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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-α-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.

Introduction

The inter-α-trypsin inhibitors (ITIs) comprise a family of protease inhibitors found in the extracellular matrices of various organs, as well as in the blood circulation. Owing to their original isolation in complexes with hyaluronan (HA), ITIs are also referred to as SHAPs (Serum-derived HA-associated proteins) (Yoneda et al., 1990). It has been previously shown that interaction of ITIs with HA leads to stabilization of the extracellular matrix (Chen et al., 1994). ITI molecules consist of three protein chains: one ITI light chain, also referred to as bikunin, and two ITI heavy chains (ITIHs) (Enghild et al., 1991). The transfer of ITIHs onto HA requires tumour necrosis factor α-induced protein 6 (TNFAIP6), also known as TNF-stimulated gene 6 (Jessen and Odum, 2003). TNFAIP6 forms a stable complex (Rugg et al., 2005) with ITIH and HA during the transesterification reaction (Sanggaard et al., 2005). This formation of ITIH–HA complexes is thought to play an important role in the stabilization of HA-rich extracellular matrices, with TNFAIP6 acting as an essential cofactor and catalyst (Rugg et al., 2005). To date, five distinct ITIHs have been identified, encoded by five genes located on two different chromosomes (Diarra-Mehrpour et al., 1989; Himmelfarb et al., 2004). ITIH1, -3 and -4 have been mapped to chromosome 3p2.11–12, and ITIH2 and -5 are located on chromosome 10p14–15 (Bost et al., 1998; Himmelfarb et al., 2004). ITIH1, -2 and -3 are synthesized primarily in liver as polypeptide precursors, which undergo extensive post-translational processing; they all contain a conserved cleavage site, which enables them to form covalent bonds to bikunin via glycosaminoglycan (Enghild et al., 1991).

Various studies (Kobayashi et al., 1995; Bourguignon et al., 1999; Paris et al., 2002; Zhang et al., 2004) have shown involvement of ITIs in tumour biology using an expressed sequence-tag-based bioinformatics approach (Dahl et al., 2005). We have previously identified ITIH5 as a novel gene differentially expressed in breast cancer. After cloning, mapping and determination of its genomic organization, we analysed the expression of ITIH5 at the messenger RNA level in a panel of normal human tissues and a small set of normal and malignant breast tissue samples. Initial analyses showed that ITIH5 was predominantly expressed in human female reproductive tissues and strongly downregulated in breast tumours, suggesting a potential role in breast cancer development (Himmelfarb et al., 2004). In the present study, protein expression of ITIH5 was analysed in a large set of breast tumours using tissue microarrays (TMAs). Furthermore, the epigenetic configuration of the ITIH5 promoter was comprehensively investigated in breast cancer cell lines and primary breast tumour tissue samples and the biological role of ITIH5 was evaluated with functional in vitro assays. Clinicopathological patient characteristics were statistically correlated with expression and methylation data and revealed an unfavourable prognosis in case of methylation-mediated loss of ITIH5 expression.

Results

Characterization of the anti-ITIH5-specific antibody

The specificity of the rabbit polyclonal antiserum raised against a synthetic peptide corresponding to amino acids 207–220 of human ITIH5 protein was evaluated by western blot analysis. The antibody is able to detect human ITIH5 protein expressed in ITIH5-transfected COS7 cells (Figure 1a, lane 1) as well as in ITIH5-transfected MDA-MB-231 breast cancer cell lines (Figure 1b; lanes 1, 3, 5 represent different clones). No protein was detectable in mock-transfected COS7 cells (Figure 1b; lane 2) and mock-transfected MDA-MB-231 cells (Figure 1b; lanes 2, 4, 6), arguing that the ITIH5 antibody does not detect any protein, that is not a modification of the ITIH5 protein. ITIH5 protein in the transfected cell lines was approximately 100 kDa in size, in accordance with its deduced molecular weight. Immunocytochemical staining of transfected cells showed abundant ITIH5 protein in the cytoplasm of MDA-MB-231 breast cancer cells (Figure 1c). Mock-transfected MDA-MB-231 cells did not exhibit ITIH5 staining (Figure 1d).

Figure 1
figure1

Characterization of the ITIH5 antibody. (a and b) Detection of ITIH5 protein of 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 protein approximately 100 kDa in size is seen only in the ITIH5 clones (lane 1 in (a) and lanes 1, 3, 5 in (b)), but not in the mock-transfected cells. β-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: × 200. ITIH, inter-α-trypsin inhibitor heavy chain.

