title: The extracellular matrix protein ITIH5 is a novel prognostic marker in invasive node-negative breast cancer and its aberrant expression is caused by promoter hypermethylation.
abstract: Inter-alpha-trypsin inhibitors (ITIs) are protease inhibitors stabilizing the extracellular matrix. ITIs consist of one light (bikunin) and two heavy chains (ITIHs). We have recently characterized ITIH5, a novel member of the ITIH gene family, and showed that its messenger RNA is lost in a high proportion of breast tumours. In the present study, an ITIH5-specific polyclonal antibody was generated, validated with western blot and used for immunohistochemical analysis on a tissue microarray; ITIH5 was strongly expressed in epithelial cells of normal breast (n=11/15), while it was lost or strongly reduced in 42% (92/217) of invasive breast cancers. ITIH5 expression in invasive carcinomas was associated with positive expression of oestrogen receptor (P=0.008) and histological grade (P=0.024). Correlation of ITIH5 expression with clinical outcome revealed that patients with primary tumours retaining abundant ITIH5 expression had longer recurrence-free survival (RFS; P=0.037) and overall survival (OS; P=0.044), compared to those with reduced expression (mean RFS: 102 vs 78 months; mean OS: 120 vs 105 months). Methylation-specific PCR analysis frequently showed strong methylation of the ITIH5 promoter in primary breast tumours (41%, n=109) and breast cancer cell lines (n=6). Methylation was significantly associated with mRNA loss (P<0.001; n=39), and ITIH5 expression was induced after treatment of tumour cell lines with the demethylating agent 5-aza-2'-deoxycytidine. Moreover, ITIH5 promoter methylation was significantly associated with reduced OS (P=0.008). The cellular function of ITIH5 was evaluated by forced expression of a full-length ITIH5 complementary DNA in the breast cancer cell line MDA-MB-231, which does not endogenously express ITIH5. ITIH5-expressing clones showed a 40% reduced proliferation rate compared to mock-transfected cells. Overall, these data show that promoter methylation-mediated loss of ITIH5 expression is associated with unfavourable outcome in breast cancer patients, and thus ITIH5 could be used as a prognostic marker, although this marker is not multivariate independent due to its close association with ER expression. Our data indicate that ITIH5 is a candidate class II tumour suppressor gene and could be involved in tumour progression, invasion and metastasis, as its absence is associated with increased proliferation rates and a prognostic value indicating poor clinical outcome.
authors.affiliation: Molecular Oncology Group, Institute of Pathology, University Hospital of the RWTH Aachen, Aachen, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Veeck
authors.forename: M
authors.initials: M
authors.lastname: Chorovicer
authors.forename: A
authors.initials: A
authors.lastname: Naami
authors.forename: E
authors.initials: E
authors.lastname: Breuer
authors.forename: M
authors.initials: M
authors.lastname: Zafrakas
authors.forename: N
authors.initials: N
authors.lastname: Bektas
authors.forename: M
authors.initials: M
authors.lastname: Dürst
authors.forename: G
authors.initials: G
authors.lastname: Kristiansen
authors.forename: P J
authors.initials: PJ
authors.lastname: Wild
authors.forename: A
authors.initials: A
authors.lastname: Hartmann
authors.forename: R
authors.initials: R
authors.lastname: Knuechel
authors.forename: E
authors.initials: E
authors.lastname: Dahl
mesh_terms: D000906:Antibodies / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000401:mortality / Q000473:pathology; D002277:Carcinoma / Q000175:diagnosis* / Q000401:mortality / Q000473:pathology; D019175:DNA Methylation; D015536:Down-Regulation; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D006801:Humans; D009361:Neoplasm Invasiveness; D011379:Prognosis; D011401:Promoter Regions, Genetic; D053491:Proteinase Inhibitory Proteins, Secretory / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism
chemical_list: D000906:Antibodies; D014408:Biomarkers, Tumor; C482680:ITIH5 protein, human; D053491:Proteinase Inhibitory Proteins, Secretory; D012333:RNA, Messenger