title: Reduced abundance of the E3 ubiquitin ligase E6AP contributes to decreased expression of the INK4/ARF locus in non-small cell lung cancer.
abstract: The tumor suppressor p16INK4a, one protein encoded by the INK4/ARF locus, is frequently absent in multiple cancers, including non-small cell lung cancer (NSCLC). Whereas increased methylation of the encoding gene (CDKN2A) accounts for its loss in a third of patients, no molecular explanation exists for the remainder. We unraveled an alternative mechanism for the silencing of the INK4/ARF locus involving the E3 ubiquitin ligase and transcriptional cofactor E6AP (also known as UBE3A). We found that the expression of three tumor suppressor genes encoded in the INK4/ARF locus (p15INK4b, p16INK4a, and p19ARF) was decreased in E6AP-/-mouse embryo fibroblasts. E6AP induced the expression of the INK4/ARF locus at the transcriptional level by inhibiting CDC6 transcription, a gene encoding a key repressor of the locus. Luciferase assays revealed that E6AP inhibited CDC6 expression by reducing its E2F1-dependent transcription. Chromatin immunoprecipitation analysis indicated that E6AP reduced the amount of E2F1 at the CDC6 promoter. In a subset of NSCLC samples, an E6AP-low/CDC6-high/p16INK4a-low protein abundance profile correlated with low methylation of the gene encoding p16INK4a(CDKN2A) and poor patient prognosis. These findings define a previously unrecognized tumor-suppressive role for E6AP in NSCLC, reveal an alternative silencing mechanism of the INK4/ARF locus, and reveal E6AP as a potential prognostic marker in NSCLC.
authors.affiliation: Tumour Suppression Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.|Sir Peter MacCallum Department of Oncology, University of Melbourne, Victoria 3000, Australia.
authors.forename: Cristina
authors.initials: C
authors.lastname: Gamell
authors.affiliation: Tumour Suppression Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.|Sir Peter MacCallum Department of Oncology, University of Melbourne, Victoria 3000, Australia.
authors.forename: Twishi
authors.initials: T
authors.lastname: Gulati
authors.affiliation: The Hebrew University Hadassah Medical School, Jerusalem 9112102, Israel.
authors.forename: Yaara
authors.initials: Y
authors.lastname: Levav-Cohen
authors.affiliation: Molecular Therapeutics and Biomarkers Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Young
authors.affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria 3084, Australia.
authors.forename: Hongdo
authors.initials: H
authors.lastname: Do
authors.affiliation: Biomedical Manufacturing Program, Commonwealth Scientific and Industrial Research Organization, Melbourne, Victoria 3169, Australia.
authors.forename: Pat
authors.initials: P
authors.lastname: Pilling
authors.affiliation: Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.
authors.forename: Elena
authors.initials: E
authors.lastname: Takano
authors.affiliation: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia.
authors.forename: Neil
authors.initials: N
authors.lastname: Watkins
authors.affiliation: Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Fox
authors.affiliation: Department of Anatomical Pathology, St. Vincent's Hospital, Melbourne, Victoria 3065, Australia.
authors.forename: Prudence
authors.initials: P
authors.lastname: Russell
authors.affiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
authors.forename: Doron
authors.initials: D
authors.lastname: Ginsberg
authors.affiliation: Systems Biology and Personalised Medicine, Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 2555, Australia.
authors.forename: Brendon J
authors.initials: BJ
authors.lastname: Monahan
authors.affiliation: Department of Surgery, St. Vincent's Hospital, Melbourne, Victoria 3065, Australia.|Division of Cancer Surgery, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.
authors.forename: Gavin
authors.initials: G
authors.lastname: Wright
authors.affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria 3084, Australia.
authors.forename: Alex
authors.initials: A
authors.lastname: Dobrovic
authors.affiliation: Tumour Suppression Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.|Sir Peter MacCallum Department of Oncology, University of Melbourne, Victoria 3000, Australia.
authors.forename: Sue
authors.initials: S
authors.lastname: Haupt
authors.affiliation: Sir Peter MacCallum Department of Oncology, University of Melbourne, Victoria 3000, Australia.|Molecular Therapeutics and Biomarkers Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.
authors.forename: Ben
authors.initials: B
authors.lastname: Solomon
authors.affiliation: Tumour Suppression Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia. ygal.haupt@petermac.org.|Sir Peter MacCallum Department of Oncology, University of Melbourne, Victoria 3000, Australia.|Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia.|Department of Pathology, University of Melbourne, Victoria 3800, Australia.
authors.forename: Ygal
authors.initials: Y
authors.lastname: Haupt
mesh_terms: D000818:Animals; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000235:genetics* / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D050764:Cyclin-Dependent Kinase Inhibitor p19 / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D053208:Kaplan-Meier Estimate; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism
chemical_list: C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050764:Cyclin-Dependent Kinase Inhibitor p19; D050687:E2F1 Transcription Factor; D009687:Nuclear Proteins; D044767:Ubiquitin-Protein Ligases