Papillomaviruses are small, ubiquitous DNA viruses that infect a variety of keratinizing and mucosal epithelia. While all HPVs produce benign lesions, a small subset of high risk (HR) HPVs cause lesions that can progress to cancer. A hallmark of nearly all cancers is that they have activated an enzyme, telomerase, which maintains the repetitive DNA sequences at the end of chromosomes, allowing tumor cells to replicate indefinitely. We have shown that the HR HPV E6 proteins directly activate the expression of the catalytic subunit of telomerase, hTERT. E6 coordinates the recruitment of transcriptional activators and elimination of repressors at the hTERT promoter, and further enhances TERT levels through post-transcriptional means. The overall goal of this application is to continue to explore the mechanisms by which HR HPV E6 proteins regulate telomerase activity, to determine whether these mechanisms are unique to HPV or play a role in other cancers, to explore the benefit of hTERT activation to the HPV lifecycle, and to determine whether telomerase-independent activities of hTERT influence the biology of HPVs.
Our specific aims are:: 1) To characterize mechanisms by which E6 regulates hTERT transcription. We will focus on candidates obtained in a novel screen for which there are predicted binding sites in the -725 to +61 bp promoter. Additional candidates predicted to bind but not found in the screen will be tested. We propose to determine the mechanisms by which E6 affects the recruitment or elimination of these factors 2. To examine the generalizability of hTERT regulation by E6. We propose to examine a set of transcriptional activators and repressors shown to regulate hTERT in HPV E6 -HFKs in a variety of non-HPV tumor cell lines. 3. To determine the role of hTERT in the HPV lifecycle. To determine whether HPVs require hTERT for some stage in their lifecycle we will knockdown hTERT in cells into which the entire HPV genome has been transfected. Using both proliferative and differentiation-promoting growth conditions we will measure episome maintenance, viral DNA amplification, late gene expression, and epithelial differentiation. 4. To determine if telomerase-independent functions of hTERT affect the HPV lifecycle. First, we will determine whether induction of TERT is linked to Wnt signaling in HFKs and whether Wnt signaling is important for the HPV lifecycle. Secondly, we will explore the new finding that TERT associates with RMRP RNA, which is processed into siRNAs. We will test the hypothesis that siRNAs generated by this complex regulate critical features of HPV expressing cells.

Public Health Relevance

Human papillomaviruses (HPVs) are ubiquitous sexually transmitted viral infections, that have the potential to cause anogenital and oropharyngeal cancers, resulting in 300,000 deaths annually. Despite the optimism that has accompanied the introduction of prophylactic vaccines to prevent some HPV infections, the relatively modest uptake of the vaccine, especially in the developing world, and the very high fraction of men and women who are already infected, means that HPV-associated disease will remain a significant public health problem for decades. Understanding the mechanisms by which HPVs induce telomerase, a critical factor in malignancy, and how induction of hTERT affects the HPV lifecycle may provide important targets to limit HPV neoplasia and will result in new insights into HPV pathogenesis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA064795-18
Application #
8468653
Study Section
Special Emphasis Panel (ZRG1-IDM-A (03))
Program Officer
Read-Connole, Elizabeth Lee
Project Start
1994-12-29
Project End
2016-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
18
Fiscal Year
2013
Total Cost
$501,980
Indirect Cost
$216,764
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109
Wallace, Nicholas A; Robinson, Kristin; Howie, Heather L et al. (2015) β-HPV 5 and 8 E6 disrupt homology dependent double strand break repair by attenuating BRCA1 and BRCA2 expression and foci formation. PLoS Pathog 11:e1004687
Galloway, Denise A; Laimins, Laimonis A (2015) Human papillomaviruses: shared and distinct pathways for pathogenesis. Curr Opin Virol 14:87-92
Wallace, Nicholas A; Galloway, Denise A (2015) Novel Functions of the Human Papillomavirus E6 Oncoproteins. Annu Rev Virol 2:403-23
Wallace, Nicholas A; Robinson, Kristin; Galloway, Denise A (2014) Beta human papillomavirus E6 expression inhibits stabilization of p53 and increases tolerance of genomic instability. J Virol 88:6112-27
Wallace, Nicholas A; Galloway, Denise A (2014) Manipulation of cellular DNA damage repair machinery facilitates propagation of human papillomaviruses. Semin Cancer Biol 26:30-42
Carter, Joseph J; Daugherty, Matthew D; Qi, Xiaojie et al. (2013) Identification of an overprinting gene in Merkel cell polyomavirus provides evolutionary insight into the birth of viral genes. Proc Natl Acad Sci U S A 110:12744-9
Xu, Mei; Katzenellenbogen, Rachel A; Grandori, Carla et al. (2013) An unbiased in vivo screen reveals multiple transcription factors that control HPV E6-regulated hTERT in keratinocytes. Virology 446:17-24
Wallace, Nicholas A; Gasior, Stephen L; Faber, Zachary J et al. (2013) HPV 5 and 8 E6 expression reduces ATM protein levels and attenuates LINE-1 retrotransposition. Virology 443:69-79
Wang, Myra L; Walsh, Ryan; Robinson, Kristin L et al. (2011) Gene expression signature of c-MYC-immortalized human fibroblasts reveals loss of growth inhibitory response to TGFβ. Cell Cycle 10:2540-8
Carter, Joseph J; Galloway, Denise A (2010) Stopping HPVs dead in their tracts. Cell Host Microbe 8:221-2

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