Growth Of Mammary Epithelial Cells Research Articles

Stanniocalcin 2 Regulates Autophagy and Ferroptosis in Mammary Epithelial Cells of Dairy Cows Through the Mechanistic Target of Rapamycin Complex 1 Pathway

BackgroundStanniocalcin 2 (STC2), a glycoprotein hormone, is extensively expressed in various organs and tissues, particularly in the mammary gland. STC2 plays a crucial role in enabling cells to adapt to stress conditions and avert apoptosis. The efficiency of milk production is closely linked to both the quantity and quality of mammary cells. Yet, there remains a dearth of research on the impact of STC2 on mammary cells’ activity in dairy cows. ObjectivesThe objective of this study was to investigate the effects of STC2 on the viability of mammary epithelial cells in dairy cows and to elucidate the underlying mechanisms. MethodsFirst, the Gene Expression Profiling and Interactive Analysis database was employed to perform survival analysis on STC2 expression in relation to prognosis using The Cancer Genome Atlas and GETx data. Subsequently, the basic physical and chemical properties, gene expression, and potential signaling pathways involved in the growth of dairy cow mammary epithelial cells were determined using STC2 knockdown. ResultsSTC2 knockdown significantly suppressed autophagy in mammary epithelial cells of dairy cows. Moreover, STC2 knockdown upregulated glutathione peroxidase 4 protein expression, elicited an elevation in lipid ROS concentrations, and inhibited the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, consequently repressing downstream genes involved in lipid synthesis regulated by mTORC1 and ultimately inducing ferroptosis. ConclusionsThe findings of our study suggest that STC2 suppresses autophagy and ferroptosis through the activation of mTORC1. Mechanically, STC2 exerts an inhibitory effect on ferroptosis by activating antioxidative stress-related proteins, such as glutathione peroxidase 4, to suppress lipid ROS production and stimulating the mTORC1 signaling pathway to enhance the expression of genes associated with lipid synthesis.

Bovine mammary epithelial cells can grow and express milk protein synthesis genes at reduced fetal bovine serum concentration.

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions.

Molecular Action of Tamoxifen in the Ovaries of Rats with Mammary Neoplasia

Tamoxifen (TAM) is a drug commonly used in patients with breast cancer. The anticancer effect of TAM occurs via its ability to antagonize estrogen-dependent growth of mammary epithelial cells. Previously, we demonstrated that TAM prevented the chemotherapy-induced loss of ovarian follicular reserves in both cancer-free rats and rats with cancer. Such follicular loss is a main cause of infertility in young women treated for cancer. The current study was undertaken to discover the molecules and intracellular pathways involved in the action of TAM in the ovaries of rats with mammary tumors. To meet this goal we used transcriptomic (RNA-Seq) and proteomic (2D-DIGE/MS) approaches. TAM inhibited the expression of genes and lncRNAs involved in ovarian steroidogenesis. Moreover, TAM altered the expression of genes related to primordial follicle activation or arrest. In addition, proteomic screening indicated the importance of basic metabolic processes in the ovarian actions of TAM. Although simple extrapolation of these data to humans is not possible, the results of this study emphasize the need to explore the ability of TAM to affect ovarian function in women undergoing cancer treatment.

Identification of Small Inhibitors for Human Metadherin, an Oncoprotein, through in silico Approach.

Cancer is a disease that takes lives of thousands of people each year. There are more than 100 different types of cancers known to man. This fatal disease is one of the leading causes of death today. Astrocyte elevated gene-1(AEG-1)/Metadherin (MTDH) activates multiple oncogenic signaling pathways and leads to different types of cancers. MTDH interacting with staphylococcal nuclease domain containing 1(SND1) supports the survival and growth of mammary epithelial cells under oncogenic conditions. Silencing MTDH or SND1 individually or disrupting their interaction compromises the tumorigenic potential of tumor-initiating cells. The aim of our present study was to investigate novel interactions of staphylococcal nuclease domain containing 1 (SND1) binding domain of AEG-1/MTDH with different lead compounds through molecular docking approach using MOE software. Molecular docking was done by docking the ChemBridge database against important residues of MTDH involved in interaction with SND1. After docking the whole ChemBridge database, the top 200 interactive compounds were selected based on docking scores. After applying Lipinski's rule, all the remaining chosen compounds were studied on the basis of binding affinity, binding energy, docking score and protein-ligand interactions. Finally, 10 compounds showing multiple interactions with different amino acid residues were selected as the top interacting compounds. Three compounds were selected for simulation studies after testing these compounds using topkat toxicity and ADMET studies. The simulation study indicated that compound 32538601 is a lead compound for inhibiting MTDH-SND1 complex formation. These novels, potent inhibitors of MTDH-SND1 complex can ultimately help us in controlling cancer up to some extent.

