Mammary Gland Epithelial Cells Research Articles

Modulation of Cell-Cycle Progression by Hydrogen Peroxide-Mediated Cross-Linking and Degradation of Cell-Adhesive Hydrogels.

The cell cycle is known to be regulated by features such as the mechanical properties of the surrounding environment and interaction of cells with the adhering substrates. Here, we investigated the possibility of regulating cell-cycle progression of the cells on gelatin/hyaluronic acid composite hydrogels obtained through hydrogen peroxide (H2O2)-mediated cross-linking and degradation of the polymers by varying the exposure time to H2O2 contained in the air. The stiffness of the hydrogel varied with the exposure time. Human cervical cancer cells (HeLa) and mouse mammary gland epithelial cells (NMuMG) expressing cell-cycle reporter Fucci2 showed the exposure-time-dependent different cell-cycle progressions on the hydrogels. Although HeLa/Fucci2 cells cultured on the soft hydrogel (Young’s modulus: 0.20 and 0.40 kPa) obtained through 15 min and 120 min of the H2O2 exposure showed a G2/M-phase arrest, NMuMG cells showed a G1-phase arrest. Additionally, the cell-cycle progression of NMuMG cells was not only governed by the hydrogel stiffness, but also by the low-molecular-weight HA resulting from H2O2-mediated degradation. These results indicate that H2O2-mediated cross-linking and degradation of gelatin/hyaluronic acid composite hydrogel could be used to control the cell adhesion and cell-cycle progression.

Alachlor breaks down intracellular calcium homeostasis and leads to cell cycle arrest through JNK/MAPK and PI3K/AKT signaling mechanisms in bovine mammary gland epithelial cells

Alachlor is a widely used herbicide for the cultivation of various grains employed as food for cattle. The mechanisms leading to the toxic effects of alachlor on epithelial cells of the bovine mammary gland are not well known. Thus, this study was conducted to clarify the toxicological effects of alachlor on the immortalized epithelial cell line of the bovine mammary gland (MAC-T) cells. After treatment, many factors related to cell viability, proliferation, and cellular homeostasis were evaluated. Alachlor arrested cell cycle progression by blocking the expression of cyclin and cyclin-dependent kinases, and induced the breakdown of Ca2+ homeostasis. The cytosolic and mitochondrial levels of Ca2+ were also abnormally increased after the treatment of cells with alachlor, ultimately leading to the depolarization of mitochondrial membrane potential in MAC-T cells. The signaling cascade was found to be dysregulated by the abnormal phosphorylation of signaling molecules involved in PI3K/AKT (AKT, p70S6K, and S6) and MAPK/JNK (JNK and c-Jun) pathways. In these mechanisms, exposure to alachlor led to a reduction in the viability and proliferation of MAC-T cells. Altogether, the toxic effects of alachlor can lead to abnormal conditions in epithelial cells of the bovine mammary gland, which might hinder these cells from performing their main role, such as producing milk.

Common Genomic Aberrations in Mouse and Human Breast Cancers with Concurrent P53 Deficiency and Activated PTEN-PI3K-AKT Pathway.

Simultaneous P53 loss and activation of the PTEN-restricted PI3K-AKT pathway frequently occur in aggressive breast cancers. P53 loss causes genome instability, while PTEN loss and/or activating mutations of PIK3CA and AKT promote cancer cell proliferation that also increases incidences of genomic aberrations. However, the genomic alterations associated with P53 loss and activated PTEN-PI3K-AKT signaling in breast cancer have not been defined. Spatiotemporally controlled breast cancer models with inactivation of both P53 and Pten in adult mice have not been established for studying genomic alterations. Herein, we deleted both floxed Pten and Tp53 genes in the mammary gland epithelial cells in adult mice using a RCAS virus-mediated Cre-expressing system. These mice developed small tumors in 21 weeks, and poorly differentiated larger tumors in 26 weeks. In these tumors, we identified 360 genes mutated by nonsynonymous point mutations and small insertions and deletions (NSPMs/InDels), 435 genes altered by copy number amplifications (CNAs), and 450 genes inactivated by copy number deletions (CNDs). Importantly, 22.2%, 75.9% and 27.3% of these genes were also altered in human breast tumors with P53 and PTEN losses or P53 loss and activated PI3K-AKT signaling by NSPMs/InDels, CNAs and CNDs, respectively. Therefore, inactivation of P53 and Pten in adult mice causes rapid-growing breast tumors, and these tumors recapitulate a significant number of genetic aberrations in human breast tumors with inactivated P53 and activated PTEN-PI3K-AKT signaling. Further characterization of these commonly altered genes in breast cancer should help to identify novel cancer-driving genes and molecular targets for developing therapeutics.

Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein.

Mastitis is one of the most serious diseases that causes losses in the dairy industry, seriously impairing milk production and milk quality, and even affecting human health. Menthol is a cyclic monoterpene compound obtained from the stem and leaves of peppermint, which has a variety of biological activities, including anti-inflammatory and antioxidant activity. The purpose of this study was to investigate the preventive effect of menthol on the lipopolysaccharide-induced inflammatory response in primary bovine mammary gland epithelial cells (BMECs) and its anti-inflammatory mechanism. First, BMECs were isolated and amplified from the udders of Holstein cows by enzymatic hydrolysis. BMECs were treated with menthol (10, 50, 100, 200 μM) for 1h, followed by lipopolysaccharide (5μg/ml) for 12 h. Lipopolysaccharide treatment upregulated the protein levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (INOS) and the mRNA abundance of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), while menthol was able to inhibit this effect. The inhibitory effect of menthol on proinflammatory factors was significantly reduced when autophagy was blocked using 3-Methyladenine (5μg/ml), an inhibitor of autophagy. Furthermore, lipopolysaccharide treatment reduced the expression levels of milk lipids and milk proteins, which were inhibited by menthol. In addition, menthol (200 μM) treatment was able to significantly upregulate the expression level of autophagy-related protein LC3B, downregulate the expression level of P62, promote the expression abundance of autophagy-related gene mRNA, and enhance significantly enhance autophagic flux. Interestingly, treatment of BMECs with menthol (200 μM) promoted the phosphorylation of AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1) and increased the nuclear localization of nuclear factor-E2 associated factor 2 (Nrf-2). When the AMPK pathway was blocked using compound C (10μg/ml), an inhibitor of AMPK, autophagy was significantly inhibited. Autophagy levels were significantly decreased after blocking the Nrf-2 pathway using ML385 (5μg/ml), an inhibitor of Nrf-2. Overall, the data suggest that menthol activates the AMPK-ULK1 pathway to initiate the onset of autophagy and maintains the level of autophagy through the AMPK-Nrf-2 pathway. In conclusion, the findings suggest that menthol may alleviate the inflammatory response in BMECs via the AMPK/ULK1/Nrf-2/autophagy pathway.

Aclonifen induces bovine mammary gland epithelial cell death by disrupting calcium homeostasis and inducing ROS production

Herbicides play key roles in agriculture. Aclonifen is a diphenyl ether herbicide that is widely used for sunflower, potato, corn, and wheat crops. Since it has a long half-life, it is considered persistent and can easily accumulate in the environment. Therefore, livestock and humans are at risk of exposure to aclonifen. Importantly, aclonifen is toxic to several mammals such as rats, mice, and dogs. However, the toxicity of aclonifen in cattle remains unclear. Therefore, we sought to investigate its toxicity in cattle using bovine mammary gland epithelial cells (MAC-T). We found that aclonifen induces sub-G1 phase arrest and represses MAC-T proliferation. In addition, aclonifen caused mitochondrial dysfunction, as evidenced by excessive ROS production and loss of mitochondrial membrane potential. Furthermore, cytosolic and mitochondrial calcium homeostases were disrupted after aclonifen treatment. Moreover, aclonifen treatment caused alterations in the PI3K/AKT and MAPK signaling pathways, which are involved in the regulation of cell survival and death. In conclusion, aclonifen causes MAC-T cell death through mitochondrial dysfunction and the collapse of calcium homeostasis.

