Biology subjects

Limaye, A. M.

Publications and source records attributed to Limaye, A. M..

8 recordsLinked to original sources

bioRxiv2025

Expression of MMP-11, an estrogen-suppressed gene in MCF-7 cells, is elevated upon acquisition of tamoxifen resistance

Matrix metalloproteinase-11 (MMP-11), also known as stromelysin-3, is a member of a large family of zinc-dependent proteinases. Known to be expressed in breast tumor stroma, the epithelium, and the associated mononuclear inflammatory cells, its expression is associated with invasion, metastasis, and poor clinical outcomes and survival rates. Despite the known effects of estrogen on extracellular matrix remodeling, and breast cancer metastasis, the role of MMP-11 therein is not understood. Here, we show that estrogen suppresses MMP-11 expression in MCF-7 breast cancer cells via a mechanism that involves binding of the estrogen receptor (ER) to an estrogen response element located in the intron-1 of MMP-11. ER knockdown alone upregulates MMP-11 expression, highlighting its crucial role in this regulatory axis. Additionally, the anti-estrogen tamoxifen (4-OHT) blocks estrogen-mediated MMP-11 suppression. However, prolonged 4-OHT exposure or 4-OHT resistance leads to increased MMP-11 expression. This suggests that MMP-11 may play a role in 4-OHT resistance, potentially leading to a more aggressive tumor phenotype. Our study underscores the importance of estrogen-ER signaling in breast cancer progression, emphasizing the need for further investigation into MMP-11s role in 4-OHT resistance and cancer metastasis.

cancer biology↗

bioRxiv2024

The CYP1A1 connection between enterolactone and breast cancer risk

Enterolactone (EL), a mammalian enterolignan, is a gut microbe-generated plant-lignan derivative. Although observational data are controversial, meta-analyses have affirmed that plant lignan-rich diets, or high serum EL reduce breast cancer risk, or the associated mortality, in post-menopausal women. However, the mechanistic basis is unknown. Here we show that EL antagonizes AHR to reduce CYP1A1 mRNA expression in MCF-7 breast cancer cells. Intriguingly, it increases CYP1A1 protein, a mediator of xenobiotic response, which is frequently expressed in breast tumors, and implicated in cell proliferation and survival. ELs effect on CYP1A1 expression is similar to estrogen, and mediated via ER. But, by virtue of partial ER agonism/antagonism, EL attenuates estrogen-mediated increase in CYP1A1 protein. These data suggest potential mechanisms underlying ELs beneficial effects in breast cancer. In the face of xenobiotic exposure, its AHR antagonism may reduce the generation of cancer-inducing genotoxic agents. With declining levels of estrogen in post-menopausal women, EL may antagonize estrogen-mediated induction of CYP1A1 protein, and the associated proliferation of mammary epithelial cells.

cancer biology↗

bioRxiv2024

Karanjin alters gene expression through ERα: a preliminary study

Karanjin is an abundant furanoflavonoid-constitutent of pongamia oil. Among several biological actions of karanjin, the antiproliferative effect of karanjin has gained traction in the recent years; raising speculations about its anticancer potential. In the backdrop of partial estrogen-like alteration of gene expression by karanjin in ER positive MCF-7 breast cancer cells, we present preliminary evidences supporting of the role of ER in karanjin-mediated effects.

pharmacology and toxicology↗

bioRxiv2023

Proliferative response of ERalpha-positive breast cancer cells to 10 uM enterolactone, and the associated alteration in the transcriptomic landscape.

Enterolactone (EL) is a product of gut-microbial metabolism of dietary plant lignans. Studies linking EL with breast cancer risk have bolstered investigations into its effects on the mammary epithelial cells, and the mechanisms thereof. While it binds to the estrogen receptor ; ER, its effect on the proliferation of mammary tumor cell lines is reportedly ambivalent; depending on its concentration. The genomic correlates of EL actions also remain unexplored. Here we have elaborately studied the effect of EL on proliferation of ER-positive, and ER-negative cell lines. 10 {micro}M EL significantly enhanced the growth of the ER-positive MCF-7 or T47D breast cancer cells, but not the ER-negative MDA-MB-231 or MDA-MB-453 cells. In MCF-7 cells, it significantly increased the expression of TFF1 mRNA, an estrogen-induced transcript. The binding of ER to the estrogen response element within the TFF1 locus further demonstrates the pro-estrogenic effect of 10 {micro}M EL. We further explored the genome-wide transcriptomic effect of 10 {micro}M EL. Analysis of RNA-seq data obtained from control- or 10 {micro}M EL treated-MCF-7 cells revealed modulation of expression of diverse sets of functionally related genes, which reflected cell cycle progression, rather than cell cycle arrest or apoptosis. The manner in which 10 {micro}M EL regulated the hallmark G2/M checkpoint, and estrogen-response-late genes correlated with proliferation inducing, and estrogen-like effects of EL on MCF-7 cells.

