Delta-like ligand-notch1 signaling is selectively modulated by HPV16 e6 to promote squamous cell proliferation and correlates with cervical cancer prognosis

Human papillomavirus (HPV) drives high-grade intraepithelial neoplasia and cancer; for unknown reasons, this occurs most often in the cervical transformation zone. Either mutation or HPV E6- driven inhibition of Notch1 can drive neoplastic development in stratified squamous epithelia. However, the contribution of Notch1 and its Delta-like ligands (DLL) to site susceptibility remains poorly understood. Here, we map DLL1/DLL4 expression in cell populations present in normal cervical biopsies by immunofluorescence. In vitro keratinocyte 2D monolayer models, growth assays, and organotypic raft cultures were used to assess the functional role of DLL-Notch signaling in uninfected cells and its modulation by HPV16 in neoplasia. AnRNAsequencing-based gene signature was used to suggest the cell of origin of 279 HPV-positive cervical carcinomas from The Cancer Genome Atlas and to relate this to disease prognosis. Finally, the prognostic impact of DLL4 expression was investigated in three independent cervical cancer patient cohorts. Three molecular cervical carcinoma subtypes were identified, with reserve cell tumors the most common and linked to relatively good prognosis. Reserve cells were characterized as DLL1-/DLL4+, a proliferative phenotype that is temporarily observed during squamous metaplasia and wound healing but appears to be sustained by HPV16 E6 in raft models of lowgrade and, more prominently, high-grade neoplasia. High expression of DLL4 was associated with an increased likelihood of cervical cancer-associated death and recurrence. Taken together, DLL4- Notch1 signaling reflects a proliferative cellular state transiently present during physiologic processes but inherent to cervical reserve cells, making them strongly resemble neoplastic tissue even before HPV infection has occurred.