RESUMEN

Oncogenic mutations are abundant in the tissues of healthy individuals, but rarely form tumours1-3. Yet, the underlying protection mechanisms are largely unknown. To resolve these mechanisms in mouse mammary tissue, we use lineage tracing to map the fate of wild-type and Brca1-/-;Trp53-/- cells, and find that both follow a similar pattern of loss and spread within ducts. Clonal analysis reveals that ducts consist of small repetitive units of self-renewing cells that give rise to short-lived descendants. This offers a first layer of protection as any descendants, including oncogenic mutant cells, are constantly lost, thereby limiting the spread of mutations to a single stem cell-descendant unit. Local tissue remodelling during consecutive oestrous cycles leads to the cooperative and stochastic loss and replacement of self-renewing cells. This process provides a second layer of protection, leading to the elimination of most mutant clones while enabling the minority that by chance survive to expand beyond the stem cell-descendant unit. This leads to fields of mutant cells spanning large parts of the epithelial network, predisposing it for transformation. Eventually, clone expansion becomes restrained by the geometry of the ducts, providing a third layer of protection. Together, these mechanisms act to eliminate most cells that acquire somatic mutations at the expense of driving the accelerated expansion of a minority of cells, which can colonize large areas, leading to field cancerization.

Asunto(s)

Proteína BRCA1 , Linaje de la Célula , Transformación Celular Neoplásica , Glándulas Mamarias Animales , Mutación , Proteína p53 Supresora de Tumor , Animales , Ratones , Femenino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/patología , Glándulas Mamarias Animales/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linaje de la Célula/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Transformación Celular Neoplásica/genética , Células Clonales/metabolismo , Células Clonales/citología , Carcinogénesis/genética , Carcinogénesis/patología , Autorrenovación de las Células/genética

RESUMEN

The prolactin (PRL) hormone is a major regulator of mammary gland development and lactation. However, it remains unclear whether and how PRL contributes to mammary epithelial cell proliferation and secretion. The Boer and Macheng black crossbred goats are superior in reproduction, meat, and milk, and are popular in Hubei province. To elucidate the mechanisms of PRL on mammary growth and lactation, to improve the local goat economic trade, we have performed studies on these crossbred goats during pregnancy and early lactation, and in goat mammary epithelial cells (GMECs). Here, we first found that the amino acid transporters of SNAT1 and SNAT2 expression in vivo and in vitro were closely associated with PRL levels, the proliferation and secretion of GMECs; knockdown and over-expression of SNAT1/2 demonstrated that PRL modulated the proliferation and lactation of GMECs through regulating SNAT1/2 expression. Transcriptome sequencing and qPCR assays demonstrated the effect of PRL on the transcriptional regulation of SNAT1 and SNAT2 in GMECs. Dual-luciferase reporter gene assays further verified that the binding of the potential PRL response element in the SNAT1/2 promoter regions activated SNAT1/2 transcription after PRL stimulation. Additionally, silencing of either PRLR or STAT5 nearly abolished PRL-stimulated SNAT1/2 promoter activity, suggesting PRLR-STAT5 signaling is involved in the regulation of PRL on the transcriptional activation of SNAT1/2. These results illustrated that PRL modulates the proliferation and secretion of GMECs via PRLR-STAT5-mediated regulation of the SNAT1/2 pathway. This study provides new insights into how PRL affects ruminant mammary development and lactation through regulation of amino acid transporters.

Asunto(s)

Proliferación Celular , Células Epiteliales , Cabras , Lactancia , Glándulas Mamarias Animales , Prolactina , Animales , Prolactina/metabolismo , Femenino , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Factor de Transcripción STAT5/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Embarazo , Regiones Promotoras Genéticas/genética

Asunto(s)

Neoplasias de la Mama , Glándulas Mamarias Animales , Animales , Femenino , Humanos , Ratones , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Transformación Celular Neoplásica/patología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología

