CYLD Alterations in the Tumorigenesis and Progression of Human Papillomavirus–Associated Head and Neck Cancers

Polyubiquitination of proteins has a pivotal role in the regulation of numerous cellular functions such as protein degradation, DNA repair and cell signaling. As deregulation of these processes can result in pathological conditions such as inflammatory diseases, neurodegeneration or cancer, tight regulation of the ubiquitin system is of tremendous importance. Ubiquitination by E3 ubiquitin ligases can be counteracted by the activity of several deubiquitinating enzymes (DUBs). CYLD, A20 and OTULIN have been implicated as key DUBs in the negative regulation of NF-κB transcription factor-mediated gene expression upon stimulation of cytokine receptors, antigen receptors and pattern recognition receptors, by removing distinct types of polyubiquitin chains from specific NF-κB signaling proteins. In addition, they control TNF-induced cell death signaling leading to apoptosis and necroptosis via similar mechanisms. In the case of A20, also catalytic-independent mechanisms of action have been demonstrated to have an important role. CYLD, A20 and OTULIN have largely overlapping substrates, suggesting at least partially redundant functions. However, mice deficient in one of the three DUBs show significant phenotypic differences, indicating also non-redundant functions. Here we discuss the activity and polyubiquitin chain-type specificity of CYLD, A20 and OTULIN, their specific role in NF-κB signaling and cell death, the molecular mechanisms that regulate their activity, their role in immune homeostasis and the association of defects in their activity with inflammation, autoimmunity and cancer.

The response of mammalian cells to stress is controlled by transcriptional regulatory proteins such as nuclear factor kappa B (NF-kappa B) to induce a wide variety of early response genes. In this report, we show that exposure of cells to hypoxia (0.02% O2) results in I kappa B alpha degradation, increased NF-kappa B DNA binding activity, and transactivation of a reporter gene construct containing two NF-kappa B DNA binding sites. Pretreatment of cells with protein tyrosine kinase inhibitors and the dominant negative allele of c-Raf-1 (Raf 301) inhibited I kappa B alpha degradation, NF-kappa B binding, and transactivation of kappa B reporter constructs by hypoxia. To demonstrate a direct link between changes in the phosphorylation pattern of I kappa B alpha with NF-kappa B activation, we immunoprecipitated I kappa B alpha after varying times of hypoxic exposure and found that its tyrosine phosphorylation status increased during hypoxic exposure. Inhibition of the transfer of tyrosine phosphoryl groups onto I kappa B alpha prevented I kappa B alpha degradation and NF-kappa B binding. In comparison to other activators of NF-kappa B such as phorbol myristate acetate or tumor necrosis factor, we did not detect changes in the tyrosine phosphorylation status of I kappa B alpha following treatment with either of these agents. These results suggest that tyrosine phosphorylation of I kappa B alpha during hypoxia is an important proximal step which precedes its dissociation and degradation from NF-kappa B.

MicroRNAs are increasingly recognized as playing important roles in hepatocellular carcinoma (HCC) tumorigenesis. Here we identified an essential role for miR-362-5p in the regulation of HCC development. We found that miR-362-5p was significantly up-regulated in HCCs and associated with HCC progression. Inhibition of miR-362-5p in HCC cells dramatically decreased cell proliferation, clonogenicity, migration and invasion in vitro as well as tumor growth and metastasis in vivo. We subsequently identified that CYLD was a target gene of miR-362-5p. Furthermore, knockdown of CYLD expression partially counteracted the tumor suppressive effects of miR-362-5p inhibitors. Finally, we have shown that miR-362-5p acts through CYLD to activate the NF-κB signaling pathway, which contributes to HCC progression. Taken together, our findings indicate that miR-362-5p belongs to a new class of oncomiR that regulates HCC cell aggressiveness, thus providing new insight into the molecular mechanisms underlying HCC development. This study also suggests that miR-362-5p may serve as a novel therapeutic target for miRNA based HCC therapy.