title: HOIL-1L functions as the PKCζ ubiquitin ligase to promote lung tumor growth.
abstract: BACKGROUND
  Protein kinase C zeta (PKCζ) has been reported to act as a tumor suppressor. Deletion of PKCζ in experimental cancer models has been shown to increase tumor growth. However, the mechanisms of PKCζ down-regulation in cancerous cells have not been previously described.
  
  
  OBJECTIVE
  To determine the molecular mechanisms that lead to decreased PKCζ 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 PKCζ 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 PKCζ. 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 PKCζ expression was assessed in lung adenocarcinoma and glioblastoma multiforme and documented in several other cancer types by oncogenomic analysis.
  
  
  RESULTS
  Hypoxia is a hallmark of rapidly growing solid tumors. We found that during hypoxia, PKCζ 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 PKCζ 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 PKCζ 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 PKCζ 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.
authors.affiliation: 1 Division of Pulmonary and Critical Care Medicine.
authors.forename: Markus A
authors.initials: MA
authors.lastname: Queisser
authors.forename: Laura A
authors.initials: LA
authors.lastname: Dada
authors.forename: Nimrod
authors.initials: N
authors.lastname: Deiss-Yehiely
authors.forename: Martin
authors.initials: M
authors.lastname: Angulo
authors.forename: Guofei
authors.initials: G
authors.lastname: Zhou
authors.forename: Fotini M
authors.initials: FM
authors.lastname: Kouri
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Knab
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Alexander H
authors.initials: AH
authors.lastname: Stegh
authors.forename: Malcolm M
authors.initials: MM
authors.lastname: DeCamp
authors.forename: G R Scott
authors.initials: GR
authors.lastname: Budinger
authors.forename: Navdeep S
authors.initials: NS
authors.lastname: Chandel
authors.forename: Aaron
authors.initials: A
authors.lastname: Ciechanover
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Iwai
authors.forename: Jacob I
authors.initials: JI
authors.lastname: Sznajder
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology*; D000077192:Adenocarcinoma of Lung; D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor / Q000378:metabolism*; D049109:Cell Proliferation / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology*; D051379:Mice; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology; D023041:Xenograft Model Antitumor Assays; D000099221:Protein Kinase C zeta
chemical_list: D011493:Protein Kinase C; D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases; D000099221:Protein Kinase C zeta; C504507:RBCK1 protein, human
keywords: E3 ligase; hypoxia; hypoxia-inducible factors; linear ubiquitin chain assembly complex; tumorigenesis