RESUMEN

In this study, the expression profiles of miR-148a were constructed in eight different ovine tissues, including mammary gland tissue, during six different developmental periods. The effect of miR-148a on the viability, proliferation, and milk fat synthesis of ovine mammary epithelial cells (OMECs) was investigated, and the target relationship of miR-148a with two predicted target genes was verified. The expression of miR-148a exhibited obvious tissue-specific and temporal-specific patterns. miR-148a was expressed in all eight ovine tissues investigated, with the highest expression level in mammary gland tissue (p < 0.05). Additionally, miR-148a was expressed in ovine mammary gland tissue during each of the six developmental periods studied, with its highest level at peak lactation (p < 0.05). The overexpression of miR-148a increased the viability of OMECs, the number and percentage of Edu-labeled positive OMECs, and the expression levels of two cell-proliferation marker genes. miR-148a also increased the percentage of OMECs in the S phase. In contrast, transfection with an miR-148a inhibitor produced the opposite effect compared to the miR-148a mimic. These results indicate that miR-148a promotes the viability and proliferation of OMECs in Small-tailed Han sheep. The miR-148a mimic increased the triglyceride content by 37.78% (p < 0.01) and the expression levels of three milk fat synthesis marker genes in OMECs. However, the miR-148a inhibitor reduced the triglyceride level by 87.11% (p < 0.01). These results suggest that miR-148a promotes milk fat synthesis in OMECs. The dual-luciferase reporter assay showed that miR-148a reduced the luciferase activities of DNA methyltransferase 1 (DNMT1) and peroxisome proliferator-activated receptor gamma coactivator 1-A (PPARGC1A) in wild-type vectors, suggesting that they are target genes of miR-148a. The expression of miR-148a was highly negatively correlated with PPARGC1A (r = -0.789, p < 0.001) in ovine mammary gland tissue, while it had a moderate negative correlation with DNMT1 (r = -0.515, p = 0.029). This is the first study to reveal the molecular mechanisms of miR-148a underlying the viability, proliferation, and milk fat synthesis of OMECs in sheep.

Asunto(s)

Proliferación Celular , Supervivencia Celular , ADN (Citosina-5-)-Metiltransferasa 1 , Células Epiteliales , Glándulas Mamarias Animales , MicroARNs , Leche , Animales , MicroARNs/genética , MicroARNs/metabolismo , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/citología , Femenino , Ovinos , Leche/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Lactancia/genética , Lactancia/metabolismo , Regulación de la Expresión Génica

RESUMEN

Selenomethionine (SeMet) is an important nutrient, but its role in milk synthesis and the GPCR related to SeMet sensing is still largely unknown. Here, we determined the dose-dependent role of SeMet on milk protein and fat synthesis and proliferation of mammary epithelial cells (MECs), and we also uncovered the GPCR-mediating SeMet function. At 24 h postdelivery, lactating mother mice were fed a maintenance diet supplemented with 0, 5, 10, 20, 40, and 80 mg/kg SeMet, and the feeding process lasted for 18 days. The 10 mg/kg group had the best increase in milk production, weight gain of offspring mice, and mammary gland weight and acinar size, whereas a higher concentration of SeMet gradually decreased the weight gain of the offspring mice and showed toxic effects. Transcriptome sequencing was performed to find the differentially expressed genes (DEGs) between the mammary gland tissues of mother mice in the 10 mg/kg SeMet treatment group and the control group. A total of 258 DEGs were screened out, including 82 highly expressed genes including GPR37 and 176 lowly expressed genes. SeMet increased milk protein and fat synthesis in HC11 cells and cell proliferation, mTOR and S6K1 phosphorylation, and expression of GPR37 in a dose-dependent manner. GPR37 knockdown decreased milk protein and fat synthesis in HC11 cells and cell proliferation and blocked SeMet stimulation on mTOR and S6K1 phosphorylation. Taken together, our data demonstrate that SeMet can promote milk protein and fat synthesis and proliferation of MECs and functions through the GPR37-mTOR-S6K1 signaling pathway.

