Proliferative response of ERα-positive breast cancer cells to 10 µM 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 α (ERα), 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 ERα-positive, and ERα-negative cell lines. 10 µM EL significantly enhanced the growth of the ERα-positive MCF-7 or T47D breast cancer cells, but not the ERα-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 ERα to the estrogen response element within the TFF1 locus further demonstrated the pro-estrogenic effect of 10 µM EL. We further explored the genome-wide transcriptomic effect of 10 µM EL using the next generation sequencing technology (RNA-seq). Analysis of RNA-seq data obtained from vehicle (0.1% DMSO)- or 10 µM EL-treated MCF-7 cells revealed modulation of expression of diverse sets of functionally related genes, which reflected cell cycle progression. The manner in which 10 µM 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.

Transcriptomic data of MCF-7 breast cancer cells treated with 10 µM enterolactone.

Enterolactone (EL) is a mammalian enterolignan produced in the intestine as a result of the microbial biotransformation of the dietary lignans. EL is a potential nutraceutical, with several health benefits, including anticancer and antimetastatic properties. Epidemiological data suggest a possible link between EL exposure and breast cancer risk. However, EL binds to estrogen receptor-α, produces estrogen-like effects on gene expression, and induces proliferation of MCF-7 breast cancer cells at a concentration of 10 µM. Here, we present RNA-seq data obtained from MCF-7 breast cancer cells treated with 10 µM EL for a period of 72 h, which captures the transcriptomic alterations associated with cell proliferation. The data are available from Gene Expression Omnibus (GEO, accession number GSE216876).

Transcriptomic data of MCF-7 breast cancer cells treated with G1, a G-protein coupled estrogen receptor (GPER) agonist.

Besides short-term non-genomic effects, the G-protein coupled estrogen receptor (GPER) also mediates long-term genomic effects of estrogen. The genomic effects of GPER activation are not completely understood. G1 is a selective GPER agonist, which is popularly used for addressing the effects of GPER activation. Here, we present transcriptomic (RNA-seq) data on MCF-7 cells treated with 100 nM, or 1 µM G1 for a period of 48 h. The data are available from GEO (accession number GSE188706).

High-level expression and optimization of pantoate-ß-alanine ligase in Bacillus megaterium for the enhanced biocatalytic production of D-pantothenic acid.

D-Pantothenic acid (DPA), also known as vitamin B5 is associated with several biological functions and its deficiency causes metabolic and energetic disorders in humans. Fortification of foods with DPA is the viable option to address this risk. DPA biological production route employs pantoate-ß-alanine ligase (PBL) as the key enzyme, which avoids the tedious and time-consuming optical resolution process. The selection of an efficient PBL enzyme is vital for the biological production of DPA. In this study, the panC gene encoding PBL from Escherichia coli, Bacillus megaterium, Corynebacterium glutamicum and Bacillus subtilis was expressed in B. megaterium. B. subtilis derived panC exhibited high PBL activity 61.62 ± 2.15 U/mL. Co-expression of phosphoenolpyruvate carboxykinase (pckA) did not improve the DPA production in B. megaterium. Biocatalytic fed-batch fermentation with externally supplemented precursor substrates (D-pantoic acid and ß-alanine) improved DPA titer to 45.56 ± 0.53 g/L. Daily dietary requirements of DPA for different age groups (including babies, small children, athletes and elderly people) is steadily increasing and the improved DPA production addressed in this study offers advantage for its application in fortification of food products meeting the emerging nutritional demand. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13197-021-05093-6.

BMP4 enhances anoikis resistance and chemoresistance of breast cancer cells through canonical BMP signaling.

Bone morphogenetic proteins (BMPs) regulate cell fate during development and mediate cancer progression. In this study, we investigated the role of BMP4 in proliferation, anoikis resistance, metastatic migration, and drug resistance of breast cancer cells. We utilized breast cancer cell lines and clinical samples representing different subtypes to understand the functional effect of BMP4 on breast cancer. The BMP pathway was inhibited with the small molecule inhibitor LDN193189 hydrochloride (LDN). BMP4 signaling enhanced the expression of stem cell genes CD44, ALDH1A3, anti-apoptotic gene BCL2 and promoted anoikis resistance in MDA-MB-231 breast cancer cells. BMP4 enhanced self-renewal and chemoresistance in MDA-MB-231 by upregulating Notch signaling while LDN treatment abrogated anoikis resistance and proliferation of anoikis resistant breast cancer cells in the osteogenic microenvironment. Conversely, BMP4 downregulated proliferation, colony-forming ability, and suppressed anoikis resistance in MCF7 and SkBR3 cells, while LDN treatment promoted tumor spheroid formation and growth. These findings indicate that BMP4 has a context-dependent role in breast cancer. Further, our data with MDA-MB-231 cells representing triple-negative breast cancer suggest that BMP inhibition might impair its metastatic spread and colonization.

