3, 3′5 Triiodo L Thyronine Induces Apoptosis in Human Breast Cancer MCF-7cells, Repressing SMP30 Expression through Negative Thyroid Response Elements

Repressing SMP30 Expression through Negative Thyroid Response Elements. PLoS ONE 6(6): e20861. doi:10.1371/journal.pone.0020861 3, 395 Triiodo L Thyronine Induces Apoptosis in Human Breast Cancer MCF-7cells, Repressing SMP30 Expression through Negative Thyroid Response Elements Pranati Sar 0 Rosalima Peter 0 Bandita Rath 0 Alok Das Mohapatra 0 Sandip K. Mishra 0 Nai Sum Wong, University of Hong Kong, Hong Kong 0 1 Cancer Biology Lab, Department of Gene Function and Regulation, Institute of Life Sciences , Chandrasekharpur, Bhubaneswar , India , 2 Vector Born Disease Lab, Department of Infectious Disease Biology, Institute of Life Sciences , Chandrasekharpur, Bhubaneswar , India Background: Thyroid hormones regulate cell proliferation, differentiation as well as apoptosis. However molecular mechanism underlying apoptosis as a result of thyroid hormone signaling is poorly understood. The antiapoptotic role of Senescence Marker Protein-30 (SMP30) has been characterized in response to varieties of stimuli as well as in knock out model. Our earlier data suggest that thyroid hormone 3, 395 Triiodo L Thyronine (T3), represses SMP30 in rat liver. Methodology/Principal Findings: In highly metastatic MCF-7, human breast cancer cell line T3 treatment repressed SMP30 expression leading to enhanced apoptosis. Analysis by flow cytometry and other techniques revealed that overexpression and silencing of SMP30 in MCF-7 resulted in decelerated and accelerated apoptosis respectively. In order to identify the cisacting elements involved in this regulation, we have analyzed hormone responsiveness of transiently transfected hSMP30 promoter deletion reporter vectors in MCF-7 cells. As opposed to the expected epigenetic outcome, thyroid hormone down regulated hSMP30 promoter activity despite enhanced recruitment of acetylated H3 on thyroid response elements (TREs). From the stand point of established epigenetic concept we have categorised these two TREs as negative response elements. Our attempt of siRNA mediated silencing of TRb, reduced the fold of repression of SMP30 gene expression. In presence of thyroid hormone, Trichostatin- A (TSA), which is a Histone deacetylase (HDAC) inhibitor further inhibited SMP30 promoter activity. The above findings are in support of categorisation of both the thyroid response element as negative response elements as usually TSA should have reversed the repressions. Conclusion: This is the first report of novel mechanistic insights into the remarkable downregulation of SMP30 gene expression by thyroid hormone which in turn induces apoptosis in MCF-7 human breast cancer cells. We believe that our study represents a good ground for future effort to develop new therapeutic approaches to challenge the progression of breast cancer. - Funding: This study was supported by funds from the Department of Biotechnology, Goverment of India. PS is supported by a research fellowship from University Grant Commission, Goverment of India, New Delhi. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The incidence of breast cancer has shown an alarming increase trend in recent years [1]. An estimated 1.7 million women will be diagnosed with breast cancer in 2020 which is a 26% increase from the current levels, mostly in the developing world [2,3]. The development and growth of many human cancers including breast cancers are known to be influenced by steroid hormones [4,5]. Abnormal responsiveness of the cells especially to estrogen hormone has been a major cause of breast cancer development and progression [6,7]. Therefore better understanding and manipulation of the endocrine milieu may provide effective palliative treatment for patients with hormone-dependent cancers [8,9,10]. Numerous environmental risk factors, pathological conditions and physiological agents as well as thyroid hormones have been proposed to influence the development of breast cancer [11]. Interestingly, Martinez et al reported that the addition of thyroid hormones at non physiological concentrations can alter mammary epithelial cells proliferation [12]. The thyroid gland releases two potent hormones, triiodothyronine (T3) and thyroxine (T4), which can influence and alter the basal metabolism or the oxygen consumption in virtually every cell in the body. However, due to conflicting results regarding the clinical correlation between breast cancer and thyroid diseases, any precise association between thyroid status and the pathogenesis of human breast cancer remains elusive [13]. Similar pathways are shared between thyroid hormone and estrogen in regulating proliferation and growth in the target cells, including cancer cells. So the aberrant signaling by these hormones needs to be evaluated in terms of regulated growth or cancer of the target cells. Receptors of these hormones are critically important in the above process of evaluation. Although the secretion of T4 from thyroid is several times greater than T3, the later is roughly two to three times more effective than the former. T3 binds to specific high affinity receptors called thyroid receptors (TRs) which belong to the super family of nuclear receptors [14] and mediate multiple effects on the phenotype, proliferation and gene expression of cultured normal mammary epithelial cells [15,16,17]. The TRs are ligand modulated transcription factors encoded by two genes, TRa and TRb, located on human chromosomes 17 and 3 respectively [11]. Dependency of human mammary neoplasia on thyroid hormones and controversial reports in literature about the relationship between the thyroid status of the patient and neoplastic illness [18,19,20] have suggested that thyroid hormone receptors (TRs) could potentially become a marker and a therapeutic target like the estrogen and progesterone receptors [21]. One of the recent studies reports substantial changes in the expression profile of TRs in breast cancer cells, suggesting a possible deregulation of TRs which could trigger breast cancer development [21,22,23]. However, only a few reports have described the presence of TRs in breast tumors [24,25] and breast cancer cell lines [26,27]. MCF-7 is one of the well defined breast cancer cell lines where presence of receptors for thyroid hormone has been well documented [26]. This finding makes it a suitable model to study the effect of thyroid hormones in breast cancer. The effect of thyroid hormone at the cellular level is mediated through TRs, by interacting with thyroid hormone response elements (TREs) in the promoters of the target genes to regulate transcription. Depending on their ability to bind to thyroid hormone, TRs can activate or suppress gene expression in a tissue specific manner through heterodimerisation with retinoid X receptors (RXRs) and interaction with the positive or negative response el (...truncated)