[Novel prognostic marker in invasive breast cancer. ITIH5 expression is abrogated by aberrant promoter methylation]. / Neuer Prognosemarker beim invasiven Mammakarzinom. ITIH5-Expression wird über aberrante Promotormethylierung inaktiviert.

We have recently characterized ITIH5 as a new extracellular matrix protein that exhibits clear expression loss in a variety of human tumour entities, including breast cancer. The aim of the present study was to decipher the molecular cause of ITIH5 expression loss in breast cancer and to learn more about the possible role of this molecule in cancer diseases. ITIH5 protein expression was found to be strongly reduced in 42% of invasive breast carcinomas-interestingly, with significant association with poor patient outcome. ITIH5 promoter methylation was frequently detected in breast cell lines and in primary carcinomas (40%), and it was functionally correlated with loss of ITIH5 mRNA expression. Moreover, ITIH5 promoter methylation was also significantly associated with poor clinical patient outcome and also with the occurrence of lymph node and distant metastases. In conclusion, we propose that ITIH5 may represent a novel metastasis repressor in human breast cancer. Both ITIH5 protein expression and ITIH5 promoter methylation may serve as prognostic biomarkers, thereby helping improve clinical patient outcome.

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.

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.