement, and an increased ability to adhere to the extracellular matrix. We also observed changes in the expression levels of E-cadherin, N-cadherin, Zeb-1 and Zeb-2, with acquisition of more epithelial phenotype, supporting the involvement of Sema4C in Mesenchymal to Epithelial transition (MET) process. We then investigated the possible downstream partners of Sema4C/Plexin-B2 signaling by analyzing cell transcriptomic profiles with micro-arrays and interestingly found significant expression changes in Sema4C-overexpressing cells in relevant genes, such as ID1, ID3, KISS1 and ALDH3A1. In particular, ID (inhibitor of DNA-Binding) proteins are known to mediate breast cancer cells to colonize lungs and induce metastatic colonization by mesenchymal to epithelial transition. We also expressed in cancer cells deletion mutants of Semaphorin 4C and observed that its function seems to be primarily mediated through a reverse signaling pathway, in which Sema4C acts as a receptor and Plexin B1/B2 as ligands. 

In vivo, upon silencing Sema4C, the primary tumors grew larger in size when compared to control tumors, while upon Sema4C overexpression the cells formed relatively small tumors but interestingly gave rise to the same number of lung metastasis. The latter retained both elevated expression of Sema4C and of E-Cadherin, highlighting the fact that Sema4C-induced E-Cadherin upregulation did not hamper the invasive and metastatic ability of cells in the primary tumor. We furthermore performed tail vein injections of Sema4C-overexpressing tumor cells and observed an increased ability to extravasate and form metastatic seeds in the lungs.

 We are currently analyzing the possible downstream partners of Sema4C/Plexin-B2 to ID1/ID3 signaling axis, and the role of these pathways in human metastatic tumors by comparing relative gene expression in patient derived samples. Overall these data suggest that Semaphorin 4C mediates mesenchymal to epithelial transition in breast cancer cells and further understanding this signaling axis could highlight its role in mediating cancer cells plasticity during the metastatic process.

Attività di ricerca

Conference:

Tumor Microenvironment, Metabolism and Metastasis September 28-30, 2015

German Cancer Research Center (DKFZ) Communication Center, Lecture Hall DKFZ-ZMBH Alliance Forum organized by the DKFZ-ZMBH Alliance 

Abstract: Semaphorin 4C and Plexin B1/B2 signaling controls mesenchymal to epithelial transition in cancer cells.

 Sreeharsha Gurrapu,Michael Rehman,Luca Tamagnone.

 Affiliation- Candiolo Cancer Center – IRCCS, Candiolo , Italy & Dept. of Oncology, University of Torino, Italy. 

 
 Semaphorin 4C (Sema4C) belongs to a secreted and membrane bound subtype of semaphorin signals. Sema4C has been previously involved in development and cancer. Multiple breast cancer cell lines showed high levels of semaphorin4C and Plexin B1 and Plexin B2.In this study, we found that Sema4C overexpression could induce mesenchymal-epithelial transition in aggressive MDA-MB231 breast carcinoma cells, while silencing endogenous Sema4C expression had opposite effect. Sema4C-expressing MDA-MB231 underwent striking morphological changes, and molecular analysis revealed a “switch” from N- to E-Cadherin expression. In addition, Sema4c reduced the expression of vimentin and fibronectin, mesenchymal-specific markers. Both PlexinB1 and PlexinB2 can act as Sema4C-Receptors. Furthermore, RNAi-mediated silencing of either PlexinB1 or B2 reversed the effects induced by Sema4C, suggesting their requirement in the signaling axis. Our findings support the idea that Sema4C-PlexinB1/B2 axis is a novel regulator of mesenchymal-epithelial transition in breast cancer cells.