HOIL-1L functions as the PKCζ ubiquitin ligase to promote lung tumor growth

Markus A. Queisser, Laura Dada, Nimrod Deiss-Yehiely, Martin Angulo, Guofei Zhou, Foteini Kouri, Lawrence M. Knab, Jing Liu, Alexander H Stegh, Malcom McAvoy DeCamp Jr, GR Scott Budinger, Navdeep Chandel, Aaron Ciechanover, Kazuhiro Iwai, Jacob I Sznajder*

*Corresponding author for this work

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Rationale: 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. Objectives: 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. Measurements and Main 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 tumormodel and lung cancermodel, 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.

Original languageEnglish (US)
Pages (from-to)688-698
Number of pages11
JournalAmerican journal of respiratory and critical care medicine
Volume190
Issue number6
DOIs
StatePublished - Sep 15 2014

Fingerprint

Ligases
Ubiquitin
Heme
Lung
Growth
Neoplasms
Ubiquitination
protein kinase C zeta
Western Blotting
Survival
Glioblastoma
RNA Interference

Keywords

  • E3 ligase
  • Hypoxia
  • Hypoxia-inducible factors
  • Linear ubiquitin chain assembly complex
  • Tumorigenesis

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Critical Care and Intensive Care Medicine

Cite this

Queisser, Markus A. ; Dada, Laura ; Deiss-Yehiely, Nimrod ; Angulo, Martin ; Zhou, Guofei ; Kouri, Foteini ; Knab, Lawrence M. ; Liu, Jing ; Stegh, Alexander H ; DeCamp Jr, Malcom McAvoy ; Budinger, GR Scott ; Chandel, Navdeep ; Ciechanover, Aaron ; Iwai, Kazuhiro ; Sznajder, Jacob I. / HOIL-1L functions as the PKCζ ubiquitin ligase to promote lung tumor growth. In: American journal of respiratory and critical care medicine. 2014 ; Vol. 190, No. 6. pp. 688-698.
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title = "HOIL-1L functions as the PKCζ ubiquitin ligase to promote lung tumor growth",
abstract = "Rationale: 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. Objectives: 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. Measurements and Main 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 tumormodel and lung cancermodel, 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.",
keywords = "E3 ligase, Hypoxia, Hypoxia-inducible factors, Linear ubiquitin chain assembly complex, Tumorigenesis",
author = "Queisser, {Markus A.} and Laura Dada and Nimrod Deiss-Yehiely and Martin Angulo and Guofei Zhou and Foteini Kouri and Knab, {Lawrence M.} and Jing Liu and Stegh, {Alexander H} and {DeCamp Jr}, {Malcom McAvoy} and Budinger, {GR Scott} and Navdeep Chandel and Aaron Ciechanover and Kazuhiro Iwai and Sznajder, {Jacob I}",
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doi = "10.1164/rccm.201403-0463OC",
language = "English (US)",
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pages = "688--698",
journal = "American Journal of Respiratory and Critical Care Medicine",
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Queisser, MA, Dada, L, Deiss-Yehiely, N, Angulo, M, Zhou, G, Kouri, F, Knab, LM, Liu, J, Stegh, AH, DeCamp Jr, MM, Budinger, GRS, Chandel, N, Ciechanover, A, Iwai, K & Sznajder, JI 2014, 'HOIL-1L functions as the PKCζ ubiquitin ligase to promote lung tumor growth', American journal of respiratory and critical care medicine, vol. 190, no. 6, pp. 688-698. https://doi.org/10.1164/rccm.201403-0463OC

HOIL-1L functions as the PKCζ ubiquitin ligase to promote lung tumor growth. / Queisser, Markus A.; Dada, Laura; Deiss-Yehiely, Nimrod; Angulo, Martin; Zhou, Guofei; Kouri, Foteini; Knab, Lawrence M.; Liu, Jing; Stegh, Alexander H; DeCamp Jr, Malcom McAvoy; Budinger, GR Scott; Chandel, Navdeep; Ciechanover, Aaron; Iwai, Kazuhiro; Sznajder, Jacob I.

In: American journal of respiratory and critical care medicine, Vol. 190, No. 6, 15.09.2014, p. 688-698.

Research output: Contribution to journalArticle

TY - JOUR

T1 - HOIL-1L functions as the PKCζ ubiquitin ligase to promote lung tumor growth

AU - Queisser, Markus A.

AU - Dada, Laura

AU - Deiss-Yehiely, Nimrod

AU - Angulo, Martin

AU - Zhou, Guofei

AU - Kouri, Foteini

AU - Knab, Lawrence M.

AU - Liu, Jing

AU - Stegh, Alexander H

AU - DeCamp Jr, Malcom McAvoy

AU - Budinger, GR Scott

AU - Chandel, Navdeep

AU - Ciechanover, Aaron

AU - Iwai, Kazuhiro

AU - Sznajder, Jacob I

PY - 2014/9/15

Y1 - 2014/9/15

N2 - Rationale: 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. Objectives: 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. Measurements and Main 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 tumormodel and lung cancermodel, 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.

AB - Rationale: 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. Objectives: 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. Measurements and Main 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 tumormodel and lung cancermodel, 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.

KW - E3 ligase

KW - Hypoxia

KW - Hypoxia-inducible factors

KW - Linear ubiquitin chain assembly complex

KW - Tumorigenesis

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