genomics↗

bioRxiv2022

The G-protein-coupled estrogen receptor, a gene co-expressed with ERα in breast tumors, is regulated by estrogen-ERα signalling in ERα positive breast cancer cells.

PurposeThe purpose of this study was to assess the relationship between GPER, a seven transmembrane G-protein coupled estrogen receptor, and ER in breast tumors, and to make inroads into the mechanistic basis and clinical significance. MethodsTCGA-BRCA data was mined to examine the relationship between GPER and ER expression. GPER mRNA, and protein expression were analyzed in ER-positive or -negative breast tumors from two cohorts using immunohistochemistry, western blotting, or RT-qPCR. The Kaplan-Meier Plotter was employed for survival analysis. The influence of estrogen in vivo was studied by examining GPER expression levels in estrus or diestrus mouse mammary tissues, and the impact of 17{beta}-estradiol (E2) administration in juvenile or adult mice. The effect of E2, or propylpyrazoletriol (PPT, an ER agonist) stimulation on GPER expression was studied in MCF-7 and T47D cells, with or without tamoxifen or ER knockdown. ER-binding to the GPER locus was explored by analysing ChIP-seq data (ERP000380), in silico prediction of estrogen response elements, and chromatin immunoprecipitation assay. ResultsClinical data revealed significant positive association between GPER and ER expression in breast tumors. The median GPER expression in ER-positive tumors was significantly higher than ER-negative tumors. High GPER expression was significantly associated with longer overall survival of patients with ER-positive tumors. In vivo experiments showed a positive effect of E2 on GPER expression. E2 induced GPER expression in MCF-7 and T47D cells; an effect mimicked by PPT. Tamoxifen or ER-knockdown blocked the induction of GPER. Estrogen-mediated induction was associated with increased ER occupancy in the upstream region of GPER. ConclusionGPER expression is positively associated with ER in breast tumors, and a transcriptional target of the estrogen-ER signalling axis. More in-depth studies are required to establish the significance of GPER-ER co-expression, and their interplay in breast tumor development, progression, and treatment.

cancer biology↗

bioRxiv2021

Global transcriptome analysis reveals partial estrogen-like effects of karanjin in MCF-7 breast cancer cells

Karanjin, an abundantly occurring furanoflavonoid in edible and non-edible legumes, exerts diverse biological effects in vivo, and in vitro. Its potential as an anticancer agent is also gaining traction following recent demonstrations of its anti-proliferative, cell cycle inhibitory, and pro-apoptotic effects. However, the universality of its anticancer potential is yet to be scrutinized, particularly so because flavonoids can act as selective estrogen receptor modulators (SERMs). Even the genomic correlates of its biological activities are yet to be examined in hormone responsive cells. This paper presents the early and direct transcriptomic footprint of 10 M karanjin in MCF-7 breast cancer cells, using next generation sequencing technology (RNA-seq). We show that karanjin-modulated gene-expression repertoire is enriched in several hallmark gene sets, which include early estrogen-response, and G2/M checkpoint genes. Genes modulated by karanjin overlapped with those modulated by 1 nM 17{beta}-estradiol (E2), or 1 M tamoxifen. Karanjin altered the expression of selected estrogen-regulated genes in a cell-type, and concentration dependent manner. It downmodulated the expression of ER protein in MCF-7 cells. Furthermore, ER knockdown negatively impacted karanjins ability to modulate the expression of selected E2 target genes. Our data suggest that karanjin exerts its effects on ER-positive breast cancer cells, at least in part, via ER. The apparent SERM-like effects of karanjin pose a caveat to the anticancer potential of karanjin. In-depth studies on cell-type and concentration-dependent effects of karanjin may bring out its true potential in endocrine therapies.

