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-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.

Tumor Necrosis Factor-stimulated Gene 6 (TSG-6)-mediated Interactions with the Inter-α-inhibitor Heavy Chain 5 Facilitate Tumor Growth Factor β1 (TGFβ1)-dependent Fibroblast to Myofibroblast Differentiation

“…HC5 plays a role in tumor suppression with dysregulated or reduced expression of HC5 directly linked to tumor development (22,40,41) and its overexpression leading to suppression of cell migration and colony spreading (42). A recent study also found that HC5 was the major heavy chain expressed in skin, predominantly due to its production by dermal fibroblasts (43).…”

Epigenetic inactivation of the novel candidate tumor suppressor geneITIH5in colon cancer predicts unfavorable overall survival in the CpG island methylator phenotype

“…21 We could also demonstrate that ITIH5 is hypermethylated in circulating free DNA (cfDNA) of colon cancer patients with a frequency of 21% (12 of 58 serum cfDNA specimen was methylated). 27 To assess the biological relevance of ITIH5 in colon cancer we initially analyzed mRNA expression in 30 tumor tissue samples and 30 adjacent normal tissues from the same patient by real-time PCR.…”

“…21,35 We previously showed that loss of ITIH5 expression in breast 21,27 and bladder 22 cancer, caused by aberrant promoter hypermethylation, is associated with unfavorable prognosis. 19,21,22 Furthermore, we demonstrated a moderate hypermethylation of the ITIH5 promoter in circulating free DNA in the blood of colon cancer patients, 27 whereas the biological relevance of ITIH5 in colon cancer remains elusive. The current study is the first to analyze in depth ITIH5 expression, as well as its potential clinical and functional impact toward colon cancer.…”

“…19 Downregulation of ITIH5 in mammary as well as bladder tumors, due to a frequent hypermethylation of the ITIH5 promoter, was shown to be associated with a higher proliferation rate and malignant progression indicating a putative tumor suppressor function in breast and bladder cancer. [20][21][22] Furthermore, the ITI heavy chains effectively stabilize the ECM 23 and have been shown to be involved in processes such as tumor invasion 24 and metastasis, 25 while the biological relevance of ITIH5 in CRC remains unknown.…”

The Transfer of Heavy Chains from Bikunin Proteins to Hyaluronan Requires Both TSG-6 and HC2

“…The HCs exist in five variations (10,27). These all contain a vault protein inter-␣-trypsin domain and a von Willebrand type-A domain (10).…”