Fluroxypyr-1-methylheptyl ester causes apoptosis of bovine mammary gland epithelial cells by regulating PI3K and MAPK signaling pathways and endoplasmic reticulum stress

Fluroxypyr-1-methylheptyl ester (FPMH) is an auxin herbicide that is widely applied to crops and pastures to block growth of post-emergence weeds. Several studies have reported the toxicity of FPMH in aquatic vertebrates. However, the adverse impacts of FPMH on mammals, including domestic animals, have not been reported. The purpose of our current study is to assess the impact of FPMH on the bovine mammary system and milk production. To evaluate the toxicity of FPMH on the mammary glands of lactating cows, the bovine mammary gland epithelial cell line, MAC-T, was exposed to various concentrations (0, 5, 7.5, 10, 15, and 20 μM) of FPMH for 24 h, and then various assessments were performed. The results showed that FPMH dose-dependently reduced MAC-T cell viability following exposure to FPMH and induced mitochondrial depolarization and apoptosis. FPMH also modulated signaling through the PI3K and MAPK pathways. In addition, the expression levels of proteins related to endoplasmic reticulum (ER) stress were upregulated, indicating induction of ER stress, and calcium homeostasis was disrupted following FPMH treatment. In conclusion, our investigation suggests that FPMH may be toxic to the bovine mammary system and may decrease dairy production.

Annexin A2-Mediated Internalization of Staphylococcus aureus into Bovine Mammary Epithelial Cells Requires Its Interaction with Clumping Factor B.

Background: Staphylococcus aureus is a leading cause of contagious mastitis in dairy cattle. Internalization of S. aureus by bovine mammary gland epithelial cells is thought to be responsible for persistent and chronic intramammary infection, but the underlying mechanisms are not fully understood. Methods: In the present study, we evaluated the role of Annexin A2 (AnxA2), a membrane-binding protein, in S. aureus invasion into bovine mammary epithelial cell line (MAC-T). In vitro binding assays were performed to co-immunoprecipitate the binding proteins of AnxA2 in the lysates of S. aureus. Results: AnxA2 mediated the internalization but not adherence of S. aureus. Engagement of AnxA2 stimulated an integrin-linked protein kinase (ILK)/p38 MAPK cascade to induce S. aureus invasion. One of the AnxA2-precipitated proteins was identified as S. aureus clumping factor B (ClfB) through use of mass spectrometry. Direct binding of ClfB to AnxA2 was further confirmed by using a pull-down assay. Pre-incubation with recombinant ClfB protein enhanced S. aureus internalization, an effect that was specially blocked by anti-AnxA2 antibody. Conclusion: Our results demonstrate that binding of ClfB to AnxA2 has a function in promoting S. aureus internalization. Targeting the interaction of ClfB and AnxA2 may confer protection against S. aureus mastitis.

Metabolic Reprogramming of Mammary Epithelial Cells during TGF-β-Induced Epithelial-to-Mesenchymal Transition.

The cytokine transforming growth factor-β (TGF-β) can induce normal breast epithelial cells to take on a mesenchymal phenotype, termed epithelial-to-mesenchymal transition (EMT). While the transcriptional and proteomic changes during TGF-β-induced EMT have been described, the metabolic rewiring that occurs in epithelial cells undergoing EMT is not well understood. Here, we quantitively analyzed the TGF-β-induced metabolic reprogramming during EMT of non-transformed NMuMG mouse mammary gland epithelial cells using nuclear magnetic resonance (NMR) spectroscopy. We found that TGF-β elevates glycolytic and tricarboxylic acid (TCA)-cycle activity and increases glutaminolysis. Additionally, TGF-β affects the hexosamine pathway, arginine-proline metabolism, the cellular redox state, and strongly affects choline metabolism during EMT. TGF-β was found to induce phosphocholine production. A kinase inhibitor RSM-93A that inhibits choline kinase α (CHKα) mitigated TGF-β-induced changes associated with EMT, i.e., increased filamentous (F)-actin stress fiber formation and N-Cadherin mesenchymal marker expression.