ITIH5 protein is downregulated in the course of breast tumour progression

Immunohistochemical analysis was applied to investigate ITIH5 protein expression in normal and malignant breast tissue using a TMA containing 217 invasive breast carcinomas, 10 ductal carcinomas in situ (DCIS) and 15 normal breast tissue samples. Intensity and quantity of immunohistochemical staining was evaluated using a semiquantitative immunoreactivity score (IRS) (Remmele and Stegner, 1987). ITIH5 protein was strongly expressed in 73% (11/15) of normal breast tissue samples (Figures 2c and d) and was localized in luminal epithelial cells of the normal breast with absence in myoepthelial cells, fibroblasts, adipocytes and endothelial cells. Subcellularly, ITIH5 was localized to the cytoplasm and the cell membrane. In DCIS (Figures 2e and f), ITIH5 expression was as prominent as in normal breast tissue. However, invasive carcinomas showed strongly reduced or complete loss (IRS2) of ITIH5 expression in 42.4% (92/217) (Figures 2g and h) and a moderate expression (IRS 3–9) in 28.1% (61/217) of cases (Figures 2i and j). Interestingly, 29.5% (64/217) of invasive breast tumours had an IRS of 12, thus presenting very abundant ITIH5 expression (Figures 2k and l).

Figure 2
figure2

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 expression in normal breast tissue. (d) Scale-up of specimen shown in (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 specimen shown in (e). (g) Invasive ductal breast cancer without detectable ITIH5 expression (IRS<2). (h) Scale-up of specimen shown in (g). (i) Invasive ductal breast cancer with moderate ITIH5 expression (IRS=4). (j) Scale-up of specimen shown in shown in (i). (k) Invasive ductal breast cancer with abundant ITIH5 expression (IRS=12). (l) Scale-up of specimen shown in (k). Magnifications: (c, e, g, i and k): × 100; (j and l): × 200; (a, b, d, f and h): × 400. IRS, immunoreactivity score; ITIH, inter-α-trypsin inhibitor heavy chain.

Correlation of ITIH5 expression with clinicopathological parameters and patient survival

Clinicopathological characteristics were correlated with ITIH5 immunohistochemistry results for descriptive data analysis (Table 1). ITIH5 immunohistochemical staining was significantly associated with positivity of oestrogen receptor (ER) (P=0.008) and low (G1/G2) histological grade (P=0.024). Recurrence-free survival (RFS) and overall survival (OS) were compared between invasive breast tumours showing strong ITIH5 expression (IRS=12) and all other invasive tumours by univariate log-rank statistics (Table 2). Strong ITIH5 expression was clearly associated with longer RFS and OS, as shown by Kaplan–Meier analysis (Figure 3). Patients with strong ITIH5 expression in the tumour had an estimated mean RFS of 102 months (95% confidence interval (CI): 88–116) compared to 78 months (95% CI: 68–88) in patients with loss of ITIH5 expression (P=0.037) (Figure 3a). Strong ITIH5 expression was also associated with longer OS of 120 months (95% CI: 108–132) compared to 105 months (95% CI: 96–114) in patients with partial or total loss of ITIH5 expression (P=0.044) (Figure 3d). In a stratified univariate analysis, the prognostic value of ITIH5 became even more pronounced in the clinically important subgroup of node-negative patients (Figures 3b and e) for RFS and OS alike. However, Cox regression models including tumour grade (G1 and G2 vs G3), pT-stage (pT1 and pT2 vs pT3 and pT4), node status (negative vs positive), hormone receptor status (ER/PR combined, double negative vs any positive) and ITIH5 (IRS 12 vs 0–9) failed significance in confirming the prognostic value of ITIH5 as an independent marker (Tables 3a and b) due to the close association with ER.

Table 1 Clinicopathological and immunohistochemical factors in relation to ITIH5 immunoreactivity
Table 2 Univariate analysis of clinicopathological factors regarding recurrence-free survival and overall survival
Figure 3
figure3

Univariate survival analysis of ITIH5 immunoreactivity according to the Kaplan–Meier (KM) method concerning recurrence-free (ac) and overall survival (df). In (a) and (d) the KM-Analysis of all cases is shown. (b) and (e) illustrate the strong prognostic value of ITIH5 in the clinically relevant group node-negative patients (pN0), which is not demonstrable in node-positive patients (pN1–3) (c) and (f). Grey line: strong ITIH5 staining (IRS=12); black line: weaker ITIH5 staining. Crosses in each graph indicate censored patients. IRS, immunoreactivity score; ITIH, inter-α-trypsin inhibitor heavy chain.

