Contact Information: Professor Dennis J. McCance University of Rochester Medical Center 601 Elmwood Ave, Box 672 (Room 3-9812) Phone: (585) 275-0101 Fax: (585) 473-9573 E-Mail: djmc@mail.rochester.edu
Research: Molecular Pathogenesis of Human Papillomaviruses.

In vivo infection with the oncogenic types 16 and 18 causes inhibition of differentiation of the epithelium resulting in a premalignant lesion. The natural target cell of HPV is the keratinocyte. In vitro studies using a novel system for keratinocyte differentiation have shown that keratinocytes transfected with HPV 16 DNA failed to differentiate normally. Morphologically, the abnormal cells observed are similar to those of dysplasia associated with HPV infection in vivo. Two early genes, E6 and E7, are sufficient to immortalize and inhibit differentiation of human keratinocytes. These two early genes interact with two cellular factors p53 and the retinoblastoma gene product (Rb). E6 binds p53 and degrades it through the ubiquitin pathway, while E7 binds RB.

We have recently shown that Rb can bind to the AP-1 family of transcription factors and activate transcription from an AP-1 regulated promoter. This may be an important pathway for keratinocyte differentiation since the AP-1 factors have been known for some time to be up-regulated during differentiation and there is increasing evidence for a role of Rb in the differentiation of other cells types including muscle and fat cells. We have extensively studied the interaction of c-Jun, a major AP-1 factor, and Rb and have found that the complex is present during the early stages of differentiation of keratinocytes, but absent in cycling cells. Interestingly HPV-16 E7 is able to inhibit c-Jun-induced transcription by Rb and the Rb binding domain of E7 is essential for this inhibition. We have found that a certain subset of AP-1 factors are active during keratinocyte differentiation and are investigating the effects of Rb and E7 on the activity of these different homo- and hetero-dimers. Using a variety of assays including ChIP (chromatin immunoprecipitation) and DAPA (DAN affinity precipitation assay), we are investigating the recruitment of AP-1 factors and pRb to keratinocyte differentiation specific promoters. In addition,, we are determining the effects of post translational modifications such as acetylation and/or phosphorylation on the AP-1/pRb complexes present during keratinocyte differentiation.

The cellular response to oncogenic Ras depends upon the presence or absence of cooperating mutations. In the absence of immortalizing oncogenes or genetic lesions, activation of the Ras/Raf pathway results in a p21^Cip1-dependent cellular arrest. HPV-16 E7 transforms primary cells in cooperation with Ras and abrogates p21^Cip1-mediated growth arrest in the presence of various antimitogenic signals. We have utilized a conditional Raf molecule to investigate the effects of E7 on p21^Cip1 function in the context of Raf-induced cellular arrest. E7 bypassed Raf-induced arrest and alleviated inhibition of cyclin E-CDK2 without suppressing Raf-specific synthesis of p21^Cip1 or derepressing p21^Cip1-associated CDK2 complexes. Activation of Raf led to nuclear accumulation of p21^Cip1, and we provide evidence that this effect is mediated by inhibition of Akt, a regulator of p21^Cip1 localization. Loss of Akt activity appears to be an important event in the cellular arrest associated with Raf-induction, since maintenance of Akt activity was necessary and sufficient to bypass Raf-induced arrest. In agreement, expression of E7 sustained Akt activity and reduced nuclear accumulation of p21^Cip1, resulting in decreased association between p21^Cip1 and cyclin E-CDK2. Taken together, these data suggest that E7 inhibits p21^Cip1 function in the context of Raf signaling by altering Raf-Akt antagonism and preventing the proper subcellular localization of p21^Cip1. We propose that E7 elicits a proliferative response to Raf signaling by targeting p21^Cip1 function via a novel mechanism. Work is continuing to investigate how E7 modulates Akt activity and which of the three Akt family members is involved.

We have previously reported that HPV-16 E6 binds to C/H1, C/H3 and the C-terminus domains of co-activators p300/CBP, causing the modulation of the transcription of certain genes controlled by NF-kB (p65 or relA) and p53. To establish the biological significance of these observations, we have focused on the transcriptional regulation of interleukin 8 (IL8), a potent chemo-attractant for T lymphocytes and neutrophils, which is also essential for the initiation of the local immune response. The IL8 promoter is regulated by NF-kB/p65 in response to TNF-a and requires the co-operation of the co-activators CBP/p300 and steroid receptor co-activator 1 (SRC-1) and the CBP-associated factor (P/CAF) for optimal activation. We show, that in the presence of HPV-16 E6, the promoter activity of IL8 was repressed. Moreover, from the mutational analysis of the IL8 promoter, we found that E6 down-regulates the IL8 promoter activity through the NF-kB/p65 binding site. This inhibition appears to result from the ability of HPV-16 E6 to compete with NF-kB/p65 and SRC-1 for binding to the N-terminus and C-terminus of CBP respectively. Reporter data also showed that E7 represses IL8 promoter activity, though to a lesser extent than E6, but like E6, the repression by E7 is through the NF-kB/p65 binding site. E7 was shown for the first time to bind to P/CAF and the binding was necessary for the down regulation of the IL8 promoter. E6 and E7 together inhibited transcription of the IL8 promoter to a greater extent than either alone. Finally, by RNase Protection Assay (RPA), we showed that the synthesis of endogenous IL8 mRNA was repressed in keratinocytes stably expressing E6 and E7. Taken together, the results provide evidence that E6 and E7 can cooperatively disrupt IL8 transcription through disruption of transcriptional active complexes and this may have important consequences for immune responses in infected hosts. We are pursuing other lines of investigation to elucidate other immune regulators that may be modulated by E6 and E7.

We are using micro-array analysis to investigate genes that are regulated by E6 and E7 during keratinocyte differentiation. These studies are at an early stage but we have found a number of genes that E6 and/or E7 up- or down-regulated during differentiation. Some of these genes are ones that we, and others have investigated over the years, validating that micro-arrays can identify physiologically relevant gene regulation. We also have identified groups of functionally related genes that are affected by the viral proteins. We are continuing to document differences in expression of these genes and hope to uncover novel pathways modulated by E6 and E7.

Nyugen, D.X, Westbrook, T.F, McCance, D.J. (2002) HPV 16 E7 maintains levels of the cdc25A tyrosine phosphatase during deregulation of cell cycle arrest. J. Virol. 76: 619-632.

Huang, S-H., McCance, D.J. (2002) Downregulation of the interleukin 8 promoter by HPV-16 E6 and E7 through effects on CBP/p300 and P/CAF. J. Virol. 76: 8710-8721.

Westbrook, T,F., Nyugen, D.X., Thrash, B., McCance, D.J. (2002) E7 Abrogates Raf-induced Arrest by mislocalization of p21^CIP1. Mol. Cell Biol. 22: 7041-7052.

Book:

Human Papillomaviruses. Ed. Dennis J. McCance. Elsevier Press, 2002.