Dr. Denise A. Galloway

Cervical carcinoma is an attractive model system to study the pathways that are disrupted in epithelial cell neoplasms for several reasons. First, a critical step in its etiology is infection with high risk papillomaviruses, resulting in persistent expression of the viral oncoproteins, E6 and E7. By identifying the targets of E6 and E7 we can reveal key regulatory pathways that control proliferation, senescence and apoptosis. These pathways are also targets of somatic mution in other epithelial tumors. Secondly, development of cervical cancer occurs over decades and the precursor intraepithelial lesions can be readily obtained. Analysis of the staged clinical lesions, coupled with ectopic expression of E6 and E7 in otherwise normal cells, allows us to distinguish the proximal consequences of E6/E7 expression from the myriad changes that result from genetic instability. Additionally the requirement for HPV infection and gene expression in anogenital malignancy provides a clear target for prophylactic and therapeutic immune intervention.

A major part of our labÂ’s effort is to understand the mechanism by which E6 and E7 contribute to the neoplastic process. E7 binds to the retinoblastoma tumor suppressor, pRb, disrupting Rb/E2F complexes. Additionally, E7 targets Rb for degradation through mechanisms that are not yet understood. We have shown that inactivating Rb is necessary but not sufficient to overcome senescence and other growth arrest signals. We have found that the C-terminus of E7 encodes Rb-independent activities that can bypass p21-mediated arrest.

E6 binds to a ubiquitin ligase, E6AP, and targets the cellular tumor suppressor p53 for rapid elimination. Surprisingly, elimination of p53 was not required for immortalization; instead our data has shown that the critical function of E6 in immortalization of epithelial cells is activation of telomerase. We have shown that E6 induces transcription of the catalytic subunit of telomerase, hTERT. TERT activation by E6 requires intact E boxes but is not due to increasing the levels of MYC. Elimination of E6AP by RNAi showed that it plays an essential role in E6 transactivation of TERT. Further we have identified a novel E6/E6AP partner, NFX1, that modulates TERT transcription. NFX1 has two isoforms: NFX1-91 binds to a X-box motif adjacent to the proximal E box and represses transcription. In E 6 expressing cells E6/E6AP targets NFX1-91 for ubiquitin mediated degradation. NFX1-123 coactivates Myc transactivation of the promoter. We are pursuing three avenues of investigation: 1) to more precisely characterize the mechanisms by which NFX1 represses and activates hTERT transcription: 2) to determine whether NFX1 plays a role in regulating TERT in non-HPV containing tumors; and 3) to investigate reasons why HPVs may induce TERT or inactivate NFX1 including seeking non-telomere dependent functions of hTERT, and identifying other genes regulated by NFX1.

We are also interested in mechanisms of cellular senescence that have tumor suppressive effects. We have shown that ras-induced senescence is determined by the levels of p16INK4A, with cells that have low levels of p16 responding by proliferation rather than arrest. We have also showed that Myc can immortalize cells, but in the absence of the Werner helicase, WRN, cells senesce in response to Myc. We are investigating the pathways that mediate senescence.

In order to better understand the development of anogenital malignancy and the natural history of HPV infection, we have developed serological assays using viral particles, made by self-assembly of the L1 capsid protein. These studies have shown that infection with genital HPVs is extremely common, that antibodies develop slowly in infected individuals, persist for decades and are likely protective against reinfection with the same type. Current efforts are focused on identifying immunoreactive epitopes that are recognized in response to natural infection and vaccination with VLPs. Our data show that there is no single immunodominant epitope and that the response is likely complex and polyclonal. Additionally we are interested in studying the role that mucosal antibody plays in limiting or preventing infection, and in collaboration with immunologists, to begin to identify and characterize the host immune response to HPV infection.