Table 3a Cox multivariate analysis for RFS and ITIH5 immunohistochemistry
Table 3b Cox multivariate analysis for OS and ITIH5 immunohistochemistry

ITIH5 downregulation in breast cancer is caused by promoter hypermethylation

Analysis of the ITIH5 gene promoter using the genomic DNA information contained in ENSEMBL contig ENSG00000123243 based on NCBI Build 36.1 showed three CpG-rich islands between genomic positions 7747928 and 7749039 (−1012 to +99 relative to the expected transcription start site) on chromosome 10p. Figure 4a gives an overview of the analysed promoter sequence and the regulatory region analysed by methylation-specific PCR (MSP).

Figure 4
figure4

ITIH5 methylation and expression analysis in breast cell lines. (a) Schematic representation of the human ITIH5 promoter region. A 1.2-kb fragment is shown. A, B and C indicate predicted binding sites of regulatory transcription factors (A: CCAAT/enhancer-binding-protein-β; B: MYC-MAX-binding site; C: GC-box elements). The CpG-rich islands (black bars) are shown relative to transcriptional start (+1). Double-head arrow represents the amplified region by methylation-specific PCR (b) Methylation status of the ITIH5 promoter in nonmalignant (MCF12A) and malignant (BT20, MCF7, SKBR3, T47D, MDA-MB-231 and Hs578-T) breast cell lines. Bands labelled with U and M indicate unmethylated and methylated alleles, respectively. (c) Expression of ITIH5 mRNA in breast cell lines. Cells with a methylated promoter lack mRNA expression, whereas unmethylated MCF12A and the very weakly methylated Hs578T cells exhibit strong expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. (d) RT–PCR showing ITIH5 mRNA expression before and after incubation with 1 μ M 5-aza-2′-deoxycytidine (DAC) and 300 nM trichostatin A (TSA). ITIH5 mRNA expression is induced after demethylating treatment. Expression of GAPDH served as a control for equal starting amounts of cDNA. (e) Semiquantitative real-time PCR of the same experiment. Grey bars represent nontreated cells, black bars indicate DAC/TSA-treated cells. Gain of ITIH5 mRNA expression is indicated as fold-change (X) relative to each baseline expression. Relative Y-axis scaling is related to nontreated BT20 cells (set to 1). Level of induction is significant (P=0.014). Error bars were derived from triplicate experiments. ITIH, inter-α-trypsin inhibitor heavy chain.

Using MSP (Herman et al., 1996), a nonmalignant (MCF12A) and six malignant breast cell lines (BT20, MCF7, SKBR3, T47D, MDA-MB-231 and Hs578T) were initially studied (Figure 4b): The nonmalignant cell line exhibited lack of ITIH5 promoter methylation in the analysed promoter region, whereas five of six malignant cell lines showed ITIH5 promoter methylation—Hs578 T was the only exception. Reverse transcription–PCR (RT–PCR) analysis showed an inverse association between promoter methylation and ITIH5 expression, that is, abundant ITIH5 expression in MCF12A and Hs578T cells and absence of ITIH5 mRNA in BT20, MCF7, SKBR3, T47D and MDA-MB-231 breast cancer cells (Figure 4c). This association was further supported by treatment of four cell lines lacking ITIH5 expression with 5-aza-2′-deoxycytidine (DAC) and trichostatin A (TSA): RT–PCR and real-time PCR analyses 24 h after treatment (Figures 4d and e) showed expression of ITIH5 in all cell lines (P=0.014, one-tailed U-test).

Consistently, analysis of 109 invasive human breast cancer and ten matching normal breast tissue samples by MSP showed ITIH5 promoter hypermethylation in 77 breast tumours (70.6%), and a very weak methylation signal in only one normal breast tissue sample. Of the methylated samples, 32/77 (29.4% of all) had only weak methylation and 45/77 (41.3% of all) had strong methylation detectable. Representative results are shown in Figure 5a.