Glucose promotes cell growth and casein synthesis via ATF4/Nrf2-Sestrin2- AMPK-mTORC1 pathway in dairy cow mammary epithelial cells

In the dairy industry, glucose (Glu) is used as bioactive substance to increase milk yield. However, the molecular regulation underneath needs further clarification. Here, the regulation and its molecular mechanism of Glu on cell growth and casein synthesis of dairy cow mammary epithelial cells (DCMECs) were investigated. When Glu was added from DCMECs, both cell growth, β-casein expression and the mechanistic target of rapamycin complex 1 (mTORC1) pathway were increased. Overexpression and silencing of mTOR revealed that Glu promoted cell growth and β-casein expression through the mTORC1 pathway. When Glu was added from DCMECs, both Adenosine 5'-monophosphate-activated protein kinase α (AMPKα) and Sestrin2 (SESN2) expression were decreased. Overexpression and silencing of AMPKα or SESN2 uncovered that AMPKα suppressed cell growth and β-casein synthesis through inhibiting mTORC1 pathway, and SESN2 suppressed cell growth and β-casein synthesis through activating AMPK pathway. When Glu was depleted from DCMECs, both activating transcription factor 4 (ATF4) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression were increased. Overexpression or silencing of ATF4 or Nrf2 demonstrated that Glu depletion promoted SESN2 expression through ATF4 and Nrf2. Together, these results indicate that in DCMECs, Glu promoted cell growth and casein synthesis via ATF4/Nrf2-SESN2-AMPK-mTORC1 pathway.

FGFR2 Controls Growth, Adhesion and Migration of Nontumorigenic Human Mammary Epithelial Cells by Regulation of Integrin β1 Degradation

The role of fibroblast growth factor receptor 2 (FGFR2), an important mediator of stromal paracrine and autocrine signals, in mammary gland morphogenesis and breast cancer has been extensively studied over the last years. However, the function of FGFR2 signalling in the initiation of mammary epithelial oncogenic transformation remains elusive. Here, FGFR2-dependent behaviour of nontumorigenic model of mammary epithelial cells was studied. In vitro analyses demonstrated that FGFR2 regulates epithelial cell communication with extracellular matrix (ECM) proteins. Silencing of FGFR2 significantly changed the phenotype of cell colonies in three-dimensional cultures, decreased integrins α2, α5 and β1 protein levels and affected integrin-driven processes, such as cell adhesion and migration. More detailed analysis revealed the FGFR2 knock-down-induced proteasomal degradation of integrin β1. Analysis of RNA-seq databases showed significantly decreased FGFR2 and ITGB1 mRNA levels in breast tumour samples, when compared to non-transformed tissues. Additionally, high risk healthy individuals were found to have disrupted correlation profiles of genes associated with FGFR2 and integrin signalling, cell adhesion/migration and ECM remodelling. Taken together, our results strongly suggest that FGFR2 loss with concomitant integrin β1 degradation is responsible for deregulation of epithelial cell-ECM interactions and this process may play an important role in the initiation of mammary gland epithelial tumorigenesis.

Identification and functional analysis of m6A in the mammary gland tissues of dairy goats at the early and peak lactation stages.

N6-methyladenosine (m6A) is the most common reversible epigenetic RNA modification in the mRNA of all higher eukaryotic organisms and plays an important role in the regulation of gene expression and cell function. In this study, m6A-modified methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptome sequencing (RNA-seq) were used to identify the key genes with m6A modification during mammary gland development and lactation in dairy goats. The results showed that m6A methylation occurred at 3,927 loci, which were significantly enriched in the 3' untranslated region (3'UTR) and the termination codon region. In the early stage and peak stage of lactation, m6A methylation occurred extensively in mammary tissues, and a total of 725 differentially expressed m6A-modified genes were obtained, all negatively correlated with mRNA expression. In addition, Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that different methylated genes were mainly involved in the growth and apoptosis of mammary epithelial cells through signaling pathways, such as the mitogen-activated protein kinase (MAPK) and phospholipase D pathways, and then affected the development and lactation of mammary gland. All in all, we identified and analyzed the methylation events related to the development and lactation regulation of mammary gland at the early and peak lactation stages, and provided a theoretical basis to reveal the physiological regulatory system of mammary gland development and lactation in dairy goats.

