Oncogene (2008) 27, 865–876 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ONCOGENOMICS 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 J Veeck1,6, M Chorovicer1,6, A Naami1, E Breuer1, M Zafrakas1, N Bektas1,MDu¨ rst2, G Kristiansen3, PJ Wild4, A Hartmann5, R Knuechel1 and E Dahl1 1Molecular Oncology Group, Institute of Pathology, University Hospital of the RWTH Aachen, Aachen, Germany; 2Department of Gynecology and Obstetrics, Friedrich-Schiller-University, Jena, Germany; 3Institute of Pathology, Universita¨tsmedizin Berlin, Berlin, Germany; 4Institute of Pathology, University of Zu¨rich, Zu¨rich, Switzerland and 5Institute of Pathology, University of Regensburg, Regensburg, Germany Inter-a-trypsin inhibitors (ITIs) are protease inhibitors promoter methylation-mediated loss of ITIH5 expression stabilizing the extracellular matrix. ITIs consist of one is associated with unfavourable outcome in breast cancer light (bikunin) and two heavy chains (ITIHs). We have patients, and thus ITIH5 could be used as a prognostic recently characterized ITIH5, a novel member of the marker, although this marker is not multivariate inde- ITIH gene family, and showed that its messenger RNA is pendent due to its close association with ER expression. lost in a high proportion of breast tumours. In the present Our data indicate that ITIH5 is a candidate class II study, an ITIH5-specific polyclonal antibody was gener- tumour suppressor gene and could be involved in tumour ated, validated with western blot and used for immuno- progression, invasion and metastasis, as its absence is histochemical analysis on a tissue microarray; ITIH5 was associated with increased proliferation rates and a strongly expressed in epithelial cells of normal breast prognostic value indicating poor clinical outcome. (n ¼ 11/15), while it was lost or strongly reduced in 42% Oncogene (2008) 27, 865–876; doi:10.1038/sj.onc.1210669; (92/217) of invasive breast cancers. ITIH5 expression in published online 23 July 2007 invasive carcinomas was associated with positive expres- sion of oestrogen receptor (P ¼ 0.008) and histological Keywords: inter-a-trypsininhibitor heavy chain(ITIH); grade (P ¼ 0.024). Correlation of ITIH5 expression with breast cancer; prognostic marker; predictive marker; tumour clinical outcome revealed that patients with primary invasion; metastasis 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 Introduction expression (mean RFS: 102 vs 78 months; mean OS: 120 vs 105 months). Methylation-specific PCR analysis The inter-a-trypsininhibitors (ITIs) comprise a family of frequently showed strong methylation of the ITIH5 protease inhibitors found in the extracellular matrices of promoter in primary breast tumours (41%, n ¼ 109) and various organs, as well as in the blood circulation. breast cancer cell lines (n ¼ 6). Methylation was signifi- Owing to their original isolation in complexes with cantly associated with mRNA loss (Po0.001; n ¼ 39), and hyaluronan (HA), ITIs are also referred to as SHAPs ITIH5 expression was induced after treatment of tumour 0 (Serum-derived HA-associated proteins) (Yoneda et al., cell lines with the demethylating agent 5-aza-2 -deoxycy- 1990). It has beenpreviously shownthat interactionof tidine. Moreover, ITIH5 promoter methylation was ITIs with HA leads to stabilizationof the extracellular significantly associated with reduced OS (P ¼ 0.008). matrix (Chen et al., 1994). ITI molecules consist of three The cellular function of ITIH5 was evaluated by forced protein chains: one ITI light chain, also referred to as ONCOGENOMICS expression of a full-length ITIH5 complementary DNA in bikunin, and two ITI heavy chains (ITIHs) (Enghild the breast cancer cell line MDA-MB-231, which does not et al., 1991). The transfer of ITIHs onto HA requires endogenously express ITIH5. ITIH5-expressing clones tumour necrosis factor a-induced protein 6 (TNFAIP6), showed a 40% reduced proliferation rate compared to also known as TNF-stimulated gene 6 (Jessen and mock-transfected cells. Overall, these data show that Odum, 2003). TNFAIP6 forms a stable complex (Rugg et al., 2005) with ITIH and HA during the transester- Correspondence: E Dahl, Molecular Oncology Group, Institute of ificationreaction(Sanggaard et al., 2005). This forma- Pathology, University Hospital Aachen, RWTH Aachen, Pauwels- tionof ITIH–HA complexes is thought to play an strasse 30, 52074 Aachen, Germany. important role in the stabilization of HA-rich extra- E-mail: [email protected] 6These two authors contributed equally to this work. cellular matrices, with TNFAIP6 acting as an essential Received 21 March 2007; revised 4 June 