Figure 5
figure5

Methylation analysis of the ITIH5 promoter in primary breast cancers and correlation with ITIH5 gene silencing. (a) Representative results of three matched pairs (T=tumour, N=normal breast epithelium) and 11 invasive breast cancer specimens are shown. Bands labelled with U represent positive results from PCR reactions amplifying unmethylated promoter sequence; bands labelled with M indicate methylated promoter sequence. Bisulphite-converted DNA from the benign breast cell line MCF12A and from in vitro methylated DNA (IVD) (Esteller et al., 1999) served as positive controls for U- and M-reactions, respectively. H2O indicates the negative control. (b) Expression levels of breast cancer specimens (n=39) were analysed by real-time PCR and grouped according to the strength of their promoter methylation. Comparing the median expression levels (grey bars) of each group indicates a coherence of methylation with expression loss. All samples were related to an mRNA sample derived from normal breast tissue containing >50% breast epithelium (set to 1 on X-axis scaling). (c) Kaplan–Meier survival curves displaying patient overall survival and (d) recurrence-free survival in relation to ITIH5 promoter methylation. ITIH, inter-α-trypsin inhibitor heavy chain.

Correlation of promoter methylation with ITIH5 mRNA expression

To further prove that promoter methylation abrogates ITIH5 expression, we analysed in parallel 39 samples by real-time PCR and also by MSP. Figure 5b illustrates the distribution of ITIH5 expression among the groups of unmethylated, weakly methylated and strongly methylated samples. Comparison of the median expression level of each group reveals a significant coherence with promoter methylation, since a stable decrease of ITIH5 mRNA with increasing promoter methylation was detected.

Correlation of ITIH5 methylation with clinicopathological parameters and patient survival

Clinicopathological characteristics were correlated with ITIH5 methylation for descriptive data analysis (Table 4). ITIH5 promoter methylation was not significantly associated with tumour stage, lymph node status or histological grade. RFS and OS were compared between methylated and unmethylated ITIH5 alleles by univariate log-rank statistics. ITIH5 methylation displayed a trend towards association with occurrence of relapse (P=0.067; data not shown) and was highly significantly associated with shorter OS (P=0.008) (Table 4) as also illustrated by Kaplan–Meier analysis (Figures 5c and d). A Cox regression model was calculated on OS, but all parameters included failed significance, although ITIH5 methylation was of borderline significance (Table 5; P=0.055).

Table 4 Clinicopathological/immunohistochemical parameters in relation to ITIH5 promoter methylation and univariate analysis of factors regarding overall survival
Table 5 Cox multivariate analysis for OS and ITIH5 methylation

Reduced proliferation rates of MDA-MB-231 breast cancer cells after forced expression of ITIH5

A full-length ITIH5 cDNA derived from normal breast tissue was cloned into the eukaryotic expression vector pBK-CMV (Himmelfarb et al., 2004). A proliferation assay was performed for two independent ITIH5-transfected clones in MDA-MB-231 cells and two independent mock-transfected clones using XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assays (Figure 6). ITIH5 protein expression in the ITIH5-transfected clones and lack of expression in the mock-transfected clones had been verified by western blot (Figure 2b, lanes 3 and 5 and lanes 2 and 4, respectively). The optical density measured at the first time point (24 h) was set equal to 1. We observed an increasing growth retardation of the ITIH5-transfected compared with the mock-transfected cells, which reached 39.8% growth reduction 96 h after plating.

Figure 6
figure6

ITIH5 expression reduces proliferation in the breast cancer cell line MDA-MB-231. Stable clones have been generated with a full-length cDNA of ITIH5 (ITIH5 clone 4 and 8) or with empty pBK-CMV vector control (Mock clone 1 and 2). An XTT assay was performed at four subsequent time points. The baseline level at 24 h for each clone was set to 1. ITIH, inter-α-trypsin inhibitor heavy chain; XTT, 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.

Discussion

Thus far, numerous studies have shown involvement of ITIs in various pathological processes, including formation of urinary tract stones (Atmani and Khan, 1999; Janssen et al., 2001), female infertility (Zhuo et al., 2001), atherosclerosis and vascular disease (Onda et al., 1999; Fujita et al., 2004), and inflammation (Balduyck et al., 2000; Yingsung et al., 2003; Pineiro et al., 2004). Furthermore, several lines of evidence suggest a potential role of ITIs in tumour biology, particularly in the metastatic cascade and tumour progression (Kobayashi et al., 1995; Bourguignon et al., 1999; Paris et al., 2002; Zhang et al., 2004).