Novel Insight into the Potential Role of Acylglycerophosphate Acyltransferases Family Members on Triacylglycerols Synthesis in Buffalo.

Acylglycerophosphate acyltransferases (AGPATs) are the rate-limiting enzymes for the de novo pathway of triacylglycerols (TAG) synthesis. Although AGPATs have been extensively explored by evolution, expression and functional studies, little is known on functional characterization of how many members of the AGPAT family are involved in TAG synthesis and their impact on the cell proliferation and apoptosis. Here, 13 AGPAT genes in buffalo were identified, of which 12 AGPAT gene pairs were orthologous between buffalo and cattle. Comparative transcriptomic analysis and real-time quantitative reverse transcription PCR (qRT-PCR) further showed that both AGPAT1 and AGPAT6 were highly expressed in milk samples of buffalo and cattle during lactation. Knockdown of AGPAT1 or AGPAT6 significantly decreased the TAG content of buffalo mammary epithelial cells (BuMECs) and bovine mammary epithelial cells (BoMECs) by regulating lipogenic gene expression (p < 0.05). Knockdown of AGPAT1 or AGPAT6 inhibited proliferation and apoptosis of BuMECs through the expression of marker genes associated with the proliferation and apoptosis (p < 0.05). Our data confirmed that both AGPAT1 and AGPAT6 could regulate TAG synthesis and growth of mammary epithelial cells in buffalo. These findings will have important implications for understanding the role of the AGPAT gene in buffalo milk performance.

MAPK1 Is Regulated by LOC102188416/miR-143-3p Axis in Dairy Goat Mammary Epithelial Cells.

MicroRNA-143-3p (miR-143-3p) is one of the miRNAs involved in the growth of goat mammary epithelial cells (GMECs). In this study, Illumina/Solexa sequencing was performed to establish the lncRNA database in Laoshan dairy goats. Using the lncRNA database, long noncoding RNAs (lncRNAs) regulated by miR-143-3p were screened. In total, 4899 lncRNAs were identified, with 173 lncRNAs being differentially expressed in all three replicates. The target genes of the differentially expressed lncRNAs were annotated in GO terms and KEGG pathways. Among the differentially expressed lncRNAs, lncRNA LOC102188416 was predicted to sponge miR-143-3p and share MAPK1 as a common target gene with miR-143-3p, which was validated by dual luciferase reporter assay system and qRT-PCR. The miR-143-3p mimic significantly lowered the relative luciferase activity of psiCHECK2-LOC102188416 wildtype vector but not mutated vector, suggesting that lncRNA LOC102188416 might be a sponge of miR-143-3p, which was verified by the promotion role of lncRNA LOC102188416 siRNA (siR-LOC102188416) in the expression of miR-143-3p. It was shown that the expression of MAPK1 was downregulated by either miR-143-3p mimic or siR-LOC102188416, indicating that miR-143-3p and lncRNA LOC102188416 had a coregulatory effect on MAPK1 expression. The co-transfection of miR-143-3p inhibitor with siR-LOC102188416 reversed the decrease of MAPK1 expression regulated by siR-LOC102188416 alone, strengthening the existence of lncRNA LOC102188416/miR-143-3p/MAPK1 axis in GMECs of Laoshan dairy goats.

Chemically and mechanically defined hyaluronan hydrogels emulate the extracellular matrix for unbiased in vivo and in vitro organoid formation and drug testing in cancer