RUNX1T1, a potential prognostic marker in breast cancer, is co-ordinately expressed with ERα, and regulated by estrogen receptor signalling in breast cancer cells.

BACKGROUND: RUNX1T1 is extensively studied in the context of AML1-RUNX1T1 fusion protein in acute myeloid leukemia. Little is known about the function of RUNX1T1 itself, although data on its function and regulation have begun to emerge from clinical, and in vitro studies. It is a putative tumor suppressor, whose expression is altered in a variety of solid tumors. Recently, reduced expression of RUNX1T1 in triple-negative breast tumors, and its influence on prognosis was reported. METHODS AND RESULTS: The Kaplan-Meier Plotter online tool was used to study the relationship between RUNX1T1 expression and survival of breast cancer patients. High RUNX1T1 expression was associated with longer overall survival (OS), relapse-free survival (RFS) and distant metastasis free survival (DMFS). RUNX1T1 expression positively and negatively influenced OS of patients with ERα-positive and ERα-negative breast tumors, respectively. It was also associated with prolonged RFS, and DMFS in tamoxifen-treated patients. Expression of RUNX1T1 and ERα mRNA was analyzed in 40 breast tumor samples, and breast cancer cell lines using RT-PCR. TCGA-BRCA data was mined to study the relationship between RUNX1T1 and ERα mRNA expression. ERα-positive breast tumors showed significantly higher RUNX1T1 mRNA expression compared to ERα-negative tumors. RUNX1T1 mRNA expression was analyzed by qRT-PCR in MCF-7 or T47D cells, which were treated with 17ß-estradiol, or the ERα agonist PPT, alone or in combination with 4-hydroxytamoxifen. Effect of ERα knockdown was also investigated. Results indicate that estrogen downmodulated RUNX1T1 mRNA expression via ERα. CONCLUSION: Higher expression of RUNX1T1 in breast tumors is associated with favourable prognosis. RUNX1T1 and ERα show co-ordinated expression in breast tumors, and breast cancer cell lines. Estrogen-ERα signalling downmodulates the expression of RUNX1T1 mRNA in ERα-positive breast cancer cells. In-depth investigations on the interaction between RUNX1T1 and ERα are warranted to unravel the role and relevance of RUNX1T1 in breast cancer.

Estrogen suppresses HOXB2 expression via ERα in breast cancer cells.

The expression of HOXB2, a homeobox transcription factor, is altered in a variety of solid tumors. Using an in vivo screen to identify regulators of breast tumor growth in murine mammary fat pads, Boimel and co-workers recently identified HOXB2 as a tumor suppressor. However, the mechanistic underpinnings of its role in breast cancer is not understood. Given the emerging interaction of estrogen-regulated gene expression and altered HOX gene expression network in the pathophysiology of breast cancer, this study addressed the relationship between estrogen signaling and HOXB2 expression. Using a mouse model and human breast cancer cell lines, we show that estrogen suppresses HOXB2 expression. Suppression of HOXB2 by PPT, a known ERα agonist, in MCF-7 and T47D cells indicated the involvement of ERα, which was confirmed by siRNA-mediated ERα knockdown experiments. In-silico analysis of the upstream promoter region revealed the presence of three putative EREs. Chromatin immunoprecipitation experiments showed that upon estrogen binding, ERα engaged with EREs in the 5' upstream region of HOXB2 in MCF-7 and T47D cells. Future investigations should address the implications of estrogen-mediated suppression on the proposed tumor suppressor function of HOXB2.

A novel transcript variant of human G-protein coupled estrogen receptor.

The G-protein coupled estrogen receptor (GPER) mediates short-term non-genomic effects of estrogen in diverse cell types and tissues. According to the NCBI nucleotide database, three variants of GPER are known. They are NM_001505.2 (GPER-v2), NM_001039966.1 (GPER-v3), and NM_001098201.1 (GPER-v4). Investigations on GPER expression are key to understand its physiological and pathological roles. However, most studies on GPER mRNA expression have considered total GPER mRNA expression regardless of the individual variants. The present study is motivated by a novel transcript observed in the UCSC Genome Browser (uc010ksd.1), which is annotated as GPER. The novel variant is similar to the known transcript variants of GPER in terms of the protein-coding sequence and the 3'UTR. However, it has a unique 5'UTR, which distinguishes it from other GPER variants. Using primers specific for uc010ksd.1, we have performed RT-PCR to show that the novel GPER transcript (hereafter referred to as GPER-v5) is expressed in human cancer cell lines, such as MCF-7, SW-620, COLO-205, and HT-29. Preliminary evidences indicate that GPER-v5 is a novel GPER mRNA variant. The expression of GPER-v5 in primary cells and tissues should be investigated before probing into its role and relevance in physiological and pathological conditions.