Live S. aureus and heat-killed S. aureus induce different inflammation-associated factors in bovine endometrial tissue in vitro

Staphylococcus aureus is majorly involved in bovine mastitis; however, it weakly induces pro-inflammatory factors in mammary gland epithelial cells. We aimed to clarify the involvement of S. aureus in other inflammation types and its relationship with inflammatory factor secretion in bovine endometritis. We used live S. aureus (LSA)- and heat-killed S. aureus (HK-SA)-treated bovine endometrial tissue in vitro. The HK-SA-treated group showed significantly higher IL-6, IL-1β, TNF-α, CXCL1/2 and TLR2 expression than the LSA-infected group. Contrastingly, the LSA-infected group showed significantly higher PTGS2, mPGES-1, and EP4 expression than the HK-SA treated group. There was no significant between-group difference in hyaluronan-binding protein 1 expression, which suggested similar inflammatory responses. H&E results indicated that LSA and HK-SA induced shedding of endometrial gland epithelial cells. The LSA-infected group showed higher high-mobility group box 1 protein expression than the HK-SA treated groups, which indicated differences in signaling pathway activation. Further, the LSA-treated group had higher JNK and p38 MAPK levels while the HK-SA-treated group had higher IκB-α levels. There was no significant between-group difference in the ERK signaling pathway. Our findings indicate that the pathogen-associated molecular patterns (PAMPs) of S. aureus activate pro-inflammatory factor expression via the TLR2-ERK-NF-κB signaling pathway. Contrastingly, LSA induced PGE2 accumulation via the TLR2/MAPKs signaling pathway. This is the first report that S. aureus and the PAMPs of S. aureus activate different signaling pathways and that LSA mainly induce PGE2 accumulation rather than cytokine secretion.

Relative virulence of Staphylococcus aureus bovine mastitis strains representing the main Canadian spa types and clonal complexes as determined using in vitro and in vivo mastitis models

Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of β-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap-), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1β and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus.

Glucose-sensitive acetylation of Seryl tRNA synthetase regulates lipid synthesis in breast cancer

Abnormally enhanced de novo lipid biosynthesis has been increasingly realized to play crucial roles in the initiation and progression of varieties of cancers including breast cancer. However, the mechanisms underlying the dysregulation of lipid biosynthesis in breast cancer remain largely unknown. Here, we reported that seryl tRNA synthetase (SerRS), a key enzyme for protein biosynthesis, could translocate into the nucleus in a glucose-dependent manner to suppress key genes involved in the de novo lipid biosynthesis. In normal mammary gland epithelial cells glucose can promote the nuclear translocation of SerRS by increasing the acetylation of SerRS at lysine 323. In SerRS knock-in mice bearing acetylation-defective lysine to arginine mutation, we observed increased body weight and adipose tissue mass. In breast cancer cells the acetylation and nuclear translocation of SerRS are greatly inhibited. Overexpression of SerRS, in particularly the acetylation-mimetic lysine to glutamine mutant, dramatically inhibits the de novo lipid synthesis and hence greatly suppresses the proliferation of breast cancer cells and the growth of breast cancer xenografts in mice. We further identified that HDAC4 and HDAC5 regulated the acetylation and nuclear translocation of SerRS. Thus, we identified a SerRS-meditated inhibitory pathway in glucose-induced lipid biosynthesis, which is dysregulated in breast cancer.