Finding a material that emulates the in vivo microenvironment to allow complex cell phenotypes in vitro and without biasing cell fate in vivo remains a significant challenge. Hyaluronan is over-expressed in breast cancer and in several other tumours. Mammary epithelial cells invade tissues through both active degradation of the matrix and ameboid-like mechanisms. Thus, we synthesized a defined, biomimetic hydrogel composed of hyaluronan (HA) and matrix metalloproteinase-cleavable (MMPx) crosslinker and used oxime crosslinking to ensure tunability of the resulting HA-MMPx hydrogel. This strategy allowed us to identify the optimal matrix to study the growth and polarization of healthy and diseased mammary epithelial cancer cells first in vitro and then in vivo. We then extended the platform to study nine different cancer types in vitro. We demonstrate that primary, patient-derived breast cancer cells from biopsies established organoids within HA-MMPx and, relative to Matrigel®, had different growth rates and responses to drugs, underscoring the importance of the extracellular environment to cell fate. We established patient-derived xenografts (PDX) using HA-MMPx in SCID mice and showed superior reproducibility compared to Matrigel®. Fascinatingly, PDX grown in HA-MMPx did not induce resident murine macrophage polarization whereas those grown in Matrigel® showed an increase in the proportion of alternatively activated cells, indicating that Matrigel® itself skewed macrophage polarization. Importantly, HA-MMPx did not bias the immune cell response in vivo and supported a diverse range of organoid phenotypes in vitro.

Orthotopic Transplantation of Mouse Mammary Epithelial Cells.

The orthotopic transplantation assay has provided important insights into mammary development, stem cell function, and tumorigenesis. Technically, it consists in grafting mammary tissue fragments, organoids, mammospheres, or isolated cells into the fat pads of prepubertal mice from which the endogenous epithelium has been surgically removed, thereby allowing growth and differentiation of mammary epithelial cells in their physiological environment. Here, we describe how is conducted transplantation of epithelial fragments and cells isolated from mouse mammary glands, report the various approaches currently used to evaluate the regeneration and self-renewal properties of mammary stem cells, and highlight the strengths and limitations of this in vivo grafting assay.

Human Breast Extracellular Matrix Microstructures and Protein Hydrogel 3D Cultures of Mammary Epithelial Cells.

Simple SummaryHuman breast tissue extracellular matrix (ECM) is a microenvironment essential for the survival and biological activities of mammary epithelial cells. The ECM structural features of human breast tissues remain poorly defined. In this study, we identified the structural and mechanical properties of human normal breast and invasive ductal carcinoma tissue ECM using histological methods and atomic force microscopy. Additionally, a protein hydrogel was generated using human breast tissue ECM and defined for its microstructural features using immunofluorescence imaging and machine learning. Furthermore, we examined the three-dimensional growth of normal mammary epithelial cells or breast cancer cells cultured on the ECM protein hydrogel, where the cells exhibited biological phenotypes like those seen in native breast tissues. Our data provide novel insights into cancer cell biology, tissue microenvironment mimicry and engineering, and native tissue ECM-based biomedical and pharmaceutical applications.Tissue extracellular matrix (ECM) is a structurally and compositionally unique microenvironment within which native cells can perform their natural biological activities. Cells grown on artificial substrata differ biologically and phenotypically from those grown within their native tissue microenvironment. Studies examining human tissue ECM structures and the biology of human tissue cells in their corresponding tissue ECM are lacking. Such investigations will improve our understanding about human pathophysiological conditions for better clinical care. We report here human normal breast tissue and invasive ductal carcinoma tissue ECM structural features. For the first time, a hydrogel was successfully fabricated using whole protein extracts of human normal breast ECM. Using immunofluorescence staining of type I collagen (Col I) and machine learning of its fibrous patterns in the polymerized human breast ECM hydrogel, we have defined the microstructural characteristics of the hydrogel and compared the microstructures with those of other native ECM hydrogels. Importantly, the ECM hydrogel supported 3D growth and cell-ECM interaction of both normal and cancerous mammary epithelial cells. This work represents further advancement toward full reconstitution of the human breast tissue microenvironment, an accomplishment that will accelerate the use of human pathophysiological tissue-derived matrices for individualized biomedical research and therapeutic development.

Chemerin Regulates Epithelial Barrier Function of Mammary Glands in Dairy Cows.