Asunto(s)

Proliferación Celular , Células Epiteliales , Glándulas Mamarias Animales , Proteínas de la Leche , Receptores Acoplados a Proteínas G , Selenometionina , Transducción de Señal , Serina-Treonina Quinasas TOR , Animales , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Femenino , Ratones , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Selenometionina/farmacología , Transducción de Señal/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/citología , Proteínas de la Leche/metabolismo , Lactancia , Humanos , Proteínas Quinasas S6 Ribosómicas 90-kDa

RESUMEN

Background: Our previous studies have successfully reported the reprogramming of fibroblasts into induced mammary epithelial cells (iMECs). However, the regulatory relationships and functional roles of MicroRNAs (miRNAs) in the progression of fibroblasts achieving the cell fate of iMECs are insufficiently understood. Methods: First, we performed pre-and post-induction miRNAs sequencing analysis by using high-throughput sequencing. Following that, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment studies were used to determine the primary roles of the significantly distinct miRNAs and targeted genes. Finally, the effect of miR-222-3p on iMECs fate reprogramming in vitro by transfecting. Results: As a result goat ear fibroblasts (GEFs) reprogramming into iMECs activates a regulatory program, involving 79 differentially expressed miRNAs. Besides, the programming process involved changes in multiple signaling pathways such as adherens junction, TGF-ß signaling pathway, GnRH secretion and the prolactin signaling pathway, etc. Furthermore, it was discovered that the expression of miR-222-3p downregulation by miR-222-3p inhibitor significantly increase the reprogramming efficiency and promoted lipid accumulation of iMECs.

Asunto(s)

Reprogramación Celular , Células Epiteliales , Fibroblastos , Cabras , MicroARNs , MicroARNs/genética , MicroARNs/metabolismo , Animales , Fibroblastos/metabolismo , Células Epiteliales/metabolismo , Femenino , Reprogramación Celular/genética , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Transducción de Señal , Células Cultivadas , Regulación hacia Abajo

RESUMEN

Macrophages have important roles in mammary gland development and tissue homeostasis, but the specific mechanisms that regulate macrophage function need further elucidation. We have identified C/EBPß as an important transcription factor expressed by multiple macrophage populations in the normal mammary gland. Mammary glands from mice with C/EBPß-deficient macrophages (Cebpb ΔM) show a significant decrease in alveolar budding during the diestrus stage of the reproductive cycle, whereas branching morphogenesis remains unchanged. Defects in alveolar budding were found to be the result of both systemic hormones and local macrophage-directed signals. RNA sequencing shows significant changes in PR-responsive genes and alterations in the Wnt landscape of mammary epithelial cells of Cebpb ΔM mice, which regulate stem cell expansion during diestrus. Cebpb ΔM macrophages demonstrate a shift from a pro-inflammatory to a tissue-reparative phenotype, and exhibit increased phagocytic capacity as compared to WT. Finally, Cebpb ΔM macrophages down-regulate Notch2 and Notch3, which normally promote stem cell expansion during alveolar budding. These results suggest that C/EBPß is an important macrophage factor that facilitates macrophage-epithelial crosstalk during a key stage of mammary gland tissue homeostasis.

Asunto(s)

Proteína beta Potenciadora de Unión a CCAAT , Ciclo Estral , Macrófagos , Glándulas Mamarias Animales , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Femenino , Ratones , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Macrófagos/metabolismo , Ciclo Estral/genética , Ratones Noqueados , Receptores Notch/metabolismo , Receptores Notch/genética , Células Epiteliales/metabolismo , Fagocitosis/genética , Ratones Endogámicos C57BL , Eliminación de Gen

RESUMEN

The luminal-to-basal transition in mammary epithelial cells (MECs) is accompanied by changes in epithelial cell lineage plasticity; however, the underlying mechanism remains elusive. Here, we report that deficiency of Frmd3 inhibits mammary gland lineage development and induces stemness of MECs, subsequently leading to the occurrence of triple-negative breast cancer. Loss of Frmd3 in PyMT mice results in a luminal-to-basal transition phenotype. Single-cell RNA sequencing of MECs indicated that knockout of Frmd3 inhibits the Notch signaling pathway. Mechanistically, FERM domain-containing protein 3 (FRMD3) promotes the degradation of Disheveled-2 by disrupting its interaction with deubiquitinase USP9x. FRMD3 also interrupts the interaction of Disheveled-2 with CK1, FOXK1/2, and NICD and decreases Disheveled-2 phosphorylation and nuclear localization, thereby impairing Notch-dependent luminal epithelial lineage plasticity in MECs. A low level of FRMD3 predicts poor outcomes for breast cancer patients. Together, we demonstrated that FRMD3 is a tumor suppressor that functions as an endogenous activator of the Notch signaling pathway, facilitating the basal-to-luminal transformation in MECs.

