HOIL-1L functions as the PKC? ubiquitin ligase to promote lung tumor growth. Journal Article

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Authors: Queisser, MA; Dada, LA; Deiss-Yehiely, N; Angulo, M; Zhou, G; Kouri, FM; Knab, LM; Liu, J; Stegh, AH; DeCamp, MM; Budinger, GR; Chandel, NS; Ciechanover, A; Iwai, K; Sznajder, JI
Article Title: HOIL-1L functions as the PKC? ubiquitin ligase to promote lung tumor growth.
Abstract: RATIONALE: Protein kinase C zeta (PKC?) has been reported to act as a tumor suppressor. Deletion of PKC? in experimental cancer models has been shown to increase tumor growth. However, the mechanisms of PKC? down-regulation in cancerous cells have not been previously described. OBJECTIVES: To determine the molecular mechanisms that lead to decreased PKC? expression and thus increased survival in cancer cells and tumor growth. METHODS: The levels of expression of heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), HOIL-1-interacting protein (HOIP), Shank-associated RH domain-interacting protein (SHARPIN), and PKC? were analyzed by Western blot and/or quantitative real-time polymerase chain reaction in different cell lines. Coimmunoprecipitation experiments were used to demonstrate the interaction between HOIL-1L and PKC?. Ubiquitination was measured in an in vitro ubiquitination assay and by Western blot with specific antibodies. The role of hypoxia-inducible factor (HIF) was determined by gain/loss-of-function experiments. The effect of HOIL-1L expression on cell death was investigated using RNA interference approaches in vitro and on tumor growth in mice models. Increased HOIL-1L and decreased PKC? expression was assessed in lung adenocarcinoma and glioblastoma multiforme and documented in several other cancer types by oncogenomic analysis. MEASUREMENTS AND MAIN RESULTS: Hypoxia is a hallmark of rapidly growing solid tumors. We found that during hypoxia, PKC? is ubiquitinated and degraded via the ubiquitin ligase HOIL-1L, a component of the linear ubiquitin chain assembly complex (LUBAC). In vitro ubiquitination assays indicate that HOIL-1L ubiquitinates PKC? at Lys-48, targeting it for proteasomal degradation. In a xenograft tumor model and lung cancer model, we found that silencing of HOIL-1L increased the abundance of PKC? and decreased the size of tumors, suggesting that lower levels of HOIL-1L promote survival. Indeed, mRNA transcript levels of HOIL-1L were elevated in tumor of patients with lung adenocarcinoma, and in a lung adenocarcinoma tissue microarray the levels of HOIL-1L were associated with high-grade tumors. Moreover, we found that HOIL-1L expression was regulated by HIFs. Interestingly, the actions of HOIL-1L were independent of LUBAC. CONCLUSIONS: These data provide first evidence of a mechanism of cancer cell adaptation to hypoxia where HIFs regulate HOIL-1L, which targets PKC? for degradation to promote tumor survival. We provided a proof of concept that silencing of HOIL-1L impairs lung tumor growth and that HOIL-1L expression predicts survival rate in cancer patients suggesting that HOIL-1L is an attractive target for cancer therapy.
Journal Title: American journal of respiratory and critical care medicine
ISSN: 1535-4970; 1073-449X
Publisher: Unknown  
Date Published: 2014