Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH) genes in multiple human solid tumors: A systematic expression analysis

Alexander Hamm,Peter J Wild,Tamra Werbowetski-Ogilvie,Ruth Knuechel,Glen Kristiansen,Uwe Heindrichs,Rolando Del Maestro,Juergen Veeck,Edgar Dahl,Nuran Bektas,Arndt Hartmann

doi:10.1186/1471-2407-8-25

Abstract

BackgroundThe inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by AMBP – and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5), contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis.MethodsWe systematically investigated differential gene expression of the ITIH gene family, as well as AMBP and the interacting partner TNFAIP6 in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas) using cDNA dot blot analysis (Cancer Profiling Array, CPA), semiquantitative RT-PCR and immunohistochemistry.ResultsWe found that ITIH genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, ITIH genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose ITIH2 expression in human breast cancer. Loss of ITIH2 expression in 70% of cases (n = 50, CPA) could be confirmed by real-time PCR in an additional set of breast cancers (n = 36). Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p < 0.001) between ITIH2 expression and estrogen receptor (ER) expression indicating that ER may be involved in the regulation of this ECM molecule.ConclusionAltogether, this is the first systematic analysis on the differential expression of ITIH genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies.

Highlights

The inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by alpha-1-microglobulin/ bikunin precursor (AMBP) – and five homologous heavy chains, contributing to extracellular matrix stability by covalent linkage to hyaluronan
The light chain is encoded by alpha-1-microglobulin/ bikunin precursor (AMBP), which codes for alpha-1microglobulin, a member of the lipocalin superfamily that is not functionally or structurally related to the ITI family[3]
Each of the gene probes was hybridized to a Northern blot containing poly A+ RNA derived from 16 different human normal tissues (Multiple Tissue Northern Blot – MTN) to determine its transcript size(s) and the specificity of the probes later used for cDNA dot blot hybridization

Summary

Introduction

The inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by AMBP – and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5), contributing to extracellular matrix stability by covalent linkage to hyaluronan. The transfer of the ITI heavy chains – due to this linkage called serum-derived hyaluronan-associated protein (SHAP)[10] – onto HA requires tumor necrosis factor alpha induced protein 6 (TNFAIP6), known as TNF stimulated gene (TSG-6) [11]. The main function of ITIH is proposed to be an essential factor in the stabilization of the ECM[1] (extensively investigated in cumulus oophorus cells[18]), based on the covalent linkage of HA to the heavy chain, producing socalled "cable-like structures"[2]

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Discussion

Conclusion