TM9SF4: ROLE IN CANCER AND DEVELOPMENT

The tm9sf4 gene encodes a member of Transmembrane 9 Protein Superfamily, characterized by the presence of nine transmembrane domains and a high degree of evolutionary conservation. It was originally identified in Dictyostelium and Drosophila as a protein involved in cell adhesion and phagocytosis. More recent studies reported that tm9sf4 is expressed in metastatic melanoma cells and it is involved in the process of tumor cell cannibalism, which might be unique to metastatic cells. Tumor cells indiscriminately phagocytize both sibling tumor cells and lymphocytes, in addition to amorphous material, thereby allowing tumor cells to escape from the immune response, promoting cell survival in a hostile microenvironment with low nutrient supplies and hypoxia conditions and allowing them to remodel and migrate through the extracellular matrix. Tm9sf4 gene was brought to our attention by LA7 cells, which represent a cancer stem cell (CSC) model system. Our preliminary data showed that the expression level of tm9sf4 gene was higher in LA7 CSCs if compared to the differentiated counterpart. Tm9sf4 downregulation by RNA interference induced LA7 CSCs to trans-differentiate to mesenchymal-like cells and caused an increase of the expression level of EMT-associated markers, suggesting that the gene is involved in epithelial to mesenchymal transition (EMT). The aim of my PhD project is to identify the function of tm9sf4 in normal and cancer cells. Based on the assumption that all vertebrates share common pathways involved in biological processes including morphogenesis-associated development of branched structures, cell migration and sprouting, we investigated Tm9sf4 protein function in zebrafish (Danio rerio), to further investigate its role in normal development. Zebrafish was recently proven to be a powerful model, to study not only human diseases, but also processes associated with oncogenesis. Additionally, EMT was suggested to be the major driver of both embryonic morphogenesis and tumor progression, involving highly conserved cellular processes and signaling pathways. Tm9sf4 expression analyses were performed by both RT-PCR on RNA from embryos at different developmental stages and whole mount in situ hybridization (WISH). Histological sections of the stained embryos were performed. In addition, loss-of-function analyses were carried out by the injection of 1-4 cell stage embryos with antisense oligonucleotide morpholinos, using the zebrafish AB line (wild type) and the double transgenic line tg(gata1:dsRed)sd2/tg(flk1:EGFP)S843. The expression of specific brain markers and EMT-associated markers was assessed by WISH and Real Time PCR on embryos previously injected with morpholino oligos (morphants). We demonstrated that during zebrafish embryogenesis the gene was expressed from oocytes to 5 days post fertilization (dpf), suggesting that the expression is both maternal and zygotic. Moreover, it was found to be mainly expressed in the central nervous system (CNS). Following tm9sf4 downregulation, the embryos displayed head necrosis and an impaired brain compartmentation. Expression analysis of specific brain markers suggested that all of the targeted sub-structures were present in morphants, while they were less well organized than in control embryos. Additionally, tm9sf4 downregulation induced an increase of the expression levels of fgf8, shha, wnt1 and a reduction of otx2 level. These findings suggested that the gene has a role in CNS development. At 24 hours post fertilization (hpf) the morphants displayed tail bending and defects in the intermediate cell mass (ICM) region, where primitive hematopoiesis and vessel development occur. At 48 hpf the circulation was significantly impaired by protein downregulation, which caused cardiac edema and blood stasis in the tail, where morphological defects were detected in intersomitic vessels, which are formed by angiogenesis. It was suggested that angiogenesis represents a model for many core biological processes, including morphogenesis-related development of branched structures and cell migration, all of which are involved in the morphogenesis of many other organ systems. We demonstrated that tm9sf4 downregulation induced an increase of zeb2 and twist2 expression levels by 24 hpf embryos, suggesting that tm9sf4 induces negative regulation of these factors, inhibiting E-cadherin transcriptional repression, resulting in its upregulation, which promotes cell-cell adhesion. This hypothesis is consistent with previous studies, which suggested a protein role in cell adhesion. Interestingly, we found the tm9sf4 gene to be most strongly expressed in the periventricular regions of the diencephalon and mesencephalon, where there are proliferating cells that are going to migrate and could undergo epithelial to mesenchymal transition. Hence, tm9sf4 could have a role in repressing EMT during zebrafish neurulation. We also demonstrated that during gastrulation Tm9sf4 protein downregulation induced a decrease of E-cadherin expression level, which could promote the loss of cell-cell adhesion. In the near future we are going to further investigate the role of tm9sf4 in EMT, by determining in which specific CNS cells the tm9sf4 gene is expressed and by studying its effect on the expression of other components of the EMT signaling pathways, both in zebrafish gastrulation and neurulation. WISH analysis of other brain markers is now in progress, to individuate the specific regions and, possibly, the pathways affected by Tm9sf4 protein downregulation. Additionally, further investigations on circulation defects will be carried out, by both loss-of-function analyses on specific transgenic lines and cardiac markers expression studies, in order to establish whether the circulation problems are caused by defects in the heart development or rather the cardiac edema is due to the defective intersomitic vessels development