Delta-Like Ligand–Notch1 Signaling Is Selectively Modulated by HPV16 E6 to Promote Squamous Cell Proliferation and Correlates with Cervical Cancer Prognosis

Abstract

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. An RNA sequencing–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 low-grade 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. Significance: This study investigates cervical cancer cell-of-origin populations and describes a DLL–Notch1 phenotype that is associated with disease prognosis and that might help identify cells that are susceptible to HPV-induced carcinogenesis.

p63 Directs Subtype-Specific Gene Expression in HPV+ Head and Neck Squamous Cell Carcinoma

“…As expected, several SEs were associated with master TFs, including p63, but we also observed SEs associated with genes important to HPV infection and HPV-induced carcinogenesis, such as WNT7A , ITGA2 , and NOTCH1 . ( Figure 3B and Supplementary Figure 6B ; Supplementary Table 3 ) ( 62 , 63 ). Motifs for TFs with known roles in HNSCC, including FOSL1, E2F1, and E2F7 ( 10 , 64 ), were also enriched at SCC104 SEs ( Figure 3C and Supplementary Figure 6C ).…”

The Reservoir of Persistent Human Papillomavirus Infection; Strategies for Elimination Using Anti-Viral Therapies

“…Activation of the canonical Notch signalling pathway is a central regulator for basal cell commitment to differentiation, while differential interaction with ligands can define cell fate during embryonic development [72]. The hrHPV16 E6 protein not only downregulates total Notch receptor expression levels in a p53-dependent manner [57], but also changes the surface ligand distribution to favour DLL4 expression [73]. These molecular modifications make sense when considered with the phenotypes of increased proliferation and a delayed commitment to differentiation mediated by hrHPV16 E6 expression.…”

Shooting at Moving and Hidden Targets—Tumour Cell Plasticity and the Notch Signalling Pathway in Head and Neck Squamous Cell Carcinomas

“…Nevertheless, many tumour-associated cells, such as fibroblasts, immune cells, endothelial cells in capillaries and blood vessels, express both Notch ligands and receptors simultaneously. This is relatively well investigated in HNSCC; in particular the expression of Jagged1 and -2 in endothelial cells/capillaries within the tumour tissue [ 293 , 294 ], or of DLL4 [ 35 , 295 , 296 ]. These cell–cell interactions specifically activate Notch signalling pathways through direct mechanical contact, mostly by heterotypic or trans-signalling.…”