In the present study we

In the present study, we introduce a novel cell surface protein, CD98, that is specifically expressed on the cells of the basal layer and, in addition, remains stably expressed after a multitude of proteolytic treatments. We show that purified CD98high cells are able to induce tumor growth upon serial transplantation in mice. Moreover, CD98high cells show a significantly higher expression of genes involved in SAR 405 control and DNA repair as compared to CD98low cells, whereas CD98low cells significantly express higher levels of genes involved in cellular differentiation, including keratinocyte differentiation. These findings support the CSC concept in HNSCC and suggest that CD98 can be exploited as a marker to enrich for CSCs in this tumor type.
Materials and methods
Results
Discussion Over the past decade, head and neck cancer is increasingly treated with chemoradiation protocols, but the variation in treatment response is a major concern. Many studies searched in vain for cellular characteristics that can be used as good predictive markers for tumor response, but the identification of the CSCs as a treatment resistant subpopulation of tumor cells provided new directions for exploration. Since CSCs are responsible for the maintenance of all cells in the tumor, a treatment specifically targeting the CSC population seems the most effective way to eradicate a tumor. New challenges lie in the characterization and isolation of these CSCs. So far, CD44 has been the most frequently used marker for squamous cancer stem cells (Joshua et al., 2012; Koukourakis et al., 2012; Perez et al., in press; Pries et al., 2008; Prince et al., 2007; Wilson et al., in press). The heterodimeric glycoprotein CD98 has been implicated in immunity as well as in cancer. Expression of the CD98 heavy chain or light chain has been investigated with respect to clinical prognosis of multiple tumor types (Esseghir et al., 2006; Ichinoe et al., 2011; Imai et al., 2009; Kaira et al., 2008, 2009a,b, 2010; Sakata et al., 2009). Nearly all studies report higher CD98 expression in progressive and metastatic tumors, which relates to a poor prognosis. Overexpression of CD98 drives both anchorage independence and tumorigenesis (Henderson et al., 2004; Shishido et al., 2000), but the mechanism behind this is not clear. It has been shown that the CD98 heavy chain mediates increased integrin signaling (Feral et al., 2005), leading to AKT phosphorylation (Armulik et al., 2004) and eventually to stimulation of cell survival, anchorage-independence and metastasis. Furthermore, the CD98 light chain (LAT-1 or LAT-2) is able to import essential amino acids (Verrey, 2003) which in turn can activate the mTOR pathway. This leads to promotion of cell growth, proliferation and protein synthesis. Thus, CD98 is important in cancer, probably mediated through increased amino acid transport and stimulation of integrin signaling. One of the characteristics of CSCs is the ability to self-renew. Expression profiling of the CD98high tumor fraction revealed higher mRNA expression of cell cycle associated genes as compared to the CD98low cells. It seems that CSCs tightly regulate cell cycle progression, more specifically the moment of cell division (via genes like PLK1 and TP53), as wells as the process of cell division. Many genes that are differentially expressed in CD98high cells are involved in proper assembly of the mitotic spindle and chromosome segregation. Examples of such genes are the aurora kinases A and B, TPX2 and KIF11. Also expression of genes responsible for maintenance of DNA integrity was high in these cells, implicating the importance of strict control mechanisms in the cells that fuel the tumor bulk. The CD98low cells showed increased expression of genes involved in cellular differentiation when compared to the CD98high cells. Genes specifically involved in keratinization, like members of the SPRR family (Gibbs et al., 1993), were found to be highly expressed in the CD98low subpopulation. This result can be easily correlated to our finding that none of the inoculations with CD98low cells resulted in the formation of a tumor, implicating that CD98low cells are presumably already differentiated and therefore incapable of producing progeny. Stem cell related genes, like OCT4, KLF4, SOX2 and Nanog, did not display significant differences when we compared CD98high versus CD98low cell populations.