Asunto(s)

Células Epiteliales , Receptores Notch , Transducción de Señal , Animales , Células Epiteliales/metabolismo , Femenino , Receptores Notch/metabolismo , Humanos , Ratones , Linaje de la Célula , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/citología , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Diferenciación Celular , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/genética

RESUMEN

Background: Innovative therapies against bacterial infections are needed. One approach is to focus on host-directed immunotherapy (HDT), with treatments that exploit natural processes of the host immune system. The goals of this type of therapy are to stimulate protective immunity while minimizing inflammation-induced tissue damage. We use non-traditional large animal models to explore the potential of the mammosphere-derived epithelial cell (MDEC) secretome, consisting of all bioactive factors released by the cells, to modulate host immune functions. MDEC cultures are enriched for mammary stem and progenitor cells and can be generated from virtually any mammal. We previously demonstrated that the bovine MDEC secretome, collected and delivered as conditioned medium (CM), inhibits the growth of bacteria in vitro and stimulates functions related to tissue repair in cultured endothelial and epithelial cells. Methods: The immunomodulatory effects of the bovine MDEC secretome on bovine neutrophils, an innate immune cell type critical for resolving bacterial infections, were determined in vitro using functional assays. The effects of MDEC CM on neutrophil molecular pathways were explored by evaluating the production of specific cytokines by neutrophils and examining global gene expression patterns in MDEC CM-treated neutrophils. Enzyme linked immunosorbent assays were used to determine the concentrations of select proteins in MDEC CM and siRNAs were used to reduce the expression of specific MDEC-secreted proteins, allowing for the identification of bioactive factors modulating neutrophil functions. Results: Neutrophils exposed to MDEC secretome exhibited increased chemotaxis and phagocytosis and decreased intracellular reactive oxygen species and extracellular trap formation, when compared to neutrophils exposed to control medium. C-X-C motif chemokine 6, superoxide dismutase, peroxiredoxin-2, and catalase, each present in the bovine MDEC secretome, were found to modulate neutrophil functions. Conclusion: The MDEC secretome administered to treat bacterial infections may increase neutrophil recruitment to the site of infection, stimulate pathogen phagocytosis by neutrophils, and reduce neutrophil-produced ROS accumulation. As a result, pathogen clearance might be improved and local inflammation and tissue damage reduced.

Asunto(s)

Células Epiteliales , Neutrófilos , Secretoma , Animales , Bovinos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/inmunología , Secretoma/metabolismo , Femenino , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Fagocitosis , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/citología , Células Cultivadas , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Milk fat synthesis has garnered significant attention due to its influence on the quality of milk. Recently, an increasing amount of proofs have elucidated that microRNAs (miRNAs) are important post-transcriptional factor involved in regulating gene expression and play a significant role in milk fat synthesis. MiR-200a was differentially expressed in the mammary gland tissue of dairy cows during different lactation periods, which indicated that miR-200a was a candidate miRNA involved in regulating milk fat synthesis. In our research, we investigated the potential function of miR-200a in regulating milk fat biosynthesis in bovine mammary epithelial cells (BMECs). We discovered that miR-200a inhibited cellular triacylglycerol (TAG) synthesis and suppressed lipid droplet formation; at the same time, miR-200a overexpression suppressed the mRNA and protein expression of milk fat metabolism-related genes, such as fatty acid synthase (FASN), peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding protein 1 (SREBP1), CCAAT enhancer binding protein alpha (CEBPα), etc. However, knocking down miR-200a displayed the opposite results. We uncovered that insulin receptor substrate 2 (IRS2) was a candidate target gene of miR-200a through the bioinformatics online program TargetScan. Subsequently, it was confirmed that miR-200a directly targeted the 3'-untranslated region (3'-UTR) of IRS2 via real-time fluorescence quantitative PCR (RT-qPCR), western blot analysis, and dual-luciferase reporter gene assay. Additionally, IRS2 knockdown in BMECs has similar effects to miR-200a overexpression. Our research set up the mechanism by which miR-200a interacted with IRS2 and discovered that miR-200a targeted IRS2 and modulated the activity of the PI3K/Akt signaling pathway, thereby taking part in regulating milk fat synthesis in BMECs. Our research results provided valuable information on the molecular mechanisms for enhancing milk quality from the view of miRNA-mRNA regulatory networks.

