The expression of the ubiquitin ligase SIAH2 (seven in absentia homolog 2) is mediated through gene copy number in breast cancer and is associated with a basal-like phenotype and p53 expression

Abstract

Introduction: The seven in absentia homolog 2 (SIAH2) protein plays a significant role in the hypoxic response by regulating the abundance of hypoxia-inducible factor-α; however, its role in breast carcinoma is unclear. We investigated the frequency and expression pattern of SIAH2 in two independent cohorts of sporadic breast cancers.

Methods: Immunohistochemical evaluation of SIAH2protein expression was conducted in normal breast tissues and in tissue microarrays comprising ductal carcinoma in situ (DCIS) and a cohort of invasive breast carcinomas. Correlation analysis was performed between SIAH2 and clinicopathological variables and intrinsic breast cancer subgroups and validated in a cohort of 293 invasive ductal carcinomas. Promoter methylation, gene copy number and mRNA expression of SIAH2 were determined in a panel of basal-like tumors and cell lines.

Results: There was a significant increase in nuclear SIAH2 expression from normal breast tissues through to DCIS and progression to invasive cancers. A significant inverse correlation was apparent between SIAH2 and estrogen receptor and progesterone receptor and a positive association with tumor grade, HER2, p53 and an intrinsic basal-like subtype. Logistic regression analysis confirmed the significant positive association between SIAH2 expression and the basal-like phenotype. No SIAH2 promoter methylation was identified, yet there was a significant correlation between SIAH2 mRNA and gene copy number. SIAH2-positive tumors were associated with a shorter relapse-free survival in univariate but not multivariate analysis.

Conclusions: SIAH2 expression is upregulated in basal-like breast cancers via copy number changes and/or transcriptional activation by p53 and is likely to be partly responsible for the enhanced hypoxic drive through abrogation of the prolyl hydroxylases.

Figure 1

Immunohistochemistry of seven in absentia homolog 2 (SIAH2) gene in normal, in situ and invasive breast carcinomas. (A) Occasional nuclear positivity (arrows) in luminal cells in the terminal duct lobular unit. (B) Moderate to strong staining of SIAH2 in the nucleus of a small proportion of the cell in a high nuclear grade ductal carcinoma in situ with comedo necrosis. (C) Occasional weak to moderate SIAH2 (arrows) staining in a luminal type ductal carcinoma. (D) Strong SIAH2 staining in all nuclei in this basal-like breast carcinoma.

Figure 2

Semiquantitative seven in absentia homolog 2 (SIAH2) gene expression in normal, ductal carcinoma in situ (DCIS) and invasive carcinoma samples.

Figure 3

Correlation between seven in absentia homolog 2 (SIAH2) gene copy number changes as assessed by the average logR array value of eight single-nucleotide polymorphisms (SNPs) which located in SIAH2 or within the flanking region of the gene and normalized expression of SIAH2 in 15 basal-like breast cancers.

Figure 4

Kaplan-Meier curves stratified by seven in absentia homolog 2 (SIAH2) gene expression for relapse-free survival in (A) all tumors (N = 246) and (B) luminal tumors (n = 144).