Primary Mammary Epithelial Cells Research Articles

Defective Mammary Epithelial Outgrowth in Transgenic EKAREV–NLS Mice: Correction via Estrogen Supplementation and Genetic Background Modification

Fluorescent biosensors offer a powerful tool for tracking and quantifying protein activity in living systems with high temporospatial resolution. However, the expression of genetically encoded fluorescent proteins can interfere with endogenous signaling pathways, potentially leading to developmental and physiological abnormalities. The EKAREV-NLS mouse model, which carries a FRET-based biosensor for monitoring extracellular signal-regulated kinase (ERK) activity, has been widely utilized both in vivo and in vitro across various cell types and organs. In this study, we report a significant defect in mammary epithelial development in EKAREV-NLS C57BL/6J female mice. Our findings reveal that these mice exhibit severely impaired mammary epithelial outgrowth, linked to systemic defects including disrupted estrous cycling, impaired ovarian follicle maturation, anovulation, and reduced reproductive fitness. Notably, estrogen supplementation was sufficient to enhance mammary epithelial growth in the EKAREV-NLS C57BL/6J females. Furthermore, outcrossing to the ICR genetic background fully restored normal mammary epithelial outgrowth, indicating that the observed phenotype is dependent on genetic background. We also confirmed the functional performance of the biosensor in hormone-supplemented and outcrossed tissues through time-lapse imaging of primary mammary epithelial cells. Our results underscore the critical need for thorough characterization of biosensor-carrying models before their application in specific research contexts. Additionally, this work highlights the influence of hormonal and genetic factors on mammary gland development and emphasizes the importance of careful consideration when selecting biosensor strains for mammary studies.

B-351 Genotoxic Producing Escherichia coli Protects and Promotes Breast Adenocarcinoma

Abstract Background Recent studies have linked certain colibactin producing subphylas of E. coli to colorectal cancer. The bacterium synthesizes colibactin via the pks genomic island containing a series of clbA to clbS genes. The genotoxin causes inter-strand DNA cross-links, which lead to double strand breaks and promotion of colorectal adenocarcinoma. The published studies have prioritized the colorectal regions of the body where E. coli is commonly found, but none have identified the same genotoxic relationship with other types of adenocarcinoma. In this study, we evaluate DNA damage and cellular changes of mammary breast tissue based on high and low levels of colibactin producing E. coli exposure. The goal is to evaluate if colibactin can affect cells in the mammary breast tissue promoting breast adenocarcinoma growth. Methods Two cell lines were acquired from ATCC: primary mammary epithelial cells (PCS-600-010) and MCF-7 breast adenocarcinoma cells (HTB-22). Both cell lines were split into six culture flasks and maintained until ∼80% confluence was reached using the ATCC cell culture protocols. Once all the flasks had reached the target confluence, they were divided into exposure groups of high and low concentrations of colibactin producing E. coli, a group of non-colibactin producing E. coli, a group exposed only to phosphate buffered saline, and a control group with no exposure. Cellular morphology was observed using light microscopy before, after exposure, and every 5 minutes up to 15 minutes. E. coli and colibactin DNA concentrations were quantified using quantitative polymerase chain reaction. Genomic DNA was extracted from each group of cells. The DNA from each group was whole genome sequenced and analyzed for genomic variation. Results The normal cells exposed to E. coli showed immediate lysing and continued to lyse after a 5 minute incubation; however, the adenocarcinoma positive cells showed no lysing up to 30 minutes incubation. Sequencing results show genomic variation between the groups and specific break points associated with the E. coli exposed groups. Conclusions Our results show that the presence of E. coli with mammary breast tissue can destroy normal tissue, promote lysis resistance, and allow adenocarcinoma cells to continue to grow. The colibactin producing E. coli does cause double stranded breaks and can lead to cell death. This new information points out a critical need to monitor the microbiome and infection levels of people at risk or in early stages of cancer to prevent adenocarcinoma growth and destruction of normal tissue. This study indicates that colibactin producing E. coli may play a larger role in tissue damage and promotion of other types of cancer. More research is needed to elucidate the molecular pathway and mechanisms of colibactin, which will lead to new diagnostics or therapeutics that monitor E. coli levels or inhibit colibactin’s toxic effects.

Establishment of an immortalized sheep mammary epithelial cell line for studying milk fat and protein synthesis.

