Inter-a-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 ¼ 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 ¼ 0.008) and histological grade (P ¼ 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 ¼ 0.037) and overall survival (OS; P ¼ 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 ¼ 109) and breast cancer cell lines (n ¼ 6). Methylation was significantly associated with mRNA loss (Po0.001; n ¼ 39), and ITIH5 expression was induced after treatment of tumour cell lines with the demethylating agent 5-aza-2 0 -deoxycytidine. Moreover, ITIH5 promoter methylation was significantly associated with reduced OS (P ¼ 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.

The GABRP gene has been previously identified by in silico analysis of four million ESTs as a candidate gene differentially expressed in breast cancer. GABRP is located on chromosome 5q34 and it encodes the p-subunit of the c-aminobutyric acid (GABA) receptor, a transmembrane protein expressed in the brain and several nonneuronal tissues. Using cDNA dot blot hybridisation (cancer profiling array), quantitative RT-PCR and non-radioisotopic in situ hybridisation (ISH), we have analysed GABRP expression in breast cancer and normal breast tissues as well as in nontumorigenic and tumorigenic breast cell lines. Analysis of the cancer profiling array revealed a more than 2-fold downregulation of GABRP (p < 0.001) in 76% of primary breast carcinomas (n 5 50) compared to corresponding normal tissues. Quantitative RT-PCR in a panel of 23 normal human tissues showed that the GABRP expression level was most abundant in the normal breast tissues compared to other human tissues. GABRP downregulation in breast cancer was confirmed by quantitative RT-PCR in cryopreserved breast tumour and normal breast tissue specimens (n 5 22), in archival formalin-fixed, paraffin-embedded tissue specimens (n 5 32), as well as in breast cancer cell lines (n 5 8). Furthermore, a significant downregulation of GABRP was noted in large (pT3-pT4) (p 5 0.044) primary breast tumours. Non-radioisotopic ISH showed strong GABRP expression in normal epithelial and benign papilloma breast cells, but no signal could be detected in invasive ductal carcinoma. Altogether, these data suggest that GABRP is progressively down-regulated with tumour-progression, and that it may be useful as a prognostic marker in breast cancer. ' 2005 Wiley-Liss, Inc. Key words: GABRP; gene expression; breast cancer; prognostic markerThe p-subunit of the g-aminobutyric acid (GABA) receptor, encoded by the gene GABRP, is a transmembrane protein expressed in the brain and in several nonneuronal tissues.1 Type A g-aminobutyric acid receptors (GABA A receptors) are heteropentamers, consisting of five subunits that form a chloride ion channel, and mediate the majority of fast inhibitory neurotransmission in the brain.2 A wide range of GABA A receptor subunit classes have been discovered in recent years:1-5 Alternative splicing further increases the repertoire of GABA A receptors.1-4 Depending on their subunit composition, GABA A receptors have different pharmacological and electrophysiological properties. The subunit diversity results in a very large number of potential subunit combinations and, thus, in a large number of possible GABA A receptor subtypes. The majority of genes encoding GABA A receptor subunits are found in four clusters on human chromosomes 4, 5, 15 and X. 2,7,8 GABRP is located on chromosome 5q34, but it is not tightly linked with the GABA A receptor gene cluster. 9 The amino acid sequence of the p-subunit is most closely related to GABA A receptor b-and d-subunits and to the GABA C receptor q-subunit, and is less similar to other GABA A receptor or glycine receptor...

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