SNPs in Mammary Gland Epithelial Cells Unraveling Potential Difference in Milk Production Between Jersey and Kashmiri Cattle Using RNA Sequencing

Deep RNA sequencing experiment was employed to detect putative single nucleotide polymorphisms (SNP) in mammary epithelial cells between two diverse cattle breeds (Jersey and Kashmiri) to understand the variations in the coding regions that reflect differences in milk production traits. The low milk-producing Kashmiri cattle are being replaced by crossbreeding practices with Jersey cattle with the aim of improving milk production. However, crossbred animals are prone to infections and various other diseases resulting in unsustainable milk production. In this study, we tend to identify high-impact SNPs from Jersey and Kashmiri cows (utilizing RNA-Seq data) to delineate key pathways mediating milk production traits in both breeds. A total of 607 (442 SNPs and 169 INDELs) and 684 (464 SNPs and 220 INDELs) high-impact variants were found specific to Jersey and Kashmir cattle, respectively. Based on our results, we conclude that in Jersey cattle, genes with high-impact SNPs were enriched in nucleotide excision repair pathway, ABC transporter, and metabolic pathways like glycerolipid metabolism, pyrimidine metabolism, and amino acid synthesis (glycine, serine, and threonine). Whereas, in Kashmiri cattle, the most enriched pathways include endocytosis pathway, innate immunity pathway, antigen processing pathway, insulin resistance pathway, and signaling pathways like TGF beta and AMPK which could be a possible defense mechanism against mammary gland infections. A varied set of SNPs in both breeds, suggests a clear differentiation at the genomic level; further analysis of high-impact SNPs are required to delineate their effect on these pathways.

Abstract NG15: Progesterone-mediated immune evasion in breast cancer

Abstract NG15: Progesterone-mediated immune evasion in breast cancer

Induction of Serum Amyloid A3 in Mouse Mammary Epithelial Cells Stimulated with Lipopolysaccharide and Lipoteichoic Acid.

Simple SummarySerum amyloid A (SAA) is an acute phase protein present in mammals and birds. Based on the amino acid sequence, SAA has been classified into isoforms SAA1–4 in mice. Previously, it was reported that after the stimulation with bacterial antigens, the expression of the Saa3 mRNA was induced more strongly than that of the Saa1 mRNA in mouse epithelia, including colonic and alveolar epithelial cells, indicating that SAA3 plays a role in the local response. However, the contribution of SAA3 to the local response in mouse mammary epithelium, where mastitis occurs due to bacterial infection, has not been completely determined yet. In this study, to clarify whether mouse SAA3 has a role in the defense against bacterial infection in mouse mammary epithelium, normal murine mammary gland (NMuMG) epithelial cells were stimulated with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). LPS and LTA significantly enhanced mRNA expression level of the Saa3 gene but not that of Saa1. Furthermore, LPS induced SAA3 protein expression more strongly than LTA. Our data indicate that SAA3 expression in mouse mammary epithelial cells was increased by the stimulation with bacterial antigens, suggesting that SAA3 is involved in the defense against bacterial infection in mouse mammary epithelium.In this study, to establish whether serum amyloid A (SAA) 3 plays a role in the defense against bacterial infection in mouse mammary epithelium, normal murine mammary gland (NMuMG) epithelial cells were stimulated with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). LPS and LTA significantly enhanced mRNA expression level of the Saa3 gene, whereas no significant change was observed in the Saa1 mRNA level. Furthermore, LPS induced SAA3 protein expression more strongly than LTA, whereas neither LPS nor LTA significantly affected SAA1 protein expression. These data indicate that the expression of SAA3 in mouse mammary epithelial cells was increased by the stimulation with bacterial antigens. SAA3 has been reported to stimulate neutrophils in the intestinal epithelium and increase interleukin-22 expression, which induces activation of the innate immune system and production of antibacterial proteins, such as antimicrobial peptides. Therefore, collectively, these data suggest that SAA3 is involved in the defense against bacterial infection in mouse mammary epithelium.