2007; accepted 13 June 2007; cofactor and catalyst (Rugg et al., 2005). To date, published online 23 July 2007 five distinct ITIHs have been identified, encoded by five ITIH5 expression in breast cancer J Veeck et al 866 genes located on two different chromosomes (Diarra- in breast cancer cell lines and primary breast tumour Mehrpour et al., 1989; Himmelfarb et al., 2004). tissue samples and the biological role of ITIH5 was ITIH1, -3 and -4 have beenmapped to chromosome evaluated with functional in vitro assays. Clinicopatho- 3p2.11–12, and ITIH2 and -5 are located onchromo- logical patient characteristics were statistically corre- some 10p14–15 (Bost et al., 1998; Himmelfarb et al., lated with expressionandmethylationdata andrevealed 2004). ITIH1, -2 and -3 are synthesized primarily in liver an unfavourable prognosis in case of methylation- as polypeptide precursors, which undergo extensive mediated loss of ITIH5 expression. post-translational processing; they all contain a con- served cleavage site, which enables them to form covalent bonds to bikunin via glycosaminoglycan (Enghild et al., 1991). Results Various studies (Kobayashi et al., 1995; Bourguignon et al., 1999; Paris et al., 2002; Zhang et al., 2004) have Characterization of the anti-ITIH5-specific antibody shown involvement of ITIs in tumour biology using an The specificity of the rabbit polyclonal antiserum raised expressed sequence-tag-based bioinformatics approach against a synthetic peptide corresponding to amino acids (Dahl et al., 2005). We have previously identified ITIH5 207–220 of humanITIH5 proteinwas evaluated by as a novel gene differentially expressed in breast cancer. western blot analysis. The antibody is able to detect After cloning, mapping and determination of its humanITIH5 proteinexpressed in ITIH5-transfected genomic organization, we analysed the expression of COS7 cells (Figure 1a, lane 1) as well as in ITIH5- ITIH5 at the messenger RNA level in a panel of normal transfected MDA-MB-231 breast cancer cell lines human tissues and a small set of normal and malignant (Figure 1b; lanes 1, 3, 5 represent different clones). No breast tissue samples. Initial analyses showed that proteinwas detectable inmock-transfected COS7 cells ITIH5 was predominantly expressed in human female (Figure 1b; lane 2) and mock-transfected MDA-MB-231 reproductive tissues and strongly downregulated in cells (Figure 1b; lanes 2, 4, 6), arguing that the ITIH5 breast tumours, suggesting a potential role in breast antibody does not detect any protein, that is not a cancer development (Himmelfarb et al., 2004). Inthe modificationof the ITIH5 protein.ITIH5 proteininthe present study, protein expression of ITIH5 was analysed transfected cell lines was approximately 100 kDa in size, ina large set of breast tumours usingtissue microarrays inaccordancewith its deduced molecular weight. (TMAs). Furthermore, the epigenetic configuration of Immunocytochemical staining of transfected cells the ITIH5 promoter was comprehensively investigated showed abundant ITIH5 protein in the cytoplasm of 12 175 ITIH5 83 62 123456 47 32 100 kDa ITIH5 β-Actin β-Actin Figure 1 Characterizationof the ITIH5 antibody.( a and b) Detectionof ITIH5 proteinof lysates from stable ITIH5 clones of COS7 cells (a) and MDA-MB-231 cells (b). Lane 2 in (a) and lanes 2, 4, 6 in (b) represent lysates from mock-transfected cells (pBK-CMV). A specific signal of the ITIH5 proteinapproximately 100 kDa insize is seenonlyinthe ITIH5 clones (lane 1 in (a) and lanes 1, 3, 5 in (b)), but not in the mock- transfected cells. b-Actin was detected to prove equal loading of protein. (c and d) Immunocytochemistry of MDA-MB-231 cells transfected either with ITIH5 cDNA (c) or with empty vector (d). The ITIH5 antibody specifically detects ITIH5 protein in the ITIH5-transfected cell line. Magnification: Â 200. ITIH, inter-a-trypsin inhibitor heavy chain. Oncogene ITIH5 expression in breast cancer J Veeck et al 867 Figure 2 Expression of ITIH5 in normal breast tissue, non-invasive and invasive breast tumours. (a) Placental tissue, known to abundantly express ITIH5 (Himmelfarb et al., 2004) served as positive control. In negative controls (b), primary ITIH5-antibody was omitted. (c) Strong ITIH5 expressioninnormalbreast tissue. ( d) Scale-up of specimenshownin( c). The magnification demonstrates abundant ITIH5 expression in luminal-epithelial breast cells. (e) Very abundant ITIH5 expression in a ductal carcinoma in situ of the breast. (f) Scale-up of specimenshownin (e). (g) Invasive ductal breast cancer without detectable ITIH5 expression (IRSo2). (h) Scale-up of specimenshownin( g). (i) Invasive ductal breast