The mammary gland, crucial for milk production in mammals, presents challenges for in vitro study due to its complex structure and limited cell lifespan. We addressed this by introducing the SV40 large T antigen into primary mammary epithelial cells (MECs) from sheep, creating an immortalized T-tag MEC line. This line, stable for over 50 passages, maintained typical epithelial cell morphology during long-term culture. Through transcriptome sequencing and validation, we discovered 3833 differentially expressed genes between MECs and T-tag MEC line, encompassing key biological processes and signaling pathways like cell cycle, p53, and cancer. The cell line, expressing MEC markers (KRT8, KRT18, proliferating cell nuclear antigen, SV40, CSN2, and acetyl-CoA carboxylase alpha), proved capable of synthesizing milk fat and protein. Despite its infinite proliferation potential, the T-tag MEC line showed no tumor formation in mice or cell migration in vitro, indicating stability. This development offers a valuable resource for studying MECs in dairy sheep, facilitating the advancement of long-term culture systems and in vitro lactation bioreactors.

Stigmasterol Activates the mTOR Signaling Pathway by Inhibiting ORP5 Ubiquitination to Promote Milk Synthesis in Bovine Mammary Epithelial Cells.

Stigmasterol (ST), a phytosterol found in food, has various biological activities. However, the effect of ST on milk synthesis in dairy cows remains unclear. Therefore, bovine primary mammary epithelial cells (BMECs) were isolated, cultured, and treated with ST to determine the effect of ST on milk synthesis. The study revealed that 10 μM ST significantly increased milk synthesis in BMECs by activating the mammalian target of rapamycin (mTOR) signaling pathway. Further investigation revealed that this activation depends on the regulatory role of oxysterol binding protein 5 (ORP5). ST induces the translocation of ORP5 from the cytoplasm to the lysosome, interacts with the mTOR, recruits mTOR to target the lysosomal surface, and promotes the activation of the mTOR signaling pathway. Moreover, ST was found to increase ORP5 protein levels by inhibiting its degradation via the ubiquitin-proteasome pathway. Specifically, the E3 ubiquitin ligase membrane-associated cycle-CH-type finger 4 (MARCH4) promotes the ubiquitination and subsequent degradation of ORP5. ST mitigates the interaction between MARCH4 and ORP5, thereby enhancing the structural stability of ORP5 and reducing its ubiquitination. In summary, ST stabilizes ORP5 by inhibiting the interaction between MARCH4 and ORP5, thereby activating mTOR signaling pathway and enhancing milk synthesis.

Polymeric Micellar Nanoparticles Enable Image-guided Drug Delivery in Solid Tumors.

We report the development of a nanotechnology to co-deliver chemocoxib A with a reactive oxygen species (ROS)-activatable and COX-2 targeted pro-fluorescent probe, fluorocoxib Q (FQ) enabling real time visualization of COX-2 and CA drug delivery into solid cancers, using a di-block PPS 135 - b -POEGA 17 copolymer, selected for its intrinsic responsiveness to elevated reactive oxygen species (ROS), a key trait of the tumor microenvironment. FQ and CA were synthesized independently, then co-encapsulated within micellar PPS 135 - b -POEGA 17 co-polymeric nanoparticles (FQ-CA-NPs), and were assessed for cargo concentration, hydrodynamic diameter, zeta potential, and ROS-dependent cargo release. The uptake of FQ-CA-NPs in mouse mammary cancer cells and cargo release was assessed by fluorescence microscopy. Intravenous delivery of FQ-CA-NPs to mice harboring orthotopic mammary tumors, followed by vital optimal imaging, was used to assess delivery to tumors in vivo . The CA-FQ-NPs exhibited a hydrodynamic diameter of 109.2 ± 4.1 nm and a zeta potential (σ) of -1.59 ± 0.3 mV. Fluorescence microscopy showed ROS-dependent cargo release by FQ-CA-NPs in 4T1 cells, decreasing growth of 4T1 breast cancer cells, but not affecting growth of primary human mammary epithelial cells (HMECs). NP-derived fluorescence was detected in mammary tumors, but not in healthy organs. Tumor LC-MS/MS analysis identified both CA (2.38 nmol/g tumor tissue) and FQ (0.115 nmol/g tumor tissue), confirming the FQ-mediated image guidance of CA delivery in solid tumors. Thus, co-encapsulation of FQ and CA into micellar nanoparticles (FQ-CA-NPs) enabled ROS-sensitive drug release and COX-2-targeted visualization of solid tumors.

