This proposal investigates the interactions of human papillomavirus type 16 (HPV16) oncoproteins and the transforming growth factor-beta (TGF-beta) signaling pathway that lead to TGF-beta resistance of HPV16-immortalized human keratinocytes (HKc/HPV16) at late stages of premalignant progression in vitro. In addition, the molecular basis for the marked inhibition of HPV16 P97 promoter activity by TGF-beta will be investigated. Our preliminary results and those of others show that the HPV16 oncoproteins E6 and E7 and the TGF-beta signaling pathway exist in a balance, in which they exert opposite effects on each other and on cell proliferation: TGF-beta inhibits HPV16 early gene transcription, lowering the levels of E6/E7 in cells immortalized by full-length HPV16 DNA. On the other hand, E6/E7 inhibit TGF-beta signaling, inducing resistance to the growth inhibitory properties of TGF-beta.
Our specific aims are designed to investigate the details of these interactions in our in vitro model for HPV16-mediated multistep carcinogenesis of HKc. Progression of HKc/HPV16 toward a malignant phenotype in culture is constantly associated with the acquisition of TGF-beta resistance culminating, at late stages, with a loss of expression of the TGF-beta receptor type I. Acute expression of HPV1 6 E6/E7 from retroviral vectors produces an increase in Smad3 levels. Smad3 levels are even higher in established HKc/HPV16 lines, and increase further during progression. Stabilization and inactivation of Smad3, or binding and inactivation of phospho-Smad3 are two of the possible mechanisms by which E7 and perhaps also E6 may inhibit TGF-beta signaling. We propose to investigate in detail the mechanisms by which HPV16 E6/E7 inhibit TGF-beta activities, beginning with a mutagenesis analysis of E6 and E7, to determine the regions/activities of E6/E7 needed for this effect. We will also investigate the basis for the loss of TGF-beta receptor type I at late stages of in vitro progression of HKc/HPV16. In addition, we have defined the TGF-beta-responsive segment of the HPV16 upstream regulatory region, determined that NF-l binding sites mediate most of TGF-beta inhibition, and formulated a plan to further investigate the inhibition of this promoter by TGF-beta.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA089502-03
Application #
6725504
Study Section
Virology Study Section (VR)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
2002-05-20
Project End
2007-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
3
Fiscal Year
2004
Total Cost
$258,100
Indirect Cost
Name
University of South Carolina at Columbia
Department
Pathology
Type
Schools of Medicine
DUNS #
111310249
City
Columbia
State
SC
Country
United States
Zip Code
29208
Xu, Hanwen; Pirisi, Lucia; Creek, Kim E (2015) Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT. Virology 474:144-53
Kowli, Sangeeta; Velidandla, Rupa; Creek, Kim E et al. (2013) TGF-? regulation of gene expression at early and late stages of HPV16-mediated transformation of human keratinocytes. Virology 447:63-73
Chen, Yi; Pirisi, Lucia; Creek, Kim E (2013) Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer. Virology 444:100-8
Altomare, Diego; Velidandla, Rupa; Pirisi, Lucia et al. (2013) Partial loss of Smad signaling during in vitro progression of HPV16-immortalized human keratinocytes. BMC Cancer 13:424
Hypes, Melissa K; Pirisi, Lucia; Creek, Kim E (2009) Mechanisms of decreased expression of transforming growth factor-beta receptor type I at late stages of HPV16-mediated transformation. Cancer Lett 282:177-86
Wan, Fang; Miao, Xijiang; Quraishi, Iram et al. (2008) Gene expression changes during HPV-mediated carcinogenesis: a comparison between an in vitro cell model and cervical cancer. Int J Cancer 123:32-40
Bheda, A; Creek, K E; Pirisi, L (2008) Loss of p53 induces epidermal growth factor receptor promoter activity in normal human keratinocytes. Oncogene 27:4315-23
Baldwin, Amy; Pirisi, Lucia; Creek, Kim E (2004) NFI-Ski interactions mediate transforming growth factor beta modulation of human papillomavirus type 16 early gene expression. J Virol 78:3953-64