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.
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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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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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DOI: https://doi.org/10.1038/sj.onc.1210669
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