Cuiru Keli Improves Postpartum Hypogalactia in Rats Through Secreted Frizzled-Related Protein 2-Wnt/β-catenin Signaling Pathway

Based on the secreted frizzled-related protein 2 (SFRP2)-Wnt/β-catenin signaling pathway, this study explored the effect and mechanism of Cuiru Keli (CRKL) in the treatment of postpartum hypogalactia. A rat model of postpartum hypogalactia was established by gavaging 2 mL of 1.6 mg/mL bromocriptine mesylate to female rats on the third day after delivery. Female rats with a delivery time difference of less than 48 hours were selected and randomly assigned to 7 groups, including a normal group (without any modeling or medication), a model group, a CRKL low-dose group of model group model rats receiving CRKL at the dose of 3 g/kg, a CRKL medium-dose group of model rats receiving CRKL at the dose of 6 g/kg, a CRKL high-dose group of model rats receiving CRKL at the dose of 9 g/kg, a positive drug group of model rats receiving domperidone at the dose of 3 mg/kg, and a negative control (NC) group of model rats receiving normal saline. Each group contained 6 rats. Except for the normal and model groups, the remaining 5 groups were continuously administered with the respective intervention drugs at the specified doses by gavage once a day for 10 days. Changes in the total litter mass of the offspring in the 7 groups within 10 days were measured, and HE staining was performed to identify pathological changes in the mammary tissue (MT). Six groups of rats (excluding the positive control group) were used to observe the pathological changes of eosinophils in pituitary tissue. ELISA was performed to determine the content of prolactin (PRL) in serum, immunohistochemical staining was used to determine the expression of prolactin receptor (PRLR) in MT, and RT-qPCR was used to determine the mRNA expression of genes related to lactation in MT. Network pharmacology and molecular docking were used to study the therapeutic effect and mechanism of CRKL on postpartum hypogalactia, particularly whether it acted through the SFRP2-Wnt/β-catenin signaling pathway. The mechanism of CRKL treatment was further validated by detecting mRNA (RT-qPCR) and protein expression (Western blot) of related pathway genes. Cell experiments were conducted using primary culture rat mammary epithelial cells (RMEC) from rat MT. RMEC were divided into four groups, including a normal group (primary culture RMEC, untreated), SFRP2 overexpression group (primary cultured RMEC treated with SFRP2 overexpression vector), SFRP2 overexpression+CRKL group (receiving treatment for SFRP2 overexpression group plus 10% drug-containing serum), and negative control group (primary culture RMEC treated with empty vector). The effect of CRKL on the expression of lactation-related genes FASN, CSN2, and GLUT1 mRNA after SFRP2 overexpression was detected by RT-qPCR. In this study, CRKL was administered at a dose of 3 g/kg in the CRKL low-dose group, 6 g/kg in the medium-dose group, and 9 g/kg in the high-dose group (P<0.05 or P<0.01). Compared with the model group, CRKL at all doses significantly increased the total litter weight gain of the offsprings within 10 days (P<0.05 or P<0.01), and effectively increased lactation (P<0.01), the area of mammary lobules, and the size and filling of acinar cavities. CRKL at all doses also increased the number of eosinophils that secreted PRL in the pituitary gland of the postpartum hypogalactia rat model, and increased the content of PRL in the serum (P<0.05 or P<0.01). CRKL promoted the secretion and expression of PRL in postpartum hypogalactic model rats. In addition, it significantly promoted the expression of genes related to milk fat, milk protein, and lactose synthesis in MT (P<0.05 or P<0.01). Network pharmacology predicted that the Wnt signaling pathway might be a key pathway for CRKL in treating postpartum hypogalactia. The molecular docking results showed that related chemical components in CRKL had good binding ability with CCND1 and SFRP2. Compared with the model group, CRKL at all doses inhibited the expression of SFRP2 gene in vivo (P<0.01) and activated the mRNA and protein expression of CCND1 and c-Myc in the Wnt/β-catenin signaling pathway in MT (P<0.05 or P<0.01). Cell experiments showed that, compared to the normal group, SFRP2 overexpression reduced the mRNA expression of milk synthesis-related genes FASN, CSN2, and GLUT1 in RMEC (P<0.01). The CCK8 results indicated that 10% of the drug-containing serum was the effective concentration administered to cells (P<0.01). After administering drug-containing serum, the expression of the lactation-related genes FASN, CSN2, and GLUT1 were up-regulated (compared with the SFRP2 overexpression group, P<0.01). CRKL alleviates postpartum hypogalactia through the SFRP2-Wnt/β-catenin signaling pathway. SFRP2 might be a potential new target for the diagnosis and treatment of postpartum hypogalactia. This reveals a new mechanism of CRKL in treating postpartum hypogalactia and promotes its clinical application.