We have previously described cloning and RNA expression analysis of a novel member of the ITIH gene family, ITIH5, in normal and malignant breast tissues (Himmelfarb et al., 2004). We have now generated a polyclonal anti-ITIH5 antibody to evaluate ITIH5 expression at the protein level employing a comprehensive TMA.

Consistent with our previously published RNA expression data, we found that ITIH5 was downregulated at the protein level in a large number of breast tumours as compared to normal breast tissue, suggesting that ITIH5 expression is lost in the course of tumour progression. Correlation with clinicopathological data of the invasive breast tumours analysed in the present study showed that strong ITIH5 immunohistochemical staining was associated with longer RFS and OS. Intriguingly, ITIH5 remains a significant prognostic factor in the clinically important subgroup of patients with node-negative tumours, as shown by univariate analysis, although this finding was multivariate insignificant when the hormone receptor status was included in the analysis. It remains to be shown in confirmatory studies, if ITIH5-positivity can help to identify those patients with very low risk of tumour recurrence.

A growing number of genes have been reported to be silenced by promoter hypermethylation in breast cancer effectively silencing expression of their corresponding proteins. They fall into several broad categories of fundamental cellular networks, such as cell cycle control or steroid receptor regulation (for a review, see Yang et al., 2001), thus representing critical genes exerting a tumour suppressive function. Few studies demonstrated that cell adhesion molecules and proteins involved in cell–matrix interactions are also target of epigenetic downregulation in breast cancer (Graff et al., 1995; Bachman et al., 1999; Shao et al., 2006). Decrease in cell adhesion and increase of proteolytic activity of extracellular matrix components are thought to contribute largely to a tumour's invasive phenotype.

To analyse the cause of downregulation of ITIH5, we investigated the epigenetic configuration of its gene promoter in breast cell lines and in primary breast carcinomas. Expression analysis of normal and malignant breast cell lines showed that methylation of the ITIH5-promoter was associated with absence, whereas demethylation was associated with presence of ITIH5 mRNA. Furthermore, treatment of cell lines with demethylating and histone acetylating agents led to restoration of ITIH5 expression. Moreover, analysis by MSP showed clear ITIH5-promoter methylation in 41% of breast tumours, matching very well with a complete loss of the ITIH5-protein in 42% of breast tumours as analysed by immunohistochemistry. Since ITIHs represent the matrix-adhesive component of ITIs, these findings support the fundamental role of loss of adhesion molecules in tumorigenesis, like it has also been shown for hypermethylation of the E-cadherin gene (Graff et al., 1995), the tissue inhibitor of metalloproteinase 3 (Bachman et al., 1999) or the betaig-h3 gene (Shao et al., 2006).

To elucidate if the ITIH5 methylation status is equally discriminatory in patient-survival estimation as the ITIH5 protein level is, we performed statistical evaluations and found that methylation of the ITIH5 promoter was also significantly associated with reduced OS (P=0.008). The biological role of ITIH5 was evaluated by a functional assay that showed a 40% reduced proliferation rate in ITIH5-transfected cells as compared to mock-transfected control cells. Taken together, these findings provide evidence that ITIH5 acts as a tumour-suppressor gene in normal breast tissue. Loss of ITIH5 expression may be involved in tumour development, particularly since aberrant promoter hypermethylation of tumour suppressor genes is a well-established mechanism of tumour progression (Das and Singal, 2004; Esteller, 2005). Moreover, ITIH5 may represent a class II tumour suppressor gene whose altered expression is caused by epigenetic changes (class II) rather than by mutation (class I) (Sager, 1989). Class II tumour suppressor genes are particularly interesting drug targets, since reversing the block of their gene expression could lead to tumour regression.

Interestingly, the results on the expression analysis of ITIH5 in normal and malignant breast tissue are in line with those of previous studies investigating in general the role of ITIs in epithelial tumours. A role of ITIs in tumour invasion has been postulated on the basis of their protease inhibitor function mediated by bikunin (Kobayashi et al., 1995), and their capacity to bind to the extracellular matrix component HA mediated by the heavy chains (Huang et al., 1993; Zhao et al., 1995). In a lung cancer mouse model, overexpression of ITI protein chains led to inhibition of tumour progression and metastatic spread (Bourguignon et al., 1999).

In conclusion, our study indicates that ITIH5 may represent a class II tumour suppressor gene whose loss of expression is associated with short RFS and OS in breast cancer patients. In the clinically very important group of patients with node-negative breast cancer, ITIH5 could be used as a predictive marker to identify those patients who would not benefit from systemic chemotherapy and thus could be spared from its adverse effects. To our knowledge, this is one of only a few studies published so far identifying a biomarker potentially valuable for prognosis prediction on both the expression level and the epigenetic level.

