The role of the cell adhesion molecule NCAM and the transcription factor Dlx2 in epithelial-mesenchymal transition (EMT) and tumor progression

Cancer belongs to the most life-threatening diseases in humans and represents in a simplified manner the destruction of healthy tissue and organs by uncontrolled cell proliferation and subsequent formation of a tumor. The most dangerous step of this disease occurs when cancer cells gain the ability to invade into the surrounding tissue and to disseminate via the blood system or the lymphatics throughout the body to form at distant sites secondary tumors, a process named metastasis. To gain motility and invasiveness, cancer cells are known to undergo an epithelial-mesenchymal transition (EMT). EMT is well known from nonpathological processes like development and wound healing and describes the temporal transition of non-invasive epithelial cells into motile, invasive mesenchymal cells. To gain new and more detailed insights into the complex process of EMT and to identify new potential markers for ongoing metastasis, we established different in vitro EMT model systems and tracked changes in global gene expression occurring during EMT. By comparing these gene expression profiles we identified the neural cell adhesion molecule (NCAM) and the homeobox transcription factor distal-less homeobox 2 (Dlx2) to be upregulated during EMT. Employing different in vitro systems such as the normal murine mammary gland (NMuMG) cells which undergo a progressive EMT upon transforming growth factor (TGFβ) treatment, in combination with transgenic and syngeneic mouse models, we investigated the role of NCAM and Dlx2 during the process of EMT. Our investigations revealed that NCAM expression is required and sufficient to induce EMT in NMuMG cells. We show that during EMT NCAM undergoes a functional switch by changing both its subcellular localization and its interactions partners. A subset of upregulated NCAM breaks down its complex formation with the fibroblast growth factor receptor (FGFR) and translocates into lipid rafts where it interacts with the member of the Src family kinase (SFK) p59Fyn. In association with p59Fyn NCAM induces the phosphorylation of focal adhesion kinase (FAK), leading to stabilization of β1-integrin-mediated focal adhesion, increased cell spreading and migration. In line with this observation, we found NCAM expression at the invasive front of human and murine tumors. In contrast, Dlx2 function is not required and its expression is not sufficient to induce EMT in NMuMG cells. Instead, we found that Dlx2 function protects from TGFβ-induced cell-cycle arrest and apoptosis by two major modifications namely, inhibition of the apoptotic, canonical TGFβ-signaling pathway and the activation of mitogenic, survival-ensuring mitogen-activated protein kinase (MAPK) -and phosphoinositide 3-kinase (PI3K) pathways. The canonical apoptotic TGFβ signaling is inhibited by transcriptional repression of the TGFβ receptor II (TGFβRII) gene, leading to reduced TGFβRII protein levels, decreased activation of the signal transducers Smad2/4 and reduced transcriptional activation of the cell-cycle inhibitors such as p21CIP1. Proliferation and survival is mediated by the cooperated activation of the MAPK and PI3K pathways triggered by epidermal growth factor receptor (EGFR). Supporting the importance of Dlx2 function during tumor development and progression, we show that (i) loss of Dlx2 function in B16 melanoma cells significantly impairs their ability to form primary tumors and metastatic lesion in the lung of transplanted syngeneic mice and (ii) expression of Dlx2 correlates significantly with invasiveness of human melanoma, lung and prostate cancers. In summary, we identified the cell-adhesion molecule NCAM and the transcription factor Dlx2 as important key players of EMT by promoting invasion and survival, respectively. Whether these genes can be used as prognostic markers for EMT-driven tumor invasion requires further investigations.