Cell lineage tracing links ERα loss in Erbb2-positive breast cancers to the arising of a highly aggressive breast cancer subtype

HER2-positive (HER2+) breast cancers (BrCs) contain approximately equal numbers of ERα+HER2+ and ERα-HER2+ cases. An enduring obstacle is the unclear cell lineage-related characteristics of these BrCs. Although ERα+HER2+ BrCs could lose ERα to become ERα-HER2+ BrCs, direct evidence is missing. To investigate ERα dependencies and their implications during BrC growth and metastasis, we generated ERαCreRFP-T mice that produce an RFP-marked ERα+ mammary gland epithelial cell (MGEC) lineage. RCAS virus-mediated expression of Erbb2, a rodent Her2 homolog, first produced comparable numbers of ERα+RFP+Erbb2+ and ERα-RFP-Erbb2+ MGECs. Early hyperplasia developed mostly from ERα+RFP+Erbb2+ cells and ERα-RFP-Erbb2+ cells in these lesions were rare. The subsequently developed ductal carcinomas in situ had 64% slow-proliferating ERα+RFP+Erbb2+ cells, 15% fast-proliferating ERα-RFP+Erbb2+ cells derived from ERα+RFP+Erbb2+ cells, and 20% fast-proliferating ERα-RFP-Erbb2+ cells. The advanced tumors had mostly ERα-RFP+Erbb2+ and ERα-RFP-Erbb2+ cells and only a very small population of ERα+RFP+Erbb2+ cells. In ERα-RFP+Erbb2+ cells, GATA3 and FoxA1 decreased expression and ERα promoter regions became methylated, consistent with the loss of ERα expression. Lung metastases consisted of mostly ERα-RFP+Erbb2+ cells, a few ERα-RFP-Erbb2+ cells, and no ERα+RFP+Erbb2+ cells. The high metastatic capacity of ERα-RFP+Erbb2+ cells was associated with ERK1/2 activation. These results show that the slow-proliferating, nonmetastatic ERα+RFP+Erbb2+ cells progressively lose ERα during tumorigenesis to become fast-proliferating, highly metastatic ERα-RFP+Erbb2+ cells. The ERα-Erbb2+ BrCs with an ERα+ origin are more aggressive than those ERα-Erbb2+ BrCs with an ERα- origin, and thus, they should be distinguished and treated differently in the future.

Estrogen Induces Transcriptomic Changes Associated with Oncogenic Phenotypes in Inflammatory Breast Cancer

Inflammatory Breast Cancer (IBC) is rare and the most aggressive subtype of all breast cancers, reporting rapid progression, poor prognosis, and a unique clinical diagnosis. Approximately ~30% of IBC cases are classified as triple negative breast cancer (TNBC), meaning they do not test positive for hormone receptors (estrogen and progesterone) and amplification of the human epidermal growth factor receptor 2 (HER2); therefore limiting the effectiveness of hormonal therapeutics. However, recent studies have reported that estrogen (E2) can induce a rapid E2-response in IBC cells to enhance migration and invasion phenotypes by activation of novel cytoplasmic and membrane localized estrogen receptors (ERα36 isoform and G protein-coupled receptor 30 (GPR30)); both suggested as possible therapeutic targets. However, the role of these receptors in the estrogen genomic signaling pathway remains elusive and there is still no data published related to an E2-induced transcriptional stimulation of genes associated with IBC's aggressive phenotype. Here, we evaluated gene expression changes related to E2 in IBC cell lines SUM149PT to identify target genes and biological pathways associated with IBC's aggressive phenotype. We hypothesized that E2 exposure can induce transcriptional stimulation of genes related to migration and invasion phenotypes in IBC cells SUM149PT. Methods We performed RNA-seq (Illumina NextSeq PE 2x75bp) in 2 study groups (ETOH-treated and E2-treated) to identify specific changes in the expression of genes associated with migration and invasion in IBC SUM149PT. Functional and enrichment analysis of differentially expressed genes were performed using Metascape. qPCR was performed to validate a subset of genes related to the enriched terms associated with the migration and invasion phenotypes. Results After E2 exposue (3hrs), we have identified 370 significantly dysregulated genes (p<0.05), including the ZNF268 gene (adj. p-val significant), which can be potential therapeutic targets. Functional annotation of these genes showed association with mammary gland epithelial cell proliferation, actin cytoskeleton organization, and transmembrane receptor protein tyrosine kinase signaling pathway. Data acquired from these experiments will contribute to the better understanding of IBC's transcriptomic signature and create new opportunities to develop novel targeted therapies.