Materials and methods

Breast cancer tissue micro array

ITIH5 protein expression was assessed using a TMA with 217 breast cancer cases that have been described previously (Dahl et al., 2006). The TMA contained one tissue core from nonselected, formalin-fixed, paraffin-embedded primary breast cancer specimens diagnosed between 1994 and 2002 at the Institute of Pathology, University of Regensburg, Germany. Histologically, all tumours were graded according to Elston and Ellis (1991). Clinical follow-up data were available for all 217 breast cancer patients with a median follow-up period of 78 months (0–148 months). The Institutional Review Board of the participating centres approved the study.

Cryoconserved patient samples

Breast tissue samples for methylation and mRNA expression analysis were obtained from patients treated by primary surgery for breast cancer at the Department of Gynecology at the University Hospitals of Aachen, Jena and Regensburg, Germany. All patients gave informed consent for retention and analysis of their tissue for research purposes. Tumour material was snap-frozen in liquid nitrogen immediately after surgery. Hematoxylin and eosin-stained sections were prepared for assessment of the percentage of tumour cells, only samples with >70% tumour cells were selected. For patient characteristics, see Supplementary Table 1.

Cell lines

All breast cell lines were obtained from the American Type Culture Collection and cultured as described previously (Veeck et al., 2006). For Hs578T, medium was additionally supplemented with 1 mM sodium pyruvate and 10 μg/ml insulin (Sigma-Aldrich, Deisenheim, Germany).

Expression constructs and transfection

The coding sequence of ITIH5 was amplified from a human normal breast tissue cDNA (Acc. No. AY238437) and cloned into pBK-CMV expression vector as described previously (Himmelfarb et al., 2004). For transfection experiments, cells were grown at 60–70% confluence. Transfection was carried out using the jetPEI transfection reagent (Biomol, Hamburg, Germany), following the manufacturer's instructions.

Generation of anti-ITIH5 antibody

An ITIH5 polyclonal antiserum was generated at Eurogentec (Herstal, Belgium) by immunizing rabbits with a synthesized peptide corresponding to amino acids 207–220 of the human ITIH5 protein. The antibody was then extracted and affinity purified.

Western blot analysis

Protein extracts were prepared from mock-transfected and ITIH5-transfected cell lines by incubation with RIPA buffer (Pierce, Rockford, IL, USA) for 30 min on ice. Approximately 20 μg of protein were resolved by sodium dodecylsulphate–polyacrylamide gel electrophoresis and blotted to nitrocellulose filters. Membranes were incubated with primary antibody (polyclonal anti-ITIH5 200 ng/ml) overnight at 4°C. After washing with phosphate-buffered saline Tween-20 (PBS-T), membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Chemicon International, Hampshire, UK; 1:30 000) for 90 min at room temperature. Antibody detection was performed with the ‘ECL plus’ western blotting detection system (Amersham Life Science, Buckinghamshire, UK).

Immunocytochemical staining

After fixation with 4% paraformaldehyde, mock- and ITIH5-transfected MDA-MB-231 cells were permeabilized using 0.05% Tween. Endogenous peroxidase activity was blocked by adding 3% H2O2 for 10 min. A polyclonal anti-ITIH5 antibody (1:200) was added for 1 h. After washing twice with PBS, cells were incubated with the 4plus Universal Immunoperoxidase Detection System (Biocarta, Hamburg, Germany), followed by 10 min incubation with a streptavidin–HRP complex. 3-amino,9-ethyl-carbazole (AEC) chromogen substrat (Romulin AEC; Biocarta) was used for antibody detection. Samples were counterstained with hemalaun and examined by phase contrast microscopy (Leica TCS) (Leica, Bensheim, Germany).

ITIH5 immunohistochemistry

Paraffin-embedded tissue sections (2 μm) were subjected to immunostaining using the Envision system (DAKO, Hamburg, Germany), following the manufacturer's instructions. Antigen retrieval was performed by pre-treatment in citrate buffer (pH 6) in a microwave oven (30 min). The sections were incubated for 1 h at room temperature with primary antibody (polyclonal anti-ITIH5, 1:200). Slides were incubated for 10 min with secondary antibody (biotinylated polylink; Biocarta). AEC chromogen substrate (Romulin AEC; Biocarta) was used for antibody detection. An experienced breast cancer pathologist (E.B.) scored the immunohistochemical staining intensity according to the scoring system suggested by Remmele and Stegner (1987).

