Genomic analysis of Metallothionein expression in breast carcinogenesis

Abstract

Breast cancer is a disease that has plagued many women globally. As the level of awareness for this mammary gland disorder is raised globally, more women are stepping forward to go for routine mammogram screening, implying a foreseeable surge in the number of breast cancer cases detected. Although the outcome need not be fatal, the physical, psychological and financial impacts inflicted extend beyond the patient. While advances in treatment are evolving at a rapid pace, mortality remains high. The rapid rise in incidence rate has prompted the rigorous search to understand its etiology and find better management strategies for this dreaded disease. Biomedical research has pointed towards a genetic origin in carcinogenesis, leading to a surge in experimental effort to uncover oncogenes responsible for this life threatening disease.

Metallothioneins (MTs) belong to a superfamily of metal-binding proteins. In human, four classes of MTs are represented by 10 function isoforms, viz. MT-1A, 1B, 1E, 1F, 1G, 1H, 1X, 2A, 3 and 4. Averaging 7 kDa in size, a single mammalian polypeptide is capable of binding 7 divalent cations through the 20 cysteine residues present and forming thiolate clusters. Inherently, MT binds to trace elements such as zinc and copper, as well as, cadmium and mercury toxic heavy metals. Dysregulation of MT has been associated with many diseases, including neurological disorders, cardiopathy, liver diseases and cancers. Previously, MT over-expression has been reported in many cases of breast cancers and the MT-2A gene, the most abundantly expressed isoform, has been implicated in tumourigenesis of the mammary glands. However, there is a paucity of information on the pathways regulated by this MT isoform in breast cancer. In this study, expression level of MT has been successfully silenced and up-regulated in MCF-7 breast cancer cells through RNA interference and stable transfected cell models respectively. These in vitro models demonstrated that over-expressing MT enhanced proliferative and migratory/invasive potentials of breast cancer cells, promoting a more aggressive phenotype. On the other hand, silencing the MT-2A isoform also induced a subset of MCF-7 cells to undergo entosis, manifesting a cell-within-cell feature. Internalized cells often underwent lysosomal degradation, supporting the notion that MT plays important role in the regulation of cell growth in breast cancers.

Through genomic analysis, candidate genes associated with MT manipulated expression were identified and the involvement of follistatin (FST), one of the candidate genes, was explored. Transwell and matrigel assays confirmed that MCF-7 cell displayed increased migratory and invasive capabilities after FST silencing. Along with other MT-associated genes, namely KLK12, BCAT1, CXCL12, MEK5, MAOB, BCHE and COL3A1, these results previewed plausible interactions between MTs and their molecular partners that could impact tumour development in luminal epithelial cells.

Moreover, evaluation conducted on clinical samples confirmed high levels of MT-2A transcript observed in invasive ductal carcinoma correlated with the percentage of tumourigenic cells within the lesion and cancer staging. Furthermore, increased MT immuno-expression was separately found to be positively associated with histological grade and lymph node metastasis in tissue samples from female patients diagnosed with invasive ductal carcinoma. These results corroborate some of the in vitro findings and could substantiate the potential of MT to be a biomarker of tumour development and prognosis in breast cancer.