Isolation and Culture of Primary Human Mammary Epithelial Cells.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

249 Defining the Role of Hedgehog Signaling in Breast Cancer Risk of non-Hispanic Black Women

OBJECTIVES/GOALS: The molecular basis of increased risk of triple negative breast cancer in non-Hispanic Black women represents a critical knowledge gap that this research is designed to address; successful completion of this work could lead to better prevention, earlier stage diagnoses, and possible discovery of novel therapeutic strategies for this population. METHODS/STUDY POPULATION: We have recently generated a living tissue cohort of 11 non-Hispanic Black and 25 non-Hispanic White women who underwent breast surgery at Mayo Clinic. Gene expression profiling of normal breast tissue from this cohort has identified a pattern of gene expression differences that have been associated with the development of basal breast cancer and are also reflective of Hedgehog (Hh) signaling. We will identify protein-based biomarkers for Hedgehog signaling within normal breast tissue using immunohistochemistry methods. We will culture primary human mammary epithelial cells and further separate luminal and myoepithelial cells using flow cytometry to then decipher Hedgehog signaling. RESULTS/ANTICIPATED RESULTS: We anticipate identifying and localizing protein-based biomarkers for Hedgehog signaling within myoepithelial cells of non-Hispanic Black women. Using our findings, we aim to create a biomarker risk model for triple negative breast cancer and validate this model within a separate and larger cohort of women to predict breast cancer risk. DISCUSSION/SIGNIFICANCE: In addition to immediate benefits from improved risk prediction, the proposed work has the potential to provide new insight into the driving forces underlying basal breast carcinogenesis and the distinct biological differences that distinguish non-Hispanic Black women from non-Hispanic White women.

Cross-Oxygen Gradients Transcriptomic Comparison Revealed the Central Role of MAPK and Hippo in Hypoxia-Mediated Mammary Proliferation Inhibition.

The role of hypoxia in terms of affecting mammary epithelial cells (MECs) proliferation is closely associated with the milk synthesis of lactating mammals. Primary bovine MECs were cultured at 1, 6, 11, 16, and 21% O2 for 24 h. The results showed that cell proliferation decreased linearly, and hypoxic inducible factor (HIF)-1α expression increased linearly along with the declining O2. The linear increase in oxidative stress resulted in the accumulation of malondialdehyde and reactive oxygen species and decreased antioxidant enzyme activities following the reduced O2. Concerning mitochondria, the dynamin-related protein 1 showed improved expression, and optin atrophy protein 1 decreased along with the decreasing O2 gradient, which led to decreased mitochondrial mass and mitophagy emerging under 1% O2. Oxygen concentration-trend RNA-seq analysis was conducted. Specifically, HIF-1-MAPK (1% O2), PI3K-Akt-MAPK (6% O2), and p53-Hippo (11 and 16% O2) were found to primarily regulate cell proliferation in response to hypoxia compared with normoxia (21%), respectively. In conclusion, our study suggests that bMEC proliferation is suppressed in low-oxygen conditions, and is exacerbated following the reduced oxygen supply. The cross-oxygen gradient comparisons suggest that MAPK and Hippo, which are core pathways of mammary cell proliferation, are repressed by hypoxia via oxidative-stress-dependent signals.

Discovery and characterization of anti-cancer peptides from a random peptide library.

We performed a forward genetic screen to discover peptides that specifically target breast cancer cells using a Penetratin tagged, random 15mer peptide library. We identified a group of novel peptides that specifically inhibited the proliferation and survival of breast cancer cells without affecting normal primary mammary epithelial cells or fibroblasts. The intrinsic apoptotic pathway is activated by these peptides in the face of abnormal expression of numerous cell cycle regulatory genes. Associated alterations in histone marks, nuclear structure, and levels of critical RNA binding proteins vary in a peptide specific manner. This study demonstrates a novel method for the discovery of new potential therapeutic peptides.