Asunto(s)

Células Epiteliales , Proteínas Sustrato del Receptor de Insulina , Glándulas Mamarias Animales , MicroARNs , Leche , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Animales , Bovinos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Leche/metabolismo , Leche/química , Células Epiteliales/metabolismo , Femenino , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Triglicéridos/metabolismo , Triglicéridos/biosíntesis , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Grasas/metabolismo , Lactancia/genética

RESUMEN

The origin of breast cancer (BC) has traditionally been a focus of medical research. It is widely acknowledged that BC originates from immortal mammary stem cells and that these stem cells participate in two division modes: symmetric cell division (SCD) and asymmetrical cell division (ACD). Although both of these modes are key to the process of breast development and their imbalance is closely associated with the onset of BC, the molecular mechanisms underlying these phenomena deserve in-depth exploration. In this review, we first outline the molecular mechanisms governing ACD/SCD and analyze the role of ACD/SCD in various stages of breast development. We describe that the changes in telomerase activity, the role of polar proteins, and the stimulation of ovarian hormones subsequently lead to two distinct consequences: breast development or carcinogenesis. Finally, gene mutations, abnormalities in polar proteins, modulation of signal-transduction pathways, and alterations in the microenvironment disrupt the balance of BC stem cell division modes and cause BC. Important regulatory factors such as mammalian Inscuteable mInsc, Numb, Eya1, PKCα, PKCθ, p53, and IL-6 also play significant roles in regulating pathways of ACD/SCD and may constitute key targets for future research on stem cell division, breast development, and tumor therapy.

Asunto(s)

Neoplasias de la Mama , Carcinogénesis , Glándulas Mamarias Humanas , Humanos , Femenino , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Animales , Glándulas Mamarias Humanas/crecimiento & desarrollo , Glándulas Mamarias Humanas/patología , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/metabolismo , Carcinogénesis/patología , Carcinogénesis/metabolismo , Carcinogénesis/genética , Células Madre/metabolismo , Células Madre/citología , División Celular , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/patología , Glándulas Mamarias Animales/metabolismo , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Transducción de Señal

RESUMEN

Iron overload causes mitochondrial damage, and then activates mitophagy, which may directly trigger and amplify ferroptosis. Our objective was to investigate whether Escherichia coli (E. coli) isolated from clinical bovine mastitis induces ferroptosis in bovine mammary epithelial cells (bMECs) and if so, the underlying regulatory mechanism. E. coli infection caused mitochondrial damage, mitophagy, and ferroptosis. Rapamycin and chloroquine increased and suppressed ferroptosis, respectively, in E. coli-treated bMECs. Moreover, E. coli infection activated the Wnt/ß-catenin pathway, but foscenvivint alleviated it. In conclusion, E. coli infection induced ferroptosis through activation of the Wnt/ß-catenin pathway-promoted mitophagy, and it also suppressed GPX4 expression.

Asunto(s)

Células Epiteliales , Escherichia coli , Ferroptosis , Glándulas Mamarias Animales , Mitofagia , Vía de Señalización Wnt , Animales , Ferroptosis/efectos de los fármacos , Bovinos , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Mitofagia/efectos de los fármacos , Femenino , Escherichia coli/metabolismo , Glándulas Mamarias Animales/microbiología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/patología , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/metabolismo , Mitocondrias/metabolismo , Mastitis Bovina/microbiología , Mastitis Bovina/metabolismo

