The Role of the Mucin-Like Glycoprotein Podoplanin in the Progression of Cutaneous Squamous Cell Carcinoma

The incidence of cutaneous squamous cell carcinoma (cSCC) is increasing each year. Although early stages of cSCC can still be treated successfully, late stage tumors invade into the surrounding tissue and metastasize to distant body sites, which is associated with a poor prognosis. Strong upregulation of the transmembrane glycoprotein Podoplanin (PDPN) has been demonstrated in various tumor entities including cSCC. Enhanced PDPN expression was correlated to poor cSCC patients’ outcome, but its function in skin cancer progression is still unknown. Therefore, my study aimed to define the impact of PDPN on cSCC carcinogenesis. The descriptive analysis of differentiated cSCC patients’ material showed PDPN overexpression in tumor cells and increased expression in activated fibroblasts, particularly at the invasive front of the tumor. To functionally examine the importance of PDPN in cSCC tumor cells, PDPN was deleted in human and murine cSCC cell lines by the CRISPR/Cas9 technology followed by functional in vitro assays. Human and murine cells harboring a PDPN knockout (KO) genotype showed similar morphology and proliferative behavior as control cells in monolayer culture system. Performing both the Boyden Chamber assay and the most advanced three-dimensional organoid skin cancer model demonstrated that the loss of PDPN in human cells neither affected transmigration nor invasion. Strikingly, murine Pdpn KO cells transmigrated and invaded with less efficiency in both approaches. The invasive phenotype of murine tumor cells was further characterized to be independent of epithelial-mesenchymal transition (EMT). An initial examination of one previously described PDPN downstream pathway revealed that ezrin recruitment and cytoskeletal remodeling were not impaired upon Pdpn KO in 2D culture. Complementing these in vitro approaches, labeled murine control and Pdpn KO cells were injected orthotopically into the dermis of nude mice, to define the role of PDPN in tumor progression in vivo. Formed tumors derived from control and Pdpn KO cells displayed a well-differentiated morphology with a PDPN-positive reaction in fibroblasts in the tumor stroma recapitulating human cSCC. Smaller tumors as well as delayed tumor outgrowth were observed upon Pdpn loss, which was due to reduced tumor cell proliferation in vivo. Moreover, abrogation of Pdpn resulted in diminished EMT-independent tumor cell invasion. In conclusion, this study underscores the crucial impact of PDPN in cSCC progression. Future studies are required in order to characterize underlying molecular mechanisms that could propose potential therapeutic strategies targeting PDPN-dependent tumor cell invasion especially in late-stage cSCC patients