Simple SummaryThis study evaluated the production and the function of chemerin, known as a chemoattractant and an antimicrobial protein, with regard to mammary epithelial defense in cows. The result demonstrated that mammary epithelial cells express chemerin protein. Chemerin treatment promoted the proliferation of bovine mammary epithelial cells and protected epithelial cells from oxidative stress. Meanwhile, mammary chemerin production was elevated by mastitis, which was possibly attributed to inflammatory cytokines. Therefore, the present study demonstrates the supportive ability of chemerin for mammary epithelial tissue and its regulation by inflammatory stimuli, suggesting its role in mammary epithelial defense against pathogen infection in cows.Epithelial barrier function in the mammary gland acts as a forefront of the defense mechanism against mastitis, which is widespread and a major disorder in dairy production. Chemerin is a chemoattractant protein with potent antimicrobial ability, but its role in the mammary gland remains unelucidated. The aim of this study was to determine the function of chemerin in mammary epithelial tissue of dairy cows in lactation or dry-off periods. Mammary epithelial cells produced chemerin protein, and secreted chemerin was detected in milk samples. Chemerin treatment promoted the proliferation of cultured bovine mammary epithelial cells and protected the integrity of the epithelial cell layer from hydrogen peroxide (H2O2)-induced damage. Meanwhile, chemerin levels were higher in mammary tissue with mastitis. Tumor necrosis factor alpha (TNF-α) strongly upregulated the expression of the chemerin-coding gene (RARRES2) in mammary epithelial cells. Therefore, chemerin was suggested to support mammary epithelial cell growth and epithelial barrier function and to be regulated by inflammatory stimuli. Our results may indicate chemerin as a novel therapeutic target for diseases in the bovine mammary gland.

Transcriptome analysis of triple negative breast cancer based on public database

Triple negative breast cancer (TNBC) is a pathological type with high malignancy in breast cancer. Because the estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 appear histologically negative, the treatment effect of the existing drug is not ideal. Exploring the TNBC related pathological markers is one of hot spots in the research field of breast cancer. TNBC related gene sets are obtained from gene expression omnibus (GEO) database for transcriptome analysis, including principal component analysis, screening differentially expressed genes (DEGs), gene function enrichment analysis and signal pathway enrichment analysis, protein interaction network construction, weighted gene coexpression network analysis (WGCNA), etc. The results show that there are significantly differentially expressed genes (DEGs) between TNBC and non TNBC population. These DEGs are mainly enriched in the biological processes related to epidermal growth and differentiation, growth and development of mammary epithelial cells, and in molecular functions related to transcription factor binding and estrogen signal transduction pathways. The results of protein interaction network and weighted gene coexpression network analysis are integrated and TFF1, FOXA1, AGR2 and AGR3 are predicted as the key genes involved in the pathological phenotype of TNBC. The study of transcriptome analysis based on TNBC gene set in public database may provide certain theoretical research basis to reveal the potential biomarkers for pathogenesis of TNBC, and the derived disease diagnosis and therapeutic targets or molecular regulation mechanisms.

Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis.

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.

Bedaquiline, an FDA-approved drug, inhibits mitochondrial ATP production and metastasis in vivo, by targeting the gamma subunit (ATP5F1C) of the ATP synthase

Here, we provide evidence that high ATP production by the mitochondrial ATP-synthase is a new therapeutic target for anticancer therapy, especially for preventing tumor progression. More specifically, we isolated a subpopulation of ATP-high cancer cells which are phenotypically aggressive and demonstrate increases in proliferation, stemness, anchorage-independence, cell migration, invasion and multi-drug resistance, as well as high antioxidant capacity. Clinically, these findings have important implications for understanding treatment failure and cancer cell dormancy. Using bioinformatic analysis of patient samples, we defined a mitochondrial-related gene signature for metastasis, which features the gamma-subunit of the mitochondrial ATP-synthase (ATP5F1C). The relationship between ATP5F1C protein expression and metastasis was indeed confirmed by immunohistochemistry. Next, we used MDA-MB-231 cells as a model system to functionally validate these findings. Importantly, ATP-high MDA-MB-231 cells showed a nearly fivefold increase in metastatic capacity in vivo. Consistent with these observations, ATP-high cells overexpressed (i) components of mitochondrial complexes I–V, including ATP5F1C, and (ii) markers associated with circulating tumor cells (CTCs) and metastasis, such as EpCAM and VCAM1. Knockdown of ATP5F1C expression significantly reduced ATP-production, anchorage-independent growth, and cell migration, as predicted. Similarly, therapeutic administration of the FDA-approved drug, Bedaquiline, downregulated ATP5F1C expression in vitro and prevented spontaneous metastasis in vivo. In contrast, Bedaquiline had no effect on the growth of non-tumorigenic mammary epithelial cells (MCF10A) or primary tumors in vivo. Taken together, our results suggest that mitochondrial ATP depletion is a new therapeutic strategy for metastasis prophylaxis, to avoid treatment failure. In summary, we conclude that mitochondrial ATP5F1C is a promising new biomarker and molecular target for future drug development, for the prevention of metastatic disease progression.