Synthesis and Characterization of Potent and Safe Ciprofloxacin-Loaded Ag/TiO2/CS Nanohybrid against Mastitis Causing E. coli

To improve the efficacy of existing classes of antibiotics (ciprofloxacin), allow dose reduction, and minimize related toxicity, this study was executed because new target-oriented livestock antimicrobials are greatly needed to battle infections caused by multidrug-resistant (MDR) strains. The present study aims to green synthesize a biocompatible nanohybrid of ciprofloxacin (CIP)-Ag/TiO2/chitosan (CS). Silver and titanium nanoparticles were green synthesized using Moringa concanensis leaves extract. The incorporation of silver (Ag) nanoparticles onto the surface of titanium oxide nanoparticles (TiO2NPs) was done by the wet chemical impregnation method, while the encapsulation of chitosan (CS) around Ag/TiO2 conjugated with ciprofloxacin (CIP) was done by the ionic gelation method. The synthesized nanohybrid (CIP-Ag/TiO2/CS) was characterized using standard techniques. The antibacterial potential, killing kinetics, cytotoxicity, drug release profile, and minimum inhibitory concentration (MIC) were determined. Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) revealed spherical agglomerated nanoparticles (NPs) of Ag/TiO2 with particle sizes of 47–75 nm, and those of the CIP-Ag/TiO2/CS nanohybrid were in range of 20–80 nm. X-ray diffractometer (XRD) patterns of the hetero system transmitted diffraction peaks of anatase phase of TiO2 and centered cubic metallic Ag crystals. Fourier Transform Infrared spectroscopy (FTIR) confirmed the Ti-O-Ag linkage in the nanohybrid. The zeta potential of CIP-Ag/TiO2/CS nanohybrid was found (67.45 ± 1.8 mV), suggesting stable nanodispersion. The MIC of CIP-Ag/TiO2/CS was 0.0512 μg/mL, which is much lower than the reference value recorded by the global CLSI system (Clinical Laboratory Standards Institute). The CIP-Ag/TiO2/CS nanohybrid was found to be effective against mastitis causing MDR E. coli; killing kinetics showed an excellent reduction of E. coli cells at 6 h of treatment. Flow cytometry further confirmed antibacterial potential by computing 67.87% late apoptosis feature at 6 h of treatment; antibiotic release kinetic revealed a sustained release of CIP. FESEM and TEM confirmed the structural damages in MDR E. coli (multidrug-resistant Escherichia coli). The CIP-Ag/TiO2/CS nanohybrid was found to be biocompatible, as more than 93.08% of bovine mammary gland epithelial cells remained viable. The results provide the biological backing for the development of nanohybrid antibiotics at a lower MIC value to treat infectious diseases of cattle and improve the efficacy of existing classes of antibiotics by conjugation with nanoparticles.

Abstract LT021: Targeting AXL favors an anti-tumorigenic tumor microenvironment that enhances immunotherapy responses by decreasing HIF-1a levels in cancer cells

Abstract Breast cancer-related deaths are predominantly associated to complications arising from metastases. Hypoxia is an important biological process that fuels metastasis and contributes to deregulate the tumor microenvironment (TME) of solid tumors. Hypoxia also promotes resistance to therapies by a plethora of mechanisms including enhancing tumoral angiogenesis, metabolic reprogramming, epithelial-to-mesenchymal transition (EMT) and immune evasion. The expression of the receptor tyrosine kinase AXL, a hypoxia-induced gene, is associated with poor clinical prognosis and metastasis in various cancers, including breast cancer. Here, we hypothesize that genetic or pharmacological interference of AXL may alter hypoxic responses and associated modulation of the TME offering an ideal setting for immunotherapy. In a HER2 mouse model of breast cancer (MMTV-NIC), we found that the mammary gland epithelial cells specific deletion of AXL led to a reduction of lung metastases and improved the TME by reducing the hypoxic response in tumor cells. When breast cancer cells were subjected to hypoxic conditions, we found that AXL inhibition reduced the levels of the hypoxia transcription factor HIF-1a which resulted in an altered hypoxic response. Specifically, AXL deletion led to a reduction of hypoxia-induced EMT and invasion, and a reduction of the secretion of key cytokines for macrophages recruitment and polarization. We demonstrate in vivo that AXL pharmacological inhibition in the MMTV-NIC breast cancer model generated an anti-inflammatory TME that enhanced an anti-PD-1 immune checkpoint blockade response and decreased the metastatic burden. Collectively, these results suggest that targeting AXL could be a powerful approach to improve immunotherapy response by generating an anti-tumoral microenvironment therefore limiting the metastatic burden of breast cancer. Citation Format: Marie-Anne Goyette, Jean-Philippe Gratton, Jean-François Côté. Targeting AXL favors an anti-tumorigenic tumor microenvironment that enhances immunotherapy responses by decreasing HIF-1a levels in cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT021.