Nucleic acid extraction

Genomic DNA from cell culture and primary invasive breast tumours were isolated using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and RNA was extracted by use of TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturers' recommendations.

RT–PCR

A 1 μg portion of total RNA was reverse transcribed using the reverse transcription system (Promega, Madison, WI, USA). To improve transcription rate, we mixed oligo-dT and pdN(6)-Primers 1:2. PCR was carried out as described previously (Veeck et al., 2006) using ITIH5 and GAPDH primers as given in Supplementary Table 2.

Semiquantitative real-time PCR

Semiquantitative PCR was performed using the LightCycler system together with the LightCycler DNA Master SYBR Green I Kit (Roche Diagnostics, Mannheim, Germany) as described elsewhere (Veeck et al., 2006). Primer sequences for ITIH5 and GAPDH are listed in Supplementary Table 2. To ensure experiment accuracy, all reactions were performed in triplicates.

Bisulphite-modification and MSP

For MSP analysis, breast cell lines and cryoconserved breast cancer specimens (see Supplementary Table 1) were used. The DNA modification Kit (Chemicon, Ternecula, CA, USA) was applied according to the manufacturer's recommendations. For MSP (Herman et al., 1996), 1 μl of modified DNA was amplified using MSP primers (Supplementary Table 1), which specifically recognized either the unmethylated (product size 221 bp) or methylated (219 bp) ITIH5 promoter sequence after bisulphite conversion (Suzuki et al., 2002). For MSP reaction conditions, see Veeck et al. (2006). Normal DNA from human appendix vermiformis was treated in vitro with SssI methyltransferase (New England Biolabs, Beverly, MA, USA), to generate a positive control for methylated alleles (Esteller et al., 1999).

DAC and TSA treatment

A demethylating treatment of breast cancer cell lines was performed as previously described (Veeck et al., 2006).

Proliferation assay

The XTT proliferation assay from Biological Industries (Frankfurt, Germany) was used. Cells were plated and cultivated in a flat 96-well plate (1 × 103 cells/well). To each well, 100 μl of growth medium was added. Proliferation was assayed after 24, 48, 72 and 96 h of incubation: 50 μl of XTT reagent solution was added to each well and the plate was incubated for 4 h at 37°C. The absorbance of the samples was measured at 450 nm.

Statistical analysis

For statistical evaluation, the SPSS software version 10.0 (SPSS GmbH Software, Munich, Germany) was used. Differences were considered statistically significant when P-values were <0.05. A Mann–Whitney U-test was employed to analyse differences in expression levels. A statistical association between clinicopathological and molecular parameters was tested using a two-sided Fisher's exact test. RFS and OS were calculated according to the Kaplan–Meier method. Multivariate survival analysis was calculated using a Cox regression model including all parameters that were significantly prognostic in univariate analysis.

Conflict of interest

The authors have declared that no competing interests exist.

Abbreviations

CI:

confidence interval

DAC:

5-aza-2′-deoxycytidine

DCIS:

ductal carcinoma in situ

ER:

oestrogen receptor

IRS:

immunoreactivity score

ITI:

inter-α-trypsin inhibitor

ITIH:

inter-α-trypsin inhibitor heavy chain

MSP:

methylation-specific PCR

OS:

overall survival

PR:

progesterone receptor

RFS:

recurrence-free survival

TMA:

tissue microarray

TSA:

trichostatin A

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Acknowledgements

We thank Sonja von Serényi, Sevim Alkaya and Inge Losen for excellent technical assistance and Monika Klinkhammer-Schalke and Armin Pauer from the Tumor Registry Regensburg for continuous help in obtaining clinical follow-up data. This work is a research project within the German Human Genome Project and has been supported by the BMBF Grants 01KW0401 to ED and a grant from the RWTH Aachen (START program project ITIH5).

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Correspondence to E Dahl.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Veeck, J., Chorovicer, M., Naami, A. et al. 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. Oncogene 27, 865–876 (2008). https://doi.org/10.1038/sj.onc.1210669

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Keywords

  • inter-α-trypsin inhibitor heavy chain (ITIH)
  • breast cancer
  • prognostic marker
  • predictive marker
  • tumour invasion
  • metastasis

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