HPV and Cervical Cancer
Cervical cancer is the most common cancer for women worldwide, with a mortality rate of approximately 50 percent. This disease is almost always caused by high-risk strains of the human papillomavirus, a sexually transmitted agent, and disproportionately affects minority populations. HPV codes for E6 and E7, two oncogenes that work together to transform cells and cause cancer. Funded by an R01 award from the NIH, the Duerksen-Hughes laboratory has discovered that one of the ways in which E6 contributes to HPV oncogenicity is by helping the virus to evade the host immune response.
E6 functions, at least in part, by protecting the cells that express it from host-generated apoptotic responses such as those triggered by TNF, Fas, and TRAIL. Each of these apoptotic pathways involves several signaling molecules, and E6 works by binding to some, though not all, of them. One possible consequence of such binding is the blocking of signal transmission, such as we observed following the binding of E6 to the TNF receptor TNF R1. This prevents TNF R1 from binding to the next molecule in the pathway, TRADD. Another possible consequence is the degradation of signaling intermediates, noted following the binding of E6 to FADD or to procaspase 8. We are working to further define these interactions with a long-term goal of developing small molecule inhibitors of such binding, which would have the potential to function as therapeutic reagents.
We are also exploring the possibility that differences in the proportions of the different HPV types that infect individuals of various ethnicities could contribute to the health disparity seen in this disease, as our laboratory is also associated with the CHDMM.
Interestingly, E6 occurs in two versions due to alternative splicing. These two splicing variants offer the virus additional diversity in immune evasion mechanisms. For example, while the full-length version is required to protect cells from apoptosis triggered through Fas, both the truncated and the full-length versions can provide protection from TNF. Also, while both versions bind to procaspase 8, only the full-length version accelerates its degradation. We are currently working to further define the roles played by these two splice variants.
Finally, we have used microarray analysis to discover that E6 affects the cellular responses to apoptotic signals induced by DNA damage by changing the expression patterns of genes involved in the early response, and these studies have identified a number of potential new targets for the development of chemotherapeutic reagents. Together, our results indicate that HPV 16 has acquired a number of mechanisms designed to thwart host-triggered apoptosis and to ensure the survival of the virus and its host cell. A clear understanding of these host-virus interactions will facilitate efforts to develop novel and effective therapeutic approaches.
A second area of interest in the Duerksen-Hughes laboratory involves the ceramide-mediated signaling pathway. Ceramide serves as a signal molecule, participating in the regulation of major cellular events such as proliferation, apoptosis and differentiation. Using a tumor cell model, we found that we could increase ceramide levels by manipulating its metabolic pathways, and that these increased levels could in turn mediate cell death, suggesting this pathway as a fruitful area for future drug discovery. In addition, we are exploring the possibility that ceramide-mediated cell death may be involved in the development of Alzheimer’s Disease and other neuropathologies. Preliminary results show higher levels of ceramide in brain samples from patients with neuropathologies, suggesting that this pathway may be useful for both diagnosis and treatment.
Regulation of Splicing
We are also studying the regulation of spice activity and alternative splicing. A riboprotein complex known as the spliceosome carries out the splicing of pre-mRNA. A number of factors are involved in regulating spliceosome activity and specificity, including members of the SR splice factor family. We have found that in cells stressed by DNA damage, expression of several SR factors changes, and that in some cases, these changes are regulated, at least in part, by the p53 tumor suppressor. This link between p53 and splice factor expression may provide a molecular explanation for the fact that splicing regulation is often affected during carcinogenesis.
1. Haynes, T.-A. S., V. Filippov, M. Filippova, J. Yang, K. Zhang, and P. J. Duerksen-Hughes. DNA damage induces down-regulation of UDP-Glucose Ceramide Glucosyltransferase, increases ceramide levels and triggers apoptosis in p53-deficient cancer cells. BBA – Molecular and Cell Biology of Lipids, 1821:943-953, 2012. PMID: 22349266
2. Filippov, V., M.A. Song, K. Zhang, H. V. Vinters, S. Tung, W. M. Kirsch, J. Yang, and P. J. Duerksen-Hughes. Increased Ceramide in brains with Alzheimer’s and other neurodegenerative diseases. Journal of Alzheimer’s Disease, 29:537-547, 2012. PMID: 22258513
3. Yuan, C.-H., M. Filippova, S. S. Tungteakkhun, P. J. Duerksen-Hughes and J. L. Krstenansky. Small molecule inhibitors of the HPV16-E6 interaction with caspase 8. Bioorganic and Medicinal Chemistry Letters, 22:2125-2129. 2012. PMID: 22300659
4. Zhang, K., T.-A. S. Haynes, M. Filippova, V. Filippov and P. J. Duerksen-Hughes. Quantification of ceramide levels in mammalian cells by high performance liquid chromatography coupled to tandem mass spectrometry with multiple-reaction-monitoring mode (HPLC-MS/MS-MRM). Analytical Methods 3:1193-1197, 2011.