pharmacology and toxicology↗

bioRxiv2020

DNA methylation in the upstream CpG island of the GPER locus and its relationship with GPER expression in colon cancer cell lines

The seven-transmembrane G-protein coupled estrogen receptor (GPER) relays short-term non-genomic responses in target cells and tissues. It is a proposed tumor suppressor, which frequently undergoes down-modulation in primary tumors of the breast, ovary, and endometrium. A study by Liu and co-workers reported the loss of GPER expression in colorectal cancer and attributed it to DNA methylation-dependent silencing. The present study is based on the hypothesis that GPER expression is inversely correlated with methylation in the upstream CpG island (upCpGi) in the GPER locus. Methylation in the upCpGi was analysed by bisulfite sequencing and correlated with GPER expression in a panel of colon cancer cell lines The bisulfite sequencing results show the presence of a differentially methylated region (DMR) comprising of the downstream eight CpGs of the upCpGi. Methylation in the DMR correlated inversely with GPER expression. We compared two cell lines, namely SW620 and COLO-320DM, in terms of their viability in response to varying concentrations of G1, a GPER specific agonist, which is known to induce cell cycle arrest and apoptosis in colon cancer cell lines. SW-620 cells, which had the least methylated DMR and the highest level of GPER expression, showed significant loss of viability with 1 μM G1. On the other hand, COLO-320DM, which had the most methylated DMR and the lowest level of GPER expression, did not show a significant response to 1 μM G1. At 5 μM G1, SW620 cells showed a greater reduction in viability than COLO-320DM cells. Our study demonstrates the inverse correlation between DNA methylation in the DMR and GPER expression. GPER is a non-canonical form of estrogen receptor, and estrogen is believed to exert its oncoprotective effect in the colon via GPER. DNA methylation-dependent silencing of GPER may, at least in part, the underlying reason behind the loss of estrogen’s oncoprotective effect in the colon. Future studies should explore the utility of DNA methylation in the upCpGi, particularly the DMR, in diagnosis or prognosis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2019

Interplay of ERα binding and DNA methylation in the intron-2 determines the expression and estrogen regulation of Cystatin A in breast cancer cells

Despite advances in early detection and treatment, invasion and metastasis of breast tumors remains a major hurdle. Cystatin A (CSTA, also called stefin A), an estrogen-regulated gene in breast cancer cells, is an inhibitor of cysteine cathepsins, and a purported tumor suppressor. Loss of CSTA expression in breast tumors evidently shifts the balance in favor of cysteine cathepsins, thereby promoting extracellular matrix remodeling, tumor invasion and metastasis. However, the underlying mechanism behind the loss of CSTA expression in breast tumors is not known. Here, we have analyzed CSTA expression, and methylation of upstream and intron-2 CpG sites within the CSTA locus in human breast cancer cell lines and breast tumors of the TCGA cohort. Results showed an inverse relationship between expression and methylation. Sequence analysis revealed a potential estrogen response element (ERE) in the intron-2. Analysis of ChIP-seq data (ERP000380) and our own ChIP experiments showed that 17{beta}-estradiol (E2) enhanced ER binding to this ERE in MCF-7 cells. This ERE was located amidst the differentially methylated intron-2 CpG sites, which provoked us to examine the possible conflict between estrogen-regulation of CSTA and DNA methylation in the intron-2. We analyzed the expression of CSTA and its regulation by estrogen in MDA-MB-231 and T47D cells subjected to global demethylation by 5-azacytidine (5-aza). 5-aza, not only enhanced CSTA expression in these cell lines but also restored estrogen-regulation of CSTA in these cells. Taken together, our results indicate that DNA methylation-dependent silencing could play a significant role in the loss of CSTA expression in breast tumors. The potential of DNA methylation as an indicator of CSTA expression or as a marker of tumor progression can be explored in future investigations. Furthermore, our results indicate the convergence of ER-mediated estrogen regulation and DNA methylation in the intron-2, thereby offering a novel context to understand the role of estrogen-ER signaling axis in breast tumor invasion and metastasis.

cancer biology↗