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

The G-protein coupled estrogen receptor (GPER), a proposed tumor suppressor, relays short-term non-genomic responses in target cells and tissues. It frequently undergoes down-modulation in primary tumors of the breast, ovary, and endometrium. Liu and co-workers recently reported loss of GPER expression in colorectal cancer and attributed it to DNA methylation-dependent silencing. We hypothesized 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. Eight downstream CpGs of the upCpGi was differentially methylated across the cell lines. Methylation in this differentially methylated region (DMR) correlated inversely with GPER expression. Two cell lines, namely SW620 and COLO-320DM, were compared in terms of their viability in response to varying concentrations of G1, a GPER specific agonist. 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. 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. DNA methylation in the DMR is inversely correlated with GPER expression. DNA methylation-dependent silencing of GPER may be, at least in part, the underlying reason behind the loss of estrogen's oncoprotective effect via GPER in the colon.

Production and characterization of low molecular weight heparosan in Bacillus megaterium using Escherichia coli K5 glycosyltransferases.

Low molecular weight heparosan is an un-sulfated polysaccharide primarily used as a precursor for heparin synthesis that has recently been used in drug delivery applications. Heparosan synthesis from recombinant bacterial systems provides a safer alternative to naturally producing pathogenic bacterial systems. In this study, we engineered a functional heparosan synthesis pathway in Bacillus megaterium by the expression of E. coli K5 kfiC and kfiA glycosyltransferase genes. Upregulation of individual UDP-sugar precursor pathway genes enhanced the heparosan production, indicating that UDP-precursor sugar concentrations were limiting the biosynthesis. The engineered B. megaterium yielded a maximum heparosan concentration of 394 mg/L in batch bioreactor. The heparosan titer was further increased to 1.32 g/L in fed-batch fermentation. Nuclear magnetic resonance analysis revealed that the chemical structure of B. megaterium derived heparosan was identical to E. coli K5 heparosan. The heparosan molecular weight varied from 31 to 60 kDa, indicating its potential as a precursor for chemoenzymatic heparin biosynthesis. This study provides an efficient process to produce heparosan from non-pathogenic B. megaterium.

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 17ß-estradiol (E2) enhanced ERα 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 E2 in MDA-MB-231 and T47D cells subjected to global demethylation by 5-azacytidine (5-aza). 5-aza significantly demethylated intron-2 CpGs, and enhanced estrogen-induced ERα occupancy at the intron-2 ERE, leading to restoration of estrogen-regulation. 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 ERα-mediated estrogen regulation and DNA methylation in the intron-2, thereby offering a novel context to understand the role of estrogen-ERα signaling axis in breast tumor invasion and metastasis.

Enhanced production of optically pure d (-) lactic acid from nutritionally rich Borassus flabellifer sugar and whey protein hydrolysate based-fermentation medium.

The aim of this study is to optimize the production of optically pure d (-) lactic acid (DLA) employing a cost-effective production medium. Based on the designed biomass approach, Sporolactobacillus inulinus NBRC 13595 was found to exhibit high DLA titer (19.0 g L-1 ) and optical purity (99.6%). A cost-effective medium was constituted using Palmyra palm jaggery (PJ) from Borassus flabellifer and whey protein hydrolysate (WPH) as carbon and nitrogen sources, respectively. Plackett-Burman design indicated that PJ, WPH, and MnSO4 as significant variables influence DLA production. A rotatable central composite design and response surface methodology were used to optimize the PJ and WPH concentrations. A maximum DLA titer (170.14 g L-1 ) was predicted for 222.24 g L-1 of PJ and 11.99 g L-1 of WPH, respectively. Fermentation experimental results exhibited a maximum DLA titer (189.0 ± 8.53 g L-1 ) and productivity (5.25 ± 0.24 g L-1 H-1 ), which is the highest ever reported for DLA production from a renewable feedstock in the batch process. The present investigation substantiates that the potential application of economically viable raw feedstocks (PJ and WPH) for enhanced DLA production, which is attributed to 2.5-fold reduction in DLA production cost compared with conventional medium.