Abstract
We sought to determine the role of human papillomavirus (HPV) E6 and E7 oncogenes in nuclear beta-catenin accumulation, a hallmark of activated canonical Wnt signaling pathway. We used HPV16-positive oropharyngeal cancer cell lines 147T and 090, HPV-negative cell line 040T, and cervical cell lines SiHa (bearing integrated HPV16) and HeLa (bearing integrated HPV18) to measure the cytoplasmic and nuclear beta-catenin levels and the beta-catenin/Tcf transcriptional activity before and after E6/E7 gene silencing. Repression of HPV E6 and E7 genes induced a substantial reduction in nuclear beta-catenin levels. Luciferase assay showed that transcriptional activation of Tcf promoter by beta-catenin was lower after silencing. The protein levels of beta-catenin are tightly regulated by the ubiquitin/proteasome system. We therefore performed expression analysis of regulators of beta-catenin degradation and nuclear transport and showed that seven in absentia homologue (Siah-1) mRNA and protein levels were substantially upregulated after E6/E7 repression. Siah-1 protein promotes the degradation of beta-catenin through the ubiquitin/proteasome system. To determine whether Siah-1 is important for the proteasomal degradation of beta-catenin in HPV16-positive oropharyngeal cancer cells, we introduced a Siah-1 expression vector into 147T and 090 cells and found substantial reduction of endogenous beta-catenin in these cells. Thus, E6 and E7 are involved in beta-catenin nuclear accumulation and activation of Wnt signaling in HPV-induced cancers. In addition, we show the significance of the endogenous Siah-1-dependent ubiquitin/proteasome pathway for beta-catenin degradation and its regulation by E6/E7 viral oncoproteins in HPV16-positive oropharyngeal cancer cells.
Highlights
The high-risk human papillomavirus types 16 (HPV-16) and 18 (HPV-18) have been etiologically implicated in the majority of cervical carcinomas [1]
We have recently shown that the retrovirus-mediated delivery of short hairpin RNAs targeting HPV16 E6/E7 oncogenes in HPV16-positive oropharyngeal cancer cell lines results in apoptosis and restoration of p53 and pRb tumor suppressor pathways [10]
We examined whether the degradation of β-catenin through seven in absentia homologue 1 (Siah-1) ubiquitin ligase is functional in HPV16positive oropharyngeal cell lines 147T and 090
Summary
The high-risk human papillomavirus types 16 (HPV-16) and 18 (HPV-18) have been etiologically implicated in the majority of cervical carcinomas [1]. High-risk HPVs, especially type 16, have been associated with a subset of oropharyngeal cancers in individuals without a history of alcohol use or tobacco exposure [2,3,4]. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Weinberger: Department Surgery, Section of Otolaryngology, Medical College of Georgia, Augusta, GA. Portions of this material were presented at the 99th Annual Meeting of the American Association for Cancer Research, April 2008
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