RESUMEN

In many cancers, a stem-like cell subpopulation mediates tumor initiation, dissemination and drug resistance. Here, we report that cancer stem cell (CSC) abundance is transcriptionally regulated by C-terminally phosphorylated p27 (p27pT157pT198). Mechanistically, this arises through p27 co-recruitment with STAT3/CBP to gene regulators of CSC self-renewal including MYC, the Notch ligand JAG1, and ANGPTL4. p27pTpT/STAT3 also recruits a SIN3A/HDAC1 complex to co-repress the Pyk2 inhibitor, PTPN12. Pyk2, in turn, activates STAT3, creating a feed-forward loop increasing stem-like properties in vitro and tumor-initiating stem cells in vivo. The p27-activated gene profile is over-represented in STAT3 activated human breast cancers. Furthermore, mammary transgenic expression of phosphomimetic, cyclin-CDK-binding defective p27 (p27CK-DD) increases mammary duct branching morphogenesis, yielding hyperplasia and microinvasive cancers that can metastasize to liver, further supporting a role for p27pTpT in CSC expansion. Thus, p27pTpT interacts with STAT3, driving transcriptional programs governing stem cell expansion or maintenance in normal and cancer tissues.

Asunto(s)

Neoplasias de la Mama , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Hiperplasia , Células Madre Neoplásicas , Factor de Transcripción STAT3 , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Humanos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Animales , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Femenino , Fosforilación , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Hiperplasia/metabolismo , Ratones , Regulación Neoplásica de la Expresión Génica , Autorrenovación de las Células/genética , Línea Celular Tumoral , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Glándulas Mamarias Animales/citología , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genética

RESUMEN

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.

Asunto(s)

Células Epiteliales , Glándulas Mamarias Animales , Leche , Transducción de Señal , Serina-Treonina Quinasas TOR , Ubiquitinación , Animales , Bovinos , Serina-Treonina Quinasas TOR/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Ubiquitinación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Femenino , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/citología , Leche/química , Leche/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Esteroides/genética

RESUMEN

BACKGROUND: The cell cycle of mammary stem cells must be tightly regulated to ensure normal homeostasis of the mammary gland to prevent abnormal proliferation and susceptibility to tumorigenesis. The atypical cell cycle regulator, Spy1 can override cell cycle checkpoints, including those activated by the tumour suppressor p53 which mediates mammary stem cell homeostasis. Spy1 has also been shown to promote expansion of select stem cell populations in other developmental systems. Spy1 protein is elevated during proliferative stages of mammary gland development, is found at higher levels in human breast cancers, and promotes susceptibility to mammary tumourigenesis when combined with loss of p53. We hypothesized that Spy1 cooperates with loss of p53 to increase susceptibility to tumour initiation due to changes in susceptible mammary stem cell populations during development and drives the formation of more aggressive stem like tumours. METHODS: Using a transgenic mouse model driving expression of Spy1 within the mammary gland, mammary development and stemness were assessed. These mice were intercrossed with p53 null mice to study the tumourigenic properties of Spy1 driven p53 null tumours, as well as global changes in signaling via RNA sequencing analysis. RESULTS: We show that elevated levels of Spy1 leads to expansion of mammary stem cells, even in the presence of p53, and an increase in mammary tumour formation. Spy1-driven tumours have an increased cancer stem cell population, decreased checkpoint signaling, and demonstrate an increase in therapy resistance. Loss of Spy1 decreases tumor onset and reduces the cancer stem cell population. CONCLUSIONS: This data demonstrates the potential of Spy1 to expand mammary stem cell populations and contribute to the initiation and progression of aggressive, breast cancers with increased cancer stem cell populations.

Asunto(s)

Glándulas Mamarias Animales , Ratones Transgénicos , Proteína p53 Supresora de Tumor , Animales , Femenino , Ratones , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Glándulas Mamarias Animales/patología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Carcinogénesis/genética , Proliferación Celular , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Células Madre/metabolismo , Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica

