Inactivation of the CYLD Deubiquitinase by HPV E6 Mediates Hypoxia-Induced NF-κB Activation

Abstract

SUMMARY The biochemical mechanisms that underlie hypoxia-induced NF-κB activity have remained largely undefined. We found that prolonged hypoxia-induced NF-κB activation is restricted to cancer cell lines infected with high risk human papilloma virus (HPV) serotypes. The HPV-encoded E6 protein is necessary and sufficient for prolonged hypoxia-induced NF-κB activation in these systems. The molecular target of E6 in the NF-κB pathway is the CYLD lysine 63 (K63) deubiquitinase, a negative regulator of the NF-κB pathway. Specifically, hypoxia stimulates E6-mediated ubiquitination and proteasomal degradation of CYLD. Given the established role of NF-κB in human carcinogenesis, these findings provide a potential molecular/viral link between hypoxia and the adverse clinical outcomes observed in HPV-associated malignancies. SIGNIFICANCE Intratumoral hypoxia is a critical factor in the poor clinical outcomes of human malignancies, most notably cervix and head and neck cancers. We found that prolonged hypoxia-induced NF-κB activation is not a generalized phenomenon amongst cancer cells but rather is restricted to human papilloma (HPV)-positive cancers, such as cervix and head and neck cancers. Under hypoxic conditions, the HPV-encoded E6 protein inactivates the CYLD tumor suppressor, a negative regulator of the NF-κB pathway and thereby allows for unrestricted activation of NF-κB. Because NF-κB-induced genes promote survival, proliferation, and angiogenesis, our findings illustrate how a common human virus adapts to hypoxia and helps account for the aggressive tumor biology associated with hypoxia.

NF-κB–Dependent Plasticity of the Epithelial to Mesenchymal Transition Induced by Von Hippel-Lindau Inactivation in Renal Cell Carcinomas

“…NF-κB activity was measured by electrophoretic mobility shift assay (EMSA), NF-κB-driven reporter gene expression, and an IKKβ in vitro kinase assay as we have previously described (22). The sequences of the oligonucleotides used for the EMSAs are as follows: NF-κB, 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (wild-type) and 5′-AGTTGAGGCGACTTTCCCAGGC-3′ (mutant); Oct-1, 5′-TGTCGAATGCAAATCACTAGAA-3′ (wild-type) and 5′-TGTCGAATGCAAGCCACTAGAA-3′ (mutant).…”

The helicase protein DHX29 promotes translation initiation, cell proliferation, and tumorigenesis

“…We explored this issue using a HeLa cell-based system used previously to assess cell proliferation and tumorigenicity (23)(24)(25)(26)(27)(28)(29)(30). First, anchorage-independent cell growth was assessed in shRNA-mediated DHX29-silenced and control cells.…”

Hyperactivated JNK Is a Therapeutic Target in pVHL-Deficient Renal Cell Carcinoma

“…The 786-0, UOK121, and UMRC6 isogenic pairs of cell lines were originally tested for pVHL and HIF-a expression by Western blotting as described earlier and are retested quarterly (12). The TRAF6-DN was used as described (13). Lentiviral plasmids designed to express target-specific or non-silencing (NS) short hairpin RNA (shRNA) were obtained from Open Biosystems, as was the Twist lentiviral plasmid.…”