Abstract 3996: Genomic analysis in breast cancer cells after metallothionein-2A silencing

Abstract

Introduction Breast cancer is the most prevalent type of cancer reported in women and mammoth effort has been put in to understand its etiology. Metallothionein (MT), a divalent cation binding protein, was previously shown to be associated with histopathological parameters of breast cancer and mediated cancer progression. MTs belong to a family of ubiquitous, low-molecular weight house-keeping proteins. In human, 10 known functional isoforms of MT, namely 1A, 1B, 1E, 1F, 1G, 1H, 1X, 2A, 3 and 4, encode 4 classes of MT proteins. MT-2A has been shown to be the most abundant isoform in breast cancers and was reported to promote cell proliferation and chemo-resistance. Screening for MT-2A and its associated genes may elucidate the role it plays in the progression of breast neoplasia and help devise therapeutic strategies to combat this disease. Methods Chemically synthesized small interfering RNA (siRNA) targeting the MT-2A isoform was employed to down-regulate its expression in MCF-7 breast cancer cells and validated via real-time Polymerase Chain Reaction (PCR) and immunocytochemistry. Cell viability and drug sensitivity of transfected cells were evaluated post silencing using a tetrazolium based (MTT) assay. RNAs extracted from MT-2A silenced cells were hybridized to Affymetrix gene chips for genome-wide screening and compared to control group treated with scrambled sequence siRNA. Analysis was performed across multiple platforms such as GeneSpring, dChip and GCOS to “fish” for genes associated with MT-2A down-regulation. Candidate genes that demonstrated at least 2 fold change statistically (p<0.05) were further screened via real-time PCR to confirm their association with MT-2A silencing. Results Silencing efficiencies of 88% and 90% were achieved through the use of two different MT-2A specific siRNA duplexes (siMT-2A_1 and siMT-2A_2) respectively. Cells exhibited reduced viability (∼10% lesser) and more evidently (up to 76% lesser) after Doxorubicin administration at IC50 post MT-2A silencing. Results obtained from the cDNA microarray indicate that interferon inducible genes such as interferon induced proteins with tetratricopeptide repeats (IFITs), interferon induced transmembrane protein 1 (IFITM1) and oligoadenylate synthetase (OAS) accounted for more than 54% (12/22) of the candidate gene list and these genes showed concordant fold changes in real-time PCR analysis. Conclusion The results suggest that MT-2A may regulate interferon signaling directly or indirectly. This set of candidate genes are known to play important roles in apoptosis and chemo-resistance. The molecular interaction between MT-2A and members of the interferon inducible proteins warrants further attention and we postulate that they may work co-operatively to contribute to tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3996.

Metallothionein isoforms as double agents – Their roles in carcinogenesis, cancer progression and chemoresistance

“…MT-1F has been positively correlated with malignancy grade (Jin et al, 2001), whereas MT-1E expression has been found in estrogen receptor-negative breast cancer cell lines (Friedline et al, 1998;Jin et al, 2001). In addition, MT-2A has been identified as the most abundant in breast cancer and plays a protective role in these cancer cells, with high expression involved in cell cycle regulatory capabilities (Lai et al, 2010;Lai et al, 2011). MT overexpression can also induce other proteins, e.g., MT-2A induces the overexpression of MMP-9 with subsequent induction of the invasive phenotype of breast cancer cells (Emri et al, 2013).…”