Abstract B011: BRCA2 haploinsufficiency induces P53 degradation in primary mammary epithelial cells – a crucial early event in breast tumor promotion

Abstract The presence of heterozygous germline BRCA2 mutations is associated with an increased risk of breast and ovarian cancer. In order to design effective preventative strategies, it is critical to understand early tumor promoting events that the mammary epithelial cells undergo on their way to tumorigenesis. To investigate the impact of BRCA2 heterozygosity on tumor formation, we utilized primary BRCA2mut/+ mammary epithelial cells obtained from prophylactic breast tissue of women with BRCA2 mutations. Our findings indicate that BRCA2mut/+ cells display haploinsufficiency for repairing stalled replication forks and resolving RPA32 from these sites, indicative of impaired stalled fork repair. Moreover, they exhibit defects in RAD51 recruitment to double strand break sites indicating a defective homologous recombination dependent double strand break repair efficiency. BRCA2mut/+ cells also show increased sensitivity to both replication stress and double strand break-inducing agents. Interestingly, we find that BRCA2 heterozygous cells downregulate P53 protein levels when compared to BRCA2 wild type cells, and that this downregulation is MDM2-dependent. The decreased P53 levels appear to promote the survival of BRCA2 heterozygous cells under endogenous replication stress conditions. Notably, treatment with nutlin, a P53 protein stabilizer, leads to cell death in BRCA2 heterozygous cells but not in BRCA2 wildtype cells, highlighting the sensitivity of BRCA2 heterozygous cells to endogenous P53 protein levels. Moreover, complete loss of P53 in BRCA2 heterozygous cells results in rescue of sensitivity of these cells to both replication stress and double strand break inducing agents. Furthermore, whole genome sequencing analysis of BRCA2 heterozygous cells deleted for P53 reveal DNA mutational signatures similar to those observed in BRCA2 mutant tumors, implying that loss or downregulation of P53 is an early event driving tumorigenesis in the context of BRCA2 heterozygosity. In summary, our study uncovers the significance of BRCA2 haploinsufficiency in replication stress suppression and highlights the role of P53 loss in promoting BRCA2 heterozygosity-driven cancer. These findings contribute to a better understanding of the molecular mechanisms underlying BRCA2-associated tumorigenesis and may have implications for targeted preventative therapies in the future. Citation Format: Haohui Duan, Margaret Gillis, Deboral Dillon, Judy Garber, Shailja Pathania. BRCA2 haploinsufficiency induces P53 degradation in primary mammary epithelial cells – a crucial early event in breast tumor promotion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B011.

Genes and pathways revealed by whole transcriptome analysis of milk derived bovine mammary epithelial cells after Escherichia coli challenge

Mastitis, inflammation of the mammary gland, is the costliest disease in dairy cattle and a major animal welfare concern. Mastitis is usually caused by bacteria, of which staphylococci, streptococci and Escherichia coli are most frequently isolated from bovine mastitis. Bacteria activate the mammary immune system in variable ways, thereby influencing the severity of the disease. Escherichia coli is a common cause of mastitis in cattle causing both subclinical and clinical mastitis. Understanding of the molecular mechanisms that activate and regulate the host response would be central to effective prevention of mastitis and breeding of cows more resistant to mastitis. We used primary bovine mammary epithelial cell cultures extracted noninvasively from bovine milk samples to monitor the cellular responses to Escherichia coli challenge. Differences in gene expression between control and challenged cells were studied by total RNA-sequencing at two time points post-challenge. In total, 150 and 440 (Padj < 0.05) differentially expressed genes were identified at 3 h and 24 h post-challenge, respectively. The differentially expressed genes were mostly upregulated at 3 h (141/150) and 24 h (424/440) post-challenge. Our results are in line with known effects of E. coli infection, with a strong early inflammatory response mediated by pathogen receptor families. Among the most significantly enriched early KEGG pathways were the TNF signalling pathway, the cytokine-cytokine receptor interaction, and the NF-kappa B signalling pathway. At 24 h post-challenge, most significantly enriched were the Influenza A, the NOD-like receptor signalling, and the IL-17 signaling pathway.

Effects of lysine and methionine on mRNA expression of candidate transcription factors by primary bovine mammary epithelial cells.