RESUMEN

Understanding the normal physiology of the canine mammary gland (CMG) is crucial, as it provides a foundational reference for understanding canine mammary neoplasms. The relation between the Proliferation Index (PI) indicated by Ki-67 expression, along with the Apoptotic Index (AI) determined through Caspase-3 expression during the oestrous cycle, is inadequately documented in existing literature. This study seeks to offer insights into the interplay between PI and AI in the CMG across oestrous cycle phases. An extensive investigation was conducted on a diverse case series of bitches (n = 18). Oestrous cycle stages were determined through vaginal cytology, histological examination of the reproductive tract and serum progesterone and oestradiol concentrations. The entire mammary chain was histologically examined, and proliferation and apoptosis were assessed via double immunohistochemistry employing anti-Ki-67 and Caspase-3 antibodies. PI and AI were evaluated through a systematic random sampling approach, counting a minimum of 200 cells for each cell type. There was a significantly higher PI during early dioestrus in all mammary gland components, with a greater proportion of positive cells observed in epithelial cells compared to stromal cells. The highest PI was detected in epithelial cells within the end buds. Significant differences were found in Ki-67 labelling across the cranial mammary glands. A positive and strong correlation was noted between progesterone concentration and PI in epithelial cells. The AI remained consistently low throughout the oestrous cycle, with few differences observed across histological components. Caspase-3 labelling displayed the highest positivity in caudal mammary pairs. A negative and moderate correlation was identified between progesterone concentration and AI in interlobular mesenchymal cells. This study highlights the influence of endocrine regulation on cell proliferation indices in mammary tissue, emphasizing the need to consider these hormonal variations in toxicopathological studies involving canine mammary gland.

Asunto(s)

Apoptosis , Caspasa 3 , Proliferación Celular , Ciclo Estral , Antígeno Ki-67 , Glándulas Mamarias Animales , Progesterona , Animales , Femenino , Antígeno Ki-67/metabolismo , Perros , Apoptosis/fisiología , Glándulas Mamarias Animales/fisiología , Glándulas Mamarias Animales/citología , Caspasa 3/metabolismo , Ciclo Estral/fisiología , Progesterona/sangre , Progesterona/metabolismo , Estradiol/sangre , Estradiol/metabolismo , Células Epiteliales

RESUMEN

Conflicting data exist as to how mammary epithelial cell proliferation changes during the reproductive cycle. To study the effect of endogenous hormone fluctuations on gene expression in the mouse mammary gland, we performed bulk RNAseq analyses of epithelial and stromal cell populations that were isolated either during puberty or at different stages of the adult virgin estrous cycle. Our data confirm prior findings that proliferative changes do not occur in every mouse in every cycle. We also show that during the estrous cycle the main gene expression changes occur in adipocytes and fibroblasts. Finally, we present a comprehensive overview of the Wnt gene expression landscape in different mammary gland cell types in pubertal and adult mice. This work contributes to understanding the effects of physiological hormone fluctuations and locally produced signaling molecules on gene expression changes in the mammary gland during the reproductive cycle and should be a useful resource for future studies investigating gene expression patterns in different cell types across different developmental timepoints.

Asunto(s)

Células Epiteliales , Perfilación de la Expresión Génica , Glándulas Mamarias Animales , Maduración Sexual , Células del Estroma , Transcriptoma , Animales , Femenino , Ratones , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Células del Estroma/metabolismo , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Maduración Sexual/fisiología , Proliferación Celular , Ciclo Estral/genética

RESUMEN

Subcutaneous adipocytes are crucial for mammary gland epithelial development during pregnancy. Our and others' previous data have suggested that adipo-epithelial transdifferentiation could play a key role in the mammary gland alveolar development. In this study, we tested whether adipo-epithelial transdifferentiation occurs in vitro. Data show that, under appropriate co-culture conditions with mammary epithelial organoids (MEOs), mature adipocytes lose their phenotype and acquire an epithelial one. Interestingly, even in the absence of MEOs, extracellular matrix and diffusible growth factors are able to promote adipo-epithelial transdifferentiation. Gene and protein expression studies indicate that transdifferentiating adipocytes exhibit some characteristics of milk-secreting alveolar glands, including significantly higher expression of milk proteins such as whey acidic protein and ß-casein. Similar data were also obtained in cultured human multipotent adipose-derived stem cell adipocytes. A miRNA sequencing experiment on the supernatant highlighted mir200c, which has a well-established role in the mesenchymal-epithelial transition, as a potential player in this phenomenon. Collectively, our data show that adipo-epithelial transdifferentiation can be reproduced in in vitro models where this phenomenon can be investigated at the molecular level.

