Beta human papillomavirus E6: a menace to genomic integrity

Doctor of PhilosophyDepartment of BiologyNicholas WallaceBeta genus human papillomaviruses (β-HPV) are associated with the development of cutaneous squamous cell carcinomas (cSCC) by destabilizing the genome. In vitro and in vivo studies indicate the β-HPV E6 and E7 proteins act as co-factors with ultraviolet radiation (UV) to cause genome destabilization. However, the E6 protein from β-HPV type 8 (8 E6) induces tumor formation in mice without UV exposure, but the mechanisms driving carcinogenesis are unclear. In this dissertation, we investigated UV-independent mechanisms of HPV8 E6-induced genome destabilization. In silico screens validated by cell line characterization showed that 8 E6 deregulated the Hippo pathway by destabilizing the histone acetyltransferase, p300. Hippo pathway disruption increased cell proliferation and attenuated cell death in response to failed cytokinesis. While 8 E6 alone was unable to promote long-term proliferation after cytokinesis failure, we demonstrated that 8 E6 combined with TERT expression rescued long-term proliferation. However, this resulted in increased genomic instability in the form of ploidy changes. Furthermore, we showed 8 E6 decreased the abundance of anaphase bridge resolving helicase, Bloom syndrome protein (BLM). The diminished BLM was associated with increased segregation errors and micronuclei. 8 E6 reduced antiproliferative responses to micronuclei and time-lapse imaging revealed 8 E6 promoted cells with micronuclei to complete mitosis. Finally, whole-genome sequencing demonstrated that 8 E6 induced a mutational phenomenon known as chromothripsis, in 9 chromosomes. Overall, the findings from my dissertation provide insight into the processes by which 8 E6 induces genome instability in the absence of UV exposure

Beta-Genus Human Papillomavirus 8 E6 Destabilizes the Host Genome by Promoting p300 Degradation

The beta genus of human papillomaviruses infects cutaneous keratinocytes. Their replication depends on actively proliferating cells and, thus, they conflict with the cellular response to the DNA damage frequently encountered by these cells. This review focus on one of these viruses (HPV8) that counters the cellular response to damaged DNA and mitotic errors by expressing a protein (HPV8 E6) that destabilizes a histone acetyltransferase, p300. The loss of p300 results in broad dysregulation of cell signaling that decreases genome stability. In addition to discussing phenotypes caused by p300 destabilization, the review contains a discussion of the extent to which E6 from other β-HPVs destabilizes p300, and provides a discussion on dissecting HPV8 E6 biology using mutants

β-HPV 8E6 Attenuates ATM and ATR Signaling in Response to UV Damage

Given the high prevalence of cutaneous genus beta human papillomavirus (β-HPV) infections, it is important to understand how they manipulate their host cells. This is particularly true for cellular responses to UV damage, since our skin is continually exposed to UV. The E6 protein from β-genus HPV (β-HPV E6) decreases the abundance of two essential UV-repair kinases (ATM and ATR). Although β-HPV E6 reduces their availability, the impact on downstream signaling events is unclear. We demonstrate that β-HPV E6 decreases ATM and ATR activation. This inhibition extended to XPA, an ATR target necessary for UV repair, lowering both its phosphorylation and accumulation. β-HPV E6 also hindered POLη accumulation and foci formation, critical steps in translesion synthesis. ATM’s phosphorylation of BRCA1 is also attenuated by β-HPV E6. While there was a striking decrease in phosphorylation of direct ATM/ATR targets, events further down the cascade were not reduced. In summary, despite being incomplete, β-HPV 8E6’s hindrance of ATM/ATR has functional consequences

Cervical cancer cell lines are sensitive to sub-erythemal UV exposure

High risk human papillomavirus (HPV) infections are the causative agent in virtually every cervical cancer as well as a host of other anogenital and oropharyngeal malignancies. These viruses must activate DNA repair pathways to facilitate their replication, while avoiding the cell cycle arrest and apoptosis that can accompany DNA damage. HPV oncoproteins facilitate each of these goals, but also reduce genome stability. Our data dissect the cytotoxic and cytoprotective characteristics of HPV oncogenes in cervical cancer cells. These data show that while the transformation of keratinocytes by HPV oncogene leaves these cells more sensitive to UV, the oncogenes also protect against UV-induced apoptosis. Cisplatin and UV resistant cervical cancer cell lines were generated and probed for their sensitivity to genotoxic agents. Cervical cancer cells can acquire resistance to one DNA crosslinking agent (UV or cisplatin) without gaining broad tolerance of crosslinked DNA. Further, cisplatin resistance may or may not result in sensitivity to PARP1 inhibition