title: Gene expression analysis combined with functional genomics approach identifies ITIH5 as tumor suppressor gene in cervical carcinogenesis.
abstract: Progression from human papillomavirus-induced premalignant cervical intraepithelial neoplasia (CIN) to cervical cancer (CC) is driven by genetic and epigenetic events. Our microarray-based expression study has previously shown that inter-α-trypsin-inhibitor heavy chain 5 (ITIH5) mRNA levels in CCs were significantly lower than in high-grade precursor lesions (CIN3s). Therefore, we aimed to analyze in depth ITIH5 expression during cervical carcinogenesis in biopsy material and cell culture. Moreover, functional analyses were performed by ectopic expression of ITIH5 in different cell lines. We were able to confirm the validity of our microarray differential expression data by qPCR, demonstrating a clear ITIH5 downregulation in CC as compared with CIN2/3 or normal cervix. ITIH5 protein loss, evaluated by immunohistochemistry, was evident in 81% of CCs, whereas ITIH5 showed weak to moderate cytoplasmic staining in 91% of CIN2/3 cases. In addition, ITIH5 was strongly reduced or absent in seven CC cell lines and in three immortalized keratinocyte cell lines. Moreover, ITIH5 mRNA loss was associated with ITIH5 promoter methylation. ITIH5 expression could be restored in CC cell lines by pharmacological induction of DNA demethylation and histone acetylation. Functionally, ITIH5 overexpression significantly suppressed proliferation of SW756 cells and further resulted in a significant reduction of colony formation and cell migration in both CaSki and SW756 tumor models, but had no effect on invasion. Remarkably, ITIH5 overexpression did not influence the phenotype of HeLa cells. Taken together, ITIH5 gene silencing is a frequent event during disease progression, thereby providing evidence for a tumor suppressive role in cervical carcinogenesis.
authors.affiliation: Department of Gynecology, Jena University Hospital, Friedrich-Schiller-University, Jena, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Dittmann
authors.affiliation: Department of Gynecology, Jena University Hospital, Friedrich-Schiller-University, Jena, Germany.
authors.forename: Angelique
authors.initials: A
authors.lastname: Ziegfeld
authors.affiliation: Department of Gynecology, Jena University Hospital, Friedrich-Schiller-University, Jena, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Jansen
authors.affiliation: Institute of Pathology, Jena University Hospital, Friedrich-Schiller-University, Jena, Germany.
authors.forename: Mieczyslaw
authors.initials: M
authors.lastname: Gajda
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Kloten
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Edgar
authors.initials: E
authors.lastname: Dahl
authors.affiliation: Department of Gynecology, Jena University Hospital, Friedrich-Schiller-University, Jena, Germany.
authors.forename: Ingo B
authors.initials: IB
authors.lastname: Runnebaum
authors.affiliation: Department of Gynecology, Jena University Hospital, Friedrich-Schiller-University, Jena, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Dürst
authors.affiliation: Department of Gynecology, Jena University Hospital, Friedrich-Schiller-University, Jena, Germany.
authors.forename: Claudia
authors.identifier: 0000-0003-1679-6464
authors.initials: C
authors.lastname: Backsch
mesh_terms: D063646:Carcinogenesis / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D019175:DNA Methylation; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D023281:Genomics; D006801:Humans; D010053:Ovary / Q000378:metabolism / Q000473:pathology*; D011401:Promoter Regions, Genetic; D053491:Proteinase Inhibitory Proteins, Secretory / Q000032:analysis / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: C482680:ITIH5 protein, human; D053491:Proteinase Inhibitory Proteins, Secretory
keywords: ITIH5; cervical carcinogenesis; human papillomavirus; promoter methylation; tumor suppressor gene