Asunto(s)

Adipocitos , Transdiferenciación Celular , Células Epiteliales , Humanos , Femenino , Adipocitos/citología , Adipocitos/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/citología , Animales , MicroARNs/metabolismo , MicroARNs/genética , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Organoides/citología , Organoides/metabolismo , Técnicas de Cocultivo , Ratones , Modelos Biológicos

RESUMEN

Heat stress is becoming the major factor regarding dairy cow health and milk quality because of global warming. Circular RNAs (circRNAs) represent a special type of noncoding RNAs, which are related to regulating many biological processes. Nonetheless, little is known concerning their effects on heat-stressed bovine mammary epithelial cells (BMECs). Here, this study found a novel circRNA, circ_002033, using RNA sequencing (RNA-seq) and explored the role and underlying regulatory mechanism in proliferation, apoptosis, and oxidative damage in a heat-stressed bovine mammary epithelial cell line (MAC-T). According to the previous RNA-seq analysis, the abundance of circ_002033 in mammary gland tissue of heat-stressed cows increased relative to nonheat-stressed counterparts. This study found that the knockdown of circ_002033 promoted proliferation and alleviated apoptosis and oxidative damage in heat-stressed MAC-T. Mechanistically, circ_002033 localizes to miR-199a-5p in the cytoplasm of MAC-T to regulate mitogen-activated protein kinase kinase 11 (MAP3K11) expression. Meanwhile, miR-199a-5p and MAP3K11 are also involved in regulating the proliferation and apoptosis of heat-stressed MAC-T. Importantly, circ_002033 knockdown promoted the expression of miR-199a-5p while decreasing that of MAP3K11, thereby enhancing proliferation while alleviating apoptosis and oxidative damage in heat-stressed MAC-T. In summary, we found that circ_002033 regulates the proliferation, apoptosis, and oxidative damage of heat-stressed BMECs through the miR-199a-5p/MAP3K11 axis, providing the theoretical molecular foundation for mitigating heat stress of dairy cows.

Asunto(s)

Apoptosis , Proliferación Celular , Células Epiteliales , Respuesta al Choque Térmico , Quinasas Quinasa Quinasa PAM , Glándulas Mamarias Animales , MicroARNs , Estrés Oxidativo , ARN Circular , Animales , Bovinos , Células Epiteliales/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Femenino , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/citología , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Proteina Quinasa Quinasa Quinasa 11 Activada por Mitógeno , Línea Celular

RESUMEN

Adipose tissue is highly plastic, as illustrated mainly by the transdifferentiation of white adipocytes into beige adipocytes, depending on environmental conditions. However, during gestation and lactation in rodent, there is an amazing phenomenon of transformation of subcutaneous adipose tissue into mammary glandular tissue, known as pink adipose tissue, capable of synthesizing and secreting milk. Recent work using transgenic lineage-tracing experiments, mainly carried out in Saverio Cinti's team, has demonstrated very convincingly that this process does indeed correspond to a transdifferentiation of white adipocytes into mammary alveolar cells (pink adipocytes) during gestation and lactation. This phenomenon is reversible, since during the post-lactation phase, pink adipocytes revert to the white adipocyte phenotype. The molecular mechanisms underlying this reversible transdifferentiation remain poorly understood.

Asunto(s)

Tejido Adiposo , Lactancia , Animales , Humanos , Femenino , Tejido Adiposo/fisiología , Tejido Adiposo/metabolismo , Tejido Adiposo/citología , Lactancia/fisiología , Embarazo , Transdiferenciación Celular/fisiología , Glándulas Mamarias Animales/fisiología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Adipocitos Blancos/fisiología , Adipocitos Blancos/metabolismo , Adipocitos Blancos/citología , Plasticidad de la Célula/fisiología , Glándulas Mamarias Humanas/fisiología , Glándulas Mamarias Humanas/crecimiento & desarrollo , Glándulas Mamarias Humanas/citología , Adipocitos/fisiología , Adipocitos/citología