5. Whitaker, E. L., V. Filippov, M. Filippova, C. F. Guerrero-Juarez, and P. J. Duerksen-Hughes. Splice variants of mda-7/IL-24 differentially affect survival and induce apoptosis in U2OS cells. Cytokine, 56:272-281, 2011. PMID: 21843952
6. Tungteakkhun, S. S., M. Filippova, N. Fodor and P. J. Duerksen-Hughes. The Full Length Isoform of HPV 16 E6 and its Splice Variant E6* Bind to Different Sites on Procaspase 8 DED. J. Virol. 84:1453-1463, 2010. PMID: 19906919
7. Filippova, M., V. A. Filippov, M. Kagoda, T. Garnett, N. Fodor and P. J. Duerksen-Hughes. Complexes of Human Papillomavirus 16 E6 Proteins Form Pseudo-DISC Structures During TNF-Medicated Apoptosis. J. Virol. 83:210-227, 2009. PMID: 18842714
8. Tungteakkhun, S., M. Filippova, J. W. Neidigh, N. Fodor and P. J. Duerksen-Hughes. The Interaction Between HPV 16 E6 and FADD is Mediated by a Novel E6 Binding Domain. J. Virol. 82:9600-9614, 2008. PMID: 18632871
9. Filippov, V., E. L. Schmidt, M. Filippova and P. J. Duerksen-Hughes. Splicing and splice factor SRp55 participate in the response to DNA damage by changing isoform ratios of target genes. Gene. 420:34-41, 2008. PMID: 18571879
10. Haynes, T.-A S., P. J. Duerksen-Hughes, M. Filippova, V. Filippov and K. Zhang. C(18) Ceramide Analysis in Mammalian Cells Employing Reversed-Phase High Performance Liquid Chromatography Tandem Mass Spectrometry. Analytical Biochemistry. 378:80-86, 2008. PMID: 18423390
11. Filippov, V., M. Filippova and P. J. Duerksen-Hughes. The Early Response to DNA Damage Can Lead to Activation of Alternative Splicing Activity Resulting in CD44 Splice Pattern Changes. Cancer Research. 67:7621-7630, 2007. PMID: 17699766
12. Garnett, T. O., M. Filippova and P. J. Duerksen-Hughes. Bid is Cleaved Upstream of Caspase-8 Activation During TRAIL-Mediated Apoptosis in Human Osteosarcoma Cells. Apoptosis. 12:1299-1315, 2007. PMID: 17431792
13. Filippova, M., M. M. Johnson, M. Bautista, V. Filippov, N. Fodor, S. S. Tungteakkhun, K. Williams and P. J. Duerksen-Hughes. The Large and Small Isoforms of HPV 16 E6 Bind to and Differentially Affect Procaspase 8 Stability and Activity. J. Virology. 81:4116-4129, 2007. PMID: 17267478
14. Garnett, T., M. Filippova, and P. J. Duerksen-Hughes. Accelerated Degradation of FADD and Pro-Caspase 8 in Cells Expressing Human Papillomavirus 16 E6 Impairs TRAIL-Mediated Apoptosis. Cell Death and Differentiation. 13:1915-1926, 2006. PMID: 16528366
15. Filippova, M., T. A. Brown-Bryan, C. A. Casiano and P. J. Duerksen-Hughes. The Human Papillomavirus 16 E6 Protein Can Render Cells Either Sensitive or Resistant to TNF: Effect of Dose. Cell Death and Differentiation. 12:1622-1635, 2005. PMID: 15933739
16. Filippova, M., Parkhurst, L. and P. J. Duerksen-Hughes. HPV 16 E6 Binds to FADD and Modulates Fas-Triggered Apoptosis. J. Biol. Chem. 279:25729-25744, 2004. PMID: 15073179
17. Filippova, M., H. Song, J. L. Connolly, T. S. Dermody and P. J. Duerksen-Hughes. The Human Papillomavirus 16 E6 Protein Binds to TNF R1 and Protects Cells from TNF alphaTriggered Apoptosis. J. Biol. Chem. 277:21730-21739, 2002. PMID: 11934887
18. Yang, J. and P. J. Duerksen-Hughes. Activation of a p53 -Independent, Sphingolipid-Mediated Cytolytic Pathway in p53-Negative Mouse Fibroblast Cells Treated with N-methyl-N-nitro-N-nitrosoguanidine. J. Biol. Chem. 276:27129‑27135, 2001. PMID: 11369765