It has been established that essential amino acids (EAA) regulate protein synthesis in mammary epithelial cells by rapidly altering the phosphorylation state of translation factors. However, the long-term transcriptional response to EAA supply has been investigated much less. Eight transcription factors were selected as candidate mediators of EAA effects on mammary cell function via the amino acid response (ATF4, ATF6), mitogen-activated protein kinase (JUN, FOS, EGR1), and mechanistic target of rapamycin complex 1 (MYC, HIF1A, SREBF1). The objective was to determine if and when expression of these candidate genes was affected in primary cultures of bovine mammary epithelial cells more than 24 h after imposing an EAA deficiency, and to evaluate effects of EAA deficiency on protein synthesis, endoplasmic reticulum size, cell proliferation, and lipogenesis. Differentiated cells were cultured in 1 of 3 treatment media representing normal physiological concentrations of all amino acids (CTL), low lysine (LK), or low methionine (LM) for 24, 40, 48, or 60 h. Both LK and LM suppressed protein synthesis and activated ATF4 expression, indicating the classic amino acid response pathway had been triggered. However, there was no effect of LK or LM on endoplasmic reticulum size, possibly related to elevated ATF6 expression on LM. Expression of early response genes JUN, FOS, EGR1 and MYC was not elevated by EAA deficiency but LM decreased EGR1 expression. LM also increased expression of HIF1A. The EGR1 and HIF1A expression results are consistent with the decrease in cell proliferation rate observed. Variable responses in SREBF1 expression to LK and LM at different timepoints may have contributed to a lack of effect on lipogenesis rates. These findings indicate that EAA deficiency may inhibit mammary protein synthesis and cell proliferation through transcription factors.

Matrine Targets BTF3 to Inhibit the Growth of Canine Mammary Tumor Cells.

The canine mammary tumor model is more suitable for studying human breast cancer, and the safety concentrations of matrine and the biotin-labeled matrine probe were determined in canine primary mammary epithelial cells, and then selected canine mammary tumor cell lines CHMm and CHMp were incubated with matrine, and cell viability was detected by CCK-8. The biotin-labeled matrine probe was used to pull-down the targets of matrine in canine mammary tumor cells, and the targets were screened in combination with activity-based protein profiling (ABPP) and Genecards database, and verified by qPCR and western blot. The results showed that the maximum non-cytotoxic concentrations of matrine and biotin-labeled matrine probe in canine primary mammary epithelial cells were 250 μg/mL and 500 μg/mL, respectively. Matrine and biotin-labeled matrine probe had a proliferation inhibitory effect time-dependently on CHMm and CHMp cells within a safe concentration range, and induced autophagy in cells. Then BTF3 targets were obtained by applying ABPP and Genecards screening. Cellular thermal shift assay (CETSA) findings indicated that matrine could increase the heat stability of BTF3 protein. Pull-down employing biotin-labeled matrine probe with CHMm and CHMp cell lysates revealed that BTF3 protein was detected in the biotin-labeled matrine probe group and that BTF3 protein was significantly decreased by the addition of matrine. The qPCR and western blot findings of CHMm and CHMp cells treated with matrine revealed that matrine decreased the expression of the BTF3 gene and protein with the extension of the action time, and the impact was more substantial at the protein level, respectively.

Acylated Ghrelin Activates PI3K/mTOR Signaling Pathway by Promoting ThPOK Acetylation to Promote Milk Fat Synthesis in Bovine Mammary Epithelial Cells.

Ghrelin regulates diverse physiological activities. However, the effects of this hormone on the milk fat synthesis remain unknown. This study aimed to investigate the effect of acylated ghrelin (AG) on milk fat synthesis by modifying the expression (knockdown or overexpression) of growth hormone secretagogue receptor 1a (GHSR1a) and Th-inducing POK (ThPOK) in primary bovine mammary epithelial cells (BMECs). The results showed that AG significantly increased the triglyceride relative content from 260.83 ± 9.87 to 541.67 ± 8.38 in BMECs via GHSR1a. ThPOK functions as a key regulatory target downstream of AG, activating the PI3K and mTOR signaling pathways to promote milk fat synthesis in BMECs. Moreover, AG-regulated ThPOK by increasing the EP300 activity, which promoted ThPOK acetylation to protect it from proteasomal degradation. In conclusion, AG increases ThPOK acetylation and stabilizes ThPOK through GHSR1a, thereby activating the PI3K/mTOR signaling pathway and ultimately promoting the milk fat synthesis in BMECs.

