Molecular characterization of melanoma cancer stem cells

The contribution of our unit to the general subject of profiling the phenotype of melanoma cancer stem cells will be to investigate the expression of T-type Voltage Gated Calcium Channels VGCC) and of the sodium-calcium exchanger (NCX). These proteins are involved in controlling intracellular Ca2+ concentrations ([Ca2+]i) which are very high in proliferating cancer cells where very often they show large fluctuations. Our interest in T-type channels arises by the evidence that: 1. they are crucial to maintain cell proliferation; 2. their expression in tumors decreases upon differentiation and; 3. they are expressed by pluripotent stem cells during embryionic development. T-type channel expression is strictly cell-cycle specific being maximal in the G2/M phase (Akaike et al., 1989; Richard et al., 1992) and very high levels of these channels have been found in a number of malignant tumors such as breast cancers, gliomas, colorectal carcinomas, leukemias, retinoblastomas and prostatic carcinomas (Panner and Wurster, 2006). Intriguingly, they have been found in melanoma cell lines (Allen et al., 1997). T-type channel hyperexpression entails an increase in cell proliferation in HEK-293 cells (Wang et al., 2002), whereas antisense oligonucleotides directed against them inhibit the growth of glioma cells cultured in vitro (Panner et al., 2005). Despite all these arguments, no study has addressed up to date the hypothesis that these channel could play a role in cancer stem cell biology and their meaning in melanoma growth has never been explored. Analogously, a bunch of data support the idea that NCX could be involved in cancer growth and differentiation as observed in a mouse eritroleukemia cell line (Smith et al., 1982) and its involvement in stem cell differentiation has been proposed as well (Yanagida et al., 2004). Nevertheless, NCX role in melanoma and in its cancer stem cells remains unexplored. The aim of this project is characterizing at the molecular and functional level melanoma Cancer Stem cells (mCSCs). The main goal is to causally correlate the molecular characteristics of mCSC with their physio-pathological properties, tumorigenicity, metastatic potential and resistance to chemotherapic drugs. To this end mCSC will be isolated both from primary and metastatic melanoma. Moreover, metastatic melanoma will be further divided in photoemustine-sensitive and resistant. This will allow us to investigate the molecular mechanisms underpinning both the metastasization process and the resistance to drugs. mCSC characterization will include analysis of the genomic, transcriptomic, proteomic and miRNA profiles. Biological properties will be assessed both in vitro and in vivo, in mouse and zebrafish models. The ultimate goal is finding pharmacogenomic biomarkers for the prediction of treatment susceptibility, and, more importantly, discovering novel therapeutic targets useful to overcome drug resistance in melanoma therapy.