BackgroundThyroid 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, 3′5 Triiodo L Thyronine (T3), represses SMP30 in rat liver.Methodology/Principal FindingsIn 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 cis–acting 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 TRβ, 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.ConclusionThis 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.

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 (T 3 ), 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.

ERRβ regulates transcriptional activity of ERα as well as ERβ by modulating BCAS2 expression and leads to FST-mediated induction of nuclear fragmentation in ER-positive MCF-7 breast carcinoma cells Dharmendra K Bhargava*, Debomita Sengupta*, Sanjib Choudhary, Rosalima Peter, Dipti Ranjan Mishra, Sandip K Mishra. Corresponding Author *Equal Contribution Institute of Life Sciences (an institute under Department of Biotechnology, Govt. of INDIA), Nalco Square, Bhubaneswar, Odisha, INDIA Abstract: Estrogen related receptors (ERRs) are a group of nuclear receptors which are structurally and functionally related to Estrogen receptors, but do not bind to Estrogen. Diethylstilbestrol, which has been shown to act as a ligand of ERRs, can inhibit growth of ER-positive as well as Tamoxifen-resistant ER negative Breast Cancer cell lines and thus justifies the need of study of ERR target genes for therapeutic purpose. ERRβ has been shown to behave differently from ERRα and ERRγ in terms of regulating tumorigenesis. ERRβ expression level has been shown to be inversely co-related with S-phase fraction suggesting their role in inhibition of cellular proliferation. We have undertaken the current study to identify the common targets of Estrogen Receptors and ERR beta. Study of cross talk between ERRβ and ERα may unfold the mechanism of deregulation of ER alpha signaling leading to failure of cell cycle checkpoint induction or apoptosis in breast cancer cells. By chromatin immunoprecipitation cloning, we identified Breast Cancer Amplified Sequence 2 (BCAS2) and Follistatin (FST) to be two important target genes of ERRβ. Whereas BCAS2 is a coactivator of ER alpha as well as negative coregulator of p53, FST has been shown to enhance the ability of R30C breast carcinoma cells to undergo apoptosis and inhibit multi-organ metastasis of small cell lung carcinoma in natural-killer cell deprived SCID mice. Although, FST and BCAS2 are found to be targets of ERRβ, surprisingly FST expression is upregulated by 10 nM estrogen treatment, while BCAS2 is downregulated. This is indicative of involvement of ER alpha in the regulation of ERRβ target genes. The analysis of our western data revealed that FST activation is mediated by ERRβ. Further, our confocal analysis indicates that ERRβ is able to induce nuclear fragmentation in presence of estrogen in ER positive MCF-7 breast carcinoma cells. Additionally, our first time report on regulation of BCAS2 by ERRβ also provides direct evidence of modulation of ER α transcriptional activity by ERR beta. Our extensive study is clearly inclined towards the need of considering the prognostic importance of ERRβ in ER-positive breast tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-27. doi:1538-7445.AM2012-LB-27