RSK3 switches cell fate: from stress-induced senescence to malignant progression

BackgroundTGFβ induces several cell phenotypes including senescence, a stable cell cycle arrest accompanied by a secretory program, and epithelial-mesenchymal transition (EMT) in normal epithelial cells. During carcinogenesis cells lose the ability to undergo senescence in response to TGFβ but they maintain an EMT, which can contribute to tumor progression. Our aim was to identify mechanisms promoting TGFβ-induced senescence escape.MethodsIn vitro experiments were performed with primary human mammary epithelial cells (HMEC) immortalized by hTert. For kinase library screen and modulation of gene expression retroviral transduction was used. To characterize gene expression, RNA microarray with GSEA analysis and RT-qPCR were used. For protein level and localization, Western blot and immunofluorescence were performed. For senescence characterization crystal violet assay, Senescence Associated-β-Galactosidase activity, EdU staining were conducted. To determine RSK3 partners FLAG-baited immunoprecipitation and mass spectrometry-based proteomic analyses were performed. Proteosome activity and proteasome enrichment assays were performed. To validate the role of RSK3 in human breast cancer, analysis of METABRIC database was performed. Murine intraductal xenografts using MCF10DCIS.com cells were carried out, with histological and immunofluorescence analysis of mouse tissue sections.ResultsA screen with active kinases in HMECs upon TGFβ treatment identified that the serine threonine kinase RSK3, or RPS6KA2, a kinase mainly known to regulate cancer cell death including in breast cancer, reverted TGFβ-induced senescence. Interestingly, RSK3 expression decreased in response to TGFβ in a SMAD3-dependent manner, and its constitutive expression rescued SMAD3-induced senescence, indicating that a decrease in RSK3 itself contributes to TGFβ-induced senescence. Using transcriptomic analyses and affinity purification coupled to mass spectrometry-based proteomics, we unveiled that RSK3 regulates senescence by inhibiting the NF-κΒ pathway through the decrease in proteasome-mediated IκBα degradation. Strikingly, senescent TGFβ-treated HMECs display features of epithelial to mesenchymal transition (EMT) and during RSK3-induced senescence escaped HMECs conserve EMT features. Importantly, RSK3 expression is correlated with EMT and invasion, and inversely correlated with senescence and NF-κΒ in human claudin-low breast tumors and its expression enhances the formation of breast invasive tumors in the mouse mammary gland.ConclusionsWe conclude that RSK3 switches cell fate from senescence to malignancy in response to TGFβ signaling.

Enhancement of PPARα-Inhibited Leucine Metabolism-Stimulated β-Casein Synthesis and Fatty Acid Synthesis in Primary Bovine Mammary Epithelial Cells.

Leucine, a kind of branched-chain amino acid, plays a regulatory role in the milk production of mammalian mammary glands, but its regulatory functions and underlying molecular mechanisms remain unknown. This work showed that a leucine-enriched mixture (LEUem) supplementation increased the levels of milk protein and milk fat synthesis in primary bovine mammary epithelial cells (BMECs). RNA-seq of leucine-treated BMECs indicated alterations in lipid metabolism, translation, ribosomal structure and biogenesis, and inflammatory response signaling pathways. Meanwhile, the supplementation of leucine resulted in mTOR activation and increased the expression of BCKDHA, FASN, ACC, and SCD1. Interestingly, the expression of PPARα was independently correlated with the leucine-supplemented dose. PPARα activated by WY-14643 caused significant suppression of lipogenic genes expression. Furthermore, WY-14643 attenuated leucine-induced β-casein synthesis and enhanced the level of BCKDHA expression. Moreover, promoter analysis revealed a peroxisome-proliferator-response element (PPRE) site in the bovine BCKDHA promoter, and WY-14643 promoted the recruitment of PPARα onto the BCKDHA promoter. Together, the present data indicate that leucine promotes the synthesis of β-casein and fatty acid and that PPARα-involved leucine catabolism is the key target.

Drug mechanism enrichment analysis improves prioritization of therapeutics for repurposing

