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In Human Breast Cancer Cells TRβ Competes with ERα for Altering BCl2/Bax Ratio through SMP30-Mediated p53 Induction

Abstract

Pranati Sar, Dharmendra K Bhargava, Debomita Sengupta, Bandita Rath, Sanjib Chaudhary and Sandip K Mishra

Thyroid hormone and Estrogen regulate transcription(s) of target genes by binding to their nuclear receptors that interact with specific responsive elements -TRE and ERE, respectively. Recently, we have demonstrated that 3, 3’5 Triiodo L Thyronine (T₃) can induce apoptosis in ER positive breast cancer cells (MCF-7) through downregulation of Senescence Marker Protein-30 (SMP30) gene. SMP30, a novel age-associated protein which decreases during ageing is highly expressed in hepatocytes and in renal tubular epithelia. Earlier reports suggest that SMP30 too plays a diverse role in proliferation, survival and differentiation of the cells. SMP30 has also been reported to be downregulated by 17β-Estradiol (E₂) in prostate gland and mammary epithelial cells. Interestingly, Thyroid Receptors (TRs) and Estrogen Receptors (ERs) share a common consensus half site sequence. In this context; we hypothesize a possible competition between both the receptors in SMP30 promoter under different types of hormonal signaling. To prove this hypothesis, gel retardation and luciferase assays were conducted by taking hSMP30 promoter reporter constructs which validated our findings for the putative ERE site. Competition Chromatin Immunoprecipitation Assay (ChIP) in the above mentioned ERE showed differential TRβ binding upon thyroid/estrogen hormone treatment, while ERα showed binding mainly in control and estrogen treated sample. Although the SMP30 promoter activity was almost same in response to E₂ and T₃, but the functional consequences of down regulation of SMP30 in human breast cancer cells post E₂/T₃ treatment were different in terms of apoptosis. To unravel the mechanism behind the differential consequences of E₂/T₃ treatment, in addition to looking at the expression of regular apoptotic markers such as Bax and Cleaved PARP, we have also tried to verify the possible involvement of p53, which has been already reported to be a downstream target of SMP30.