title: MAD2 downregulation in hypoxia is independent of promoter hypermethylation.
abstract: Aberrant expression of the MAD2 protein has been linked to chromosomal instability, malignant transformation and chemoresistance. Although reduced MAD2 expression is well recognised in human cancer cell lines, the mechanism(s) underlying its downregulation remain elusive. The objective of this study was to establish the impact of hypoxia on MAD2 expression and to investigate the potential role of aberrant promoter methylation as a possible mechanism of MAD2 downregulation. For this purpose, three ovarian cancer cell lines, displaying differing levels of MAD2, were treated with chromatin modifying drugs, pre and post-hypoxia exposure and a DHPLC analysis of DNA promoter methylation carried out. We show that hypoxia induces downregulation of MAD2 expression, independently of MAD2 promoter methylation. We also show no evidence of MAD2 promoter methylation in breast and prostate cancer cells or in breast cancer clinical material. While our findings provide no evidence for MAD2 promoter methylation, we show a concomitant upregulation of p21 with downregulation of MAD2 in hypoxia. Our in vitro results were also confirmed in an ovarian cancer tissue microarray (TMA), where a reciprocal staining of MAD2 and CAIX was found in 21/60 (35%) of tumours. In summary, MAD2 downregulation may be a crucial mechanism by which hypoxic cells become chemorefractory. This stems from our previous work where we demonstrated that MAD2 downregulation induces cellular senescence, a viable cellular fate, with resultant cellular resistance to paclitaxel. Moreover, MAD2 downregulation could play a central role in the induction of chemoresistance in hypoxia, a key tumour microenvironment associated with chemoresistance.
authors.affiliation: UCD School of Medicine and Medical Science (SMMS), University College Dublin, Belfield, Dublin, Ireland. maria.prencipe@ucd.ie
authors.forename: Maria
authors.initials: M
authors.lastname: Prencipe
authors.forename: Aloysius
authors.initials: A
authors.lastname: McGoldrick
authors.forename: Antoinette S
authors.initials: AS
authors.lastname: Perry
authors.forename: Anthony
authors.initials: A
authors.lastname: O'Grady
authors.forename: Sine
authors.initials: S
authors.lastname: Phelan
authors.forename: Barbara
authors.initials: B
authors.lastname: McGrogan
authors.forename: Patricia
authors.initials: P
authors.lastname: Fitzpatrick
authors.forename: Jenny A
authors.initials: JA
authors.lastname: Watson
authors.forename: Fiona
authors.initials: F
authors.lastname: Furlong
authors.forename: Donal J
authors.initials: DJ
authors.lastname: Brennan
authors.forename: Mark
authors.initials: M
authors.lastname: Lawler
authors.forename: Elaine
authors.initials: E
authors.lastname: Kay
authors.forename: Amanda
authors.initials: A
authors.lastname: McCann
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D019175:DNA Methylation; D000077209:Decitabine; D015536:Down-Regulation; D006801:Humans; D064246:Mad2 Proteins; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D000951:Antigens, Neoplasm; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D012097:Repressor Proteins; D000077209:Decitabine; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; D001374:Azacitidine