Abstract PS17-32: Characterization of HOXB13-induced estrogen receptor reprogramming in breast cancer cells

Abstract Background: The Breast Cancer Index (BCI) is a gene expression-based signature that consists of two functional biomarker panels, HOXB13/IL17BR (H/I) and Molecular Grade Index (MGI), that interrogate important proliferation and estrogen signaling pathways in breast cancer. The BCI prognostic score reports individualized risk of overall and late distant recurrence and is based on the algorithmic combination of the H/I ratio and MGI, while the predictive component, BCI (H/I), reports a categorical prediction of high versus low likelihood of benefit from extended endocrine therapy.The homeobox transcription factor HOXB13 has previously been shown to reprogram genome-wide binding of the androgen receptor (AR) during prostate cancer tumorigenesis, where it colocalizes with FOXA1 at reprogrammed AR binding sites. The aim of the current study was to characterize the potential role of HOXB13 in estrogen receptor (ER) reprogramming by analyzing changes in the global ER binding pattern induced by transient overexpression of HOXB13 in breast cancer cells. In addition, gene ontology (GO) analysis was performed to delineate the functional role of HOXB13 in modulating estrogen signaling and response to endocrine therapy.Methods: HOXB13 was overexpressed in MCF-7 cells by electroporating HOXB13 mRNA, or eGFP mRNA as control. Cells were harvested at different time points and analyzed by western blot and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) using antibodies against ER, HOXB13, FOXA1 and H3K27ac. After aligning reads with Bowtie2, peak calling and data integration were performed using HOMER v4.10. Gene ontology (GO) analysis was performed using the Genomic Regions Enrichment of Annotations Tool (GREAT v4.0.4) (http://great.stanford.edu/) to identify annotations enriched among genes near ER genomic binding sites.Results: ChIP-seq analysis revealed substantial binding of HOXB13 to a large number of genomic binding sites compared to the eGFP control. HOXB13 overexpression in ER+ breast cancer cells significantly increased ER binding with a majority of ER binding sites being co-bound by FOXA1 and HOXB13. GO analysis of both proximal and distal genomic regions showed significant enrichment of genes associated with mammary gland development, such as “mammary gland epithelium development”, “mammary gland development”, and “mammary gland epithelial cell differentiation”.Conclusion: Findings from this analysis show that transient HOXB13 overexpression reprograms and expands the ER binding pattern in breast cancer cells. Genomic regions newly bound by ER are enriched for genes involved in mammary gland and epithelial differentiation suggesting that HOXB13 activates ER transcriptional programs that link oncogenic and developmental pathways. These results will be compared to ongoing experiments using an inducible HOXB13 expression system to assess the effects of HOXB13 expression on ER binding and function in response to estradiol and tamoxifen. Citation Format: Kai Treuner, Dominic Schenone, Christopher Benner, Catherine A Schnabel, Sven Heinz. Characterization of HOXB13-induced estrogen receptor reprogramming in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-32.

Assessing extracellular vesicles from bovine mammary gland epithelial cells cultured in FBS-free medium

Extracellular Vesicles and Circulating Nucleic Acids is an open access journal, focusing on extracellular vesicles and circulating nucleic acids including DNA, RNA, and miRNA and their therapeutic use.