Brucein D modulates MAPK signaling cascade to exert multi-faceted anti-neoplastic actions against breast cancer cells

Breast cancer is a prominent type of malignancy among women with a high rate of mortality. A number of previous studies have demonstrated the anticancer potential of brucein D (BD), a quassinoid extracted from Brucea javanica, against the cancers of the pancreas, bone, and liver. We investigated the impact of BD on apoptotic as well on mitogen-activated protein kinase (MAPK) signaling cascades in breast cancer cells. The effect of BD on p38 MAPK and JNK signaling pathways and its downstream functions was deciphered in both MDA-MB-231 and MCF-7 cell lines. We noted that BD decreased the viability of breast cancer cells without affecting the growth of healthy mammary epithelial cells (MCF-10A). Flow cytometric analysis revealed that BD can increase sub-G1 cells and enhanced annexin-V-PI stained cells. The apoptogenic impact of BD was further substantiated by cleavage of procaspase-3/8 and downregulation of antiapoptotic proteins (Bcl-xL, XIAP, and survivin). Furthermore, BD also downmodulated the migratory ability, and chemokine triggered invasion of breast cancer cells. Interestingly, the pharmacological inhibition of p38 MAPK and JNK kinases abrogated the observed anticancer actions of BD. Overall, the data indicated that BD can induce substantial apoptosis and interfere with cellular invasion by modulating MAPK signaling pathway in breast cancer cells.

Extracellular regucalcin suppresses colony formation and growth independent of tumor suppressor p53 in human mammary epithelial cells

Regucalcin plays a multifunctional role in cell regulation as a suppressor in the processes of intracellular signaling and transcription, leading to inhibition of cell growth. The downregulated expression or activity of regucalcin has been shown to contribute to the development of carcinogenesis in various types of human cancer. The wild-type tumor suppressor TP53 gene encodes for a transcriptional factor p53. This protein may play a role in cell proliferation. Loss of p53 function may induce cell transformation during carcinogenesis and tumor progression of human cancer. We investigate whether or not extracellular regucalcin suppresses the proliferation of non-tumorigenic human mammary epithelial MCF 10A cells with loss of p53 in vitro. Loss of p53 did not impact colony formation and proliferation of the cells. Interestingly, p53 loss caused decrease in the cell cycle suppressor p21, but not retinoblastoma and regucalcin, as compared with those of wild-type MCF 10A cells. Notably, extracellular regucalcin suppressed colony formation and proliferation of wild-type MCF 10A cells and p53 (-/-) cells, while it did not have an effect on cell death. Mechanistically, extracellular regucalcin decreased levels of various signaling factors including Ras, phosphatidylinositol-3 kinase, mitogen-activated protein kinase (MAPK), phospho-MAPK, and signal transducer and activator of transcription 3 in wild-type MCF 10A cells and p53 (-/-) cells. Thus, extracellular regucalcin was found to suppress the growth of MCF 10A cells with loss of p53. Extracellular regucalcin may play a role as a suppressor in the growth of human mammary epithelial cells with p53 loss, providing a novel strategy for cancer.

TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development.

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells.

Translin restricts the growth of pubertal mammary epithelial cells estrogen-independently in mice

Translin, a ubiquitous RNA/DNA-binding protein that forms a hetero-octamer together with Translin-associated factor X (TRAX), possesses endoribonuclease activity and plays a physiological role in restricting the size and differentiation of mesenchymal precursor cells. However, the precise role of Translin in epithelial cells remains unclear. Here, we show evidence that Translin restricts the growth of pubertal mammary epithelial cells. The mammary epithelia of Translin-null females exhibited retarded growth before puberty, but highly enhanced growth and DNA synthesis with increased ramification after the onset of puberty. Primary cultures of Translin-null mammary epithelial cells showed augmented DNA synthesis in a ligand-independent and ligand-enhanced manner. Translin-null ovariectomized mice implanted with slow-release estrogen pellets showed enhanced length and ramification of the mammary glands. Mammary epithelial growth was also observed in ovariectomized Translin-null mice implanted with placebo pellets. Luciferase reporter assays using embryonic fibroblasts from Translin-null mice showed unaltered estrogen receptor α function. These results indicate that Translin plays a physiological role in restricting intrinsic growth, beyond mesenchymal cells, of pubertal mammary epithelial cells.