BackgroundThere is a pressing need for improved methods to identify effective therapeutics for diseases. Many computational approaches have been developed to repurpose existing drugs to meet this need. However, these tools often output long lists of candidate drugs that are difficult to interpret, and individual drug candidates may suffer from unknown off-target effects. We reasoned that an approach which aggregates information from multiple drugs that share a common mechanism of action (MOA) would increase on-target signal compared to evaluating drugs on an individual basis. In this study, we present drug mechanism enrichment analysis (DMEA), an adaptation of gene set enrichment analysis (GSEA), which groups drugs with shared MOAs to improve the prioritization of drug repurposing candidates.ResultsFirst, we tested DMEA on simulated data and showed that it can sensitively and robustly identify an enriched drug MOA. Next, we used DMEA on three types of rank-ordered drug lists: (1) perturbagen signatures based on gene expression data, (2) drug sensitivity scores based on high-throughput cancer cell line screening, and (3) molecular classification scores of intrinsic and acquired drug resistance. In each case, DMEA detected the expected MOA as well as other relevant MOAs. Furthermore, the rankings of MOAs generated by DMEA were better than the original single-drug rankings in all tested data sets. Finally, in a drug discovery experiment, we identified potential senescence-inducing and senolytic drug MOAs for primary human mammary epithelial cells and then experimentally validated the senolytic effects of EGFR inhibitors.ConclusionsDMEA is a versatile bioinformatic tool that can improve the prioritization of candidates for drug repurposing. By grouping drugs with a shared MOA, DMEA increases on-target signal and reduces off-target effects compared to analysis of individual drugs. DMEA is publicly available as both a web application and an R package at https://belindabgarana.github.io/DMEA.

Sulforaphane prevents LPS-induced inflammation by regulating the Nrf2-mediated autophagy pathway in goat mammary epithelial cells and a mouse model of mastitis

BackgroundMastitis not only deteriorates the composition or quality of milk, but also damages the health and productivity of dairy goats. Sulforaphane (SFN) is a phytochemical isothiocyanate compound with various pharmacological effects such as anti-oxidant and anti-inflammatory. However, the effect of SFN on mastitis has yet to be elucidated. This study aimed to explore the anti-oxidant and anti-inflammatory effects and potential molecular mechanisms of SFN in lipopolysaccharide (LPS)-induced primary goat mammary epithelial cells (GMECs) and a mouse model of mastitis.ResultsIn vitro, SFN downregulated the mRNA expression of inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6), inhibited the protein expression of inflammatory mediators (cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS)) while suppressing nuclear factor kappa-B (NF-κB) activation in LPS-induced GMECs. Additionally, SFN exhibited an antioxidant effect by increasing Nrf2 expression and nuclear translocation, up-regulating antioxidant enzymes expression, and decreasing LPS-induced reactive oxygen species (ROS) production in GMECs. Furthermore, SFN pretreatment promoted the autophagy pathway, which was dependent on the increased Nrf2 level, and contributed significantly to the improved LPS-induced oxidative stress and inflammatory response. In vivo, SFN effectively alleviated histopathological lesions, suppressed the expression of inflammatory factors, enhanced immunohistochemistry staining of Nrf2, and amplified of LC3 puncta LPS-induced mastitis in mice. Mechanically, the in vitro and in vivo study showed that the anti-inflammatory and anti-oxidative stress effects of SFN were mediated by the Nrf2-mediated autophagy pathway in GMECs and a mouse model of mastitis.ConclusionsThese results indicate that the natural compound SFN has a preventive effect on LPS-induced inflammation through by regulating the Nrf2-mediated autophagy pathway in primary goat mammary epithelial cells and a mouse model of mastitis, which may improve prevention strategies for mastitis in dairy goats.

Successful editing and maintenance of lactogenic gene expression in primary bovine mammary epithelial cells

In vitro investigation of bovine lactation processes is limited by a lack of physiologically representative cell models. This deficiency is most evident through the minimal or absent expression of lactation-specific genes in cultured bovine mammary tissues. Primary bovine mammary epithelial cells (pbMECs) extracted from lactating mammary tissue and grown in culture initially express milk protein transcripts at relatively representative levels. However, expression drops dramatically after only three or four passages, which greatly reduces the utility of primary cells to model and further examine lactogenesis. To investigate the effects of alternate alleles in pbMECs including effects on transcription, we have developed methods to deliver CRISPR-Cas9 gene editing reagents to primary mammary cells, resulting in very high editing efficiencies. We have also found that culturing the cells on an imitation basement membrane composed of Matrigel, results in the restoration of a more representative lactogenic gene expression profile and the cells forming three-dimensional structures in vitro. Here, we present data from four pbMEC lines recovered from pregnant cows and detail the expression profile of five key milk synthesis genes in these MECs grown on Matrigel. Additionally, we describe an optimised method for preferentially selecting CRISPR-Cas9-edited cells conferring a knock-out of DGAT1, using fluorescence-activated cell sorting (FACS). The combination of these techniques facilitates the use of pbMECs as a model to investigate the effects of gene introgressions and genetic variation in lactating mammary tissue.