Longitudinal Keratinocyte Proliferation Induced by Human Papillomavirus 16E6 and NFX1-123 Partnership

Abstract

High-risk human papillomaviruses (HPV) cause cervical, anogenital, and head and neck cancers. Despite the availability of preventive HPV vaccines, their poor uptake leaves most men and women at risk for these cancers, many of which remain common globally and others that are increasing domestically. The HPV 16 E6 (16E6) protein plays a significant role in inducing and maintaining cellular transformation of its infected host cell; however, 16E6 itself has no enzymatic activity and carries out most of its functions through partnerships with host endogenous proteins. Previously, it was demonstrated that 16E6 binds to the host protein NFX1-123, and NFX1-123 expression is increased in HPV 16 positive cervical cancer cell lines and primary cancers compared to normal tissues. In this thesis, we quantify the growth rates of 16E6-expressing keratinocytes with endogenous or overexpressed NFX1-123 (16E6/vec and 16E6/FN123, respectively) levels by measuring population doublings and interrogate whether proteome expression changes occur early or late in longitudinal growth assays that began with higher levels of NFX1-123. Early passage 16E6/vec and 16E6/FN123 cells showed similar growth rates; however, late passage 16E6/FN123 cells had accelerated growth and greater population doublings than the 16E6/vec cells. Mass spectrometry revealed similar proteomes of both cell lines at early passages. In contrast, late passaged cells had significantly higher amounts of differentially expressed protein among 16E6/FN123 and 16E6/vec cells. This indicates a unique proteomic landscape induced by the 16E6/NFX1-123 partnership. Pathway analysis vii showed increased positive telomere regulation, DNA repair, and DNA replication pathways in the late passage 16E6/FN123 cells compared to 16E6/vec. These findings indicate that the 16E6 and NFX1-123 partnership, especially with higher levels of NFX1-123, alters the longitudinal cellular environment in a manner that may initiate a preneoplastic phenotype. Additionally, the work detailed in this dissertation provides the first evidence of NFX1-123 being a direct RNA-binding protein giving new insights into the endogenous roles of the protein.