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Development (Cambridge, England) Oct 2020The Golgi-associated RAB GTPases, RAB6A and RAB6A', regulate anterograde and retrograde transport pathways from and to the Golgi. , RAB6A/A' control several cellular...
The Golgi-associated RAB GTPases, RAB6A and RAB6A', regulate anterograde and retrograde transport pathways from and to the Golgi. , RAB6A/A' control several cellular functions including cell division, migration, adhesion and polarity. However, their role remains poorly described Here, we generated BlgCre; mice presenting a specific deletion of in the mammary luminal secretory lineage during gestation and lactation. loss severely impaired the differentiation, maturation and maintenance of the secretory tissue, compromising lactation. The mutant epithelium displayed a decreased activation of STAT5, a key regulator of the lactogenic process primarily governed by prolactin. Data obtained with a mammary epithelial cell line suggested that defective STAT5 activation might originate from a perturbed transport of the prolactin receptor, altering its membrane expression and signaling cascade. Despite the major functional defects observed upon deletion, the polarized organization of the mammary epithelial bilayer was preserved. Altogether, our data reveal a crucial role for RAB6A/A' in the lactogenic function of the mammary gland and suggest that the trafficking pathways controlled by RAB6A/A' depend on cell-type specialization and tissue context.
Topics: Animals; Blotting, Western; Cell Line; Female; Flow Cytometry; Humans; In Situ Nick-End Labeling; Mammary Glands, Human; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; STAT5 Transcription Factor; rab GTP-Binding Proteins
PubMed: 32895290
DOI: 10.1242/dev.190744 -
Developmental Cell Jan 2003Branching morphogenesis involves the restructuring of epithelial tissues into complex and organized ramified tubular networks. Early rounds of branching are controlled... (Review)
Review
Branching morphogenesis involves the restructuring of epithelial tissues into complex and organized ramified tubular networks. Early rounds of branching are controlled genetically in a hardwired fashion in many organs, whereas later, branching is stochastic, responding to environmental cues. We discuss this sequential process from formation of an organ anlage and invagination of the epithelium to branch initiation and outgrowth in several model systems including Drosophila trachea and mammalian lung, mammary gland, and kidney.
Topics: Animals; Breast; Cell Differentiation; Cell Movement; Epithelium; Humans; Kidney; Lung; Morphogenesis; Organ Specificity; Trachea
PubMed: 12530959
DOI: 10.1016/s1534-5807(02)00410-0 -
Biochimica Et Biophysica Acta. General... Jun 2021The epithelium forms a protective barrier against external biological, chemical and physical insults. So far, AFM-based, micro-mechanical measurements have only been...
BACKGROUND
The epithelium forms a protective barrier against external biological, chemical and physical insults. So far, AFM-based, micro-mechanical measurements have only been performed on single cells and confluent cells, but not yet on cells in mature layers.
METHODS
Using a combination of atomic force, fluorescence and confocal microscopy, we determined the changes in stiffness, morphology and actin distribution of human mammary epithelial cells (HMECs) as they transition from single cells to confluency to a mature layer.
RESULTS
Single HMECs have a tall, round (planoconvex) morphology, have actin stress fibers at the base, have diffuse cortical actin, and have a stiffness of 1 kPa. Confluent HMECs start to become flatter, basal actin stress fibers start to disappear, and actin accumulates laterally where cells abut. Overall stiffness is still 1 kPa with two-fold higher stiffness in the abutting regions. As HMECs mature and form multilayered structures, cells on apical surfaces become flatter (apically more level), wider, and seven times stiffer (mean, 7 kPa) than single and confluent cells. The main drivers of these changes are actin filaments, as cells show strong actin accumulation in the regions where cells adjoin, and in the apical regions.
CONCLUSIONS
HMECs stiffen, flatten and redistribute actin upon transiting from single cells to mature, confluent layers.
GENERAL SIGNIFICANCE
Our findings advance the understanding of breast ductal morphogenesis and mechanical homeostasis.
Topics: Actin Cytoskeleton; Cells, Cultured; Epithelial Cells; Female; Humans; Mammary Glands, Human; Microscopy, Atomic Force; Organogenesis
PubMed: 33689830
DOI: 10.1016/j.bbagen.2021.129891 -
Developmental Cell Oct 2020The crosstalk between tumor cells and the adjacent normal epithelium contributes to cancer progression, but its regulators have remained elusive. Here, we show that...
The crosstalk between tumor cells and the adjacent normal epithelium contributes to cancer progression, but its regulators have remained elusive. Here, we show that breast cancer cells maintained in hypoxia release small extracellular vesicles (sEVs) that activate mitochondrial dynamics, stimulate mitochondrial movements, and promote organelle accumulation at the cortical cytoskeleton in normal mammary epithelial cells. This results in AKT serine/threonine kinase (Akt) activation, membrane focal adhesion turnover, and increased epithelial cell migration. RNA sequencing profiling identified integrin-linked kinase (ILK) as the most upregulated pathway in sEV-treated epithelial cells, and genetic or pharmacologic targeting of ILK reversed mitochondrial reprogramming and suppressed sEV-induced cell movements. In a three-dimensional (3D) model of mammary gland morphogenesis, sEV treatment induced hallmarks of malignant transformation, with deregulated cell death and/or cell proliferation, loss of apical-basal polarity, and appearance of epithelial-to-mesenchymal transition (EMT) markers. Therefore, sEVs released by hypoxic breast cancer cells reprogram mitochondrial dynamics and induce oncogenic changes in a normal mammary epithelium.
Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Mammary Glands, Human; Mitochondrial Dynamics; Protein Serine-Threonine Kinases; Tumor Microenvironment
PubMed: 32780991
DOI: 10.1016/j.devcel.2020.07.014 -
Journal of Mammary Gland Biology and... Jun 2012Mass spectrometry (MS)-based label-free proteomics offers an unbiased approach to screen biomarkers related to disease progression and therapy-resistance of breast... (Review)
Review
Mass spectrometry (MS)-based label-free proteomics offers an unbiased approach to screen biomarkers related to disease progression and therapy-resistance of breast cancer on the global scale. However, multi-step sample preparation can introduce large variation in generated data, while inappropriate statistical methods will lead to false positive hits. All these issues have hampered the identification of reliable protein markers. A workflow, which integrates reproducible and robust sample preparation and data handling methods, is highly desirable in clinical proteomics investigations. Here we describe a label-free tissue proteomics pipeline, which encompasses laser capture microdissection (LCM) followed by nanoscale liquid chromatography and high resolution MS. This pipeline routinely identifies on average ∼10,000 peptides corresponding to ∼1,800 proteins from sub-microgram amounts of protein extracted from ∼4,000 LCM breast cancer epithelial cells. Highly reproducible abundance data were generated from different technical and biological replicates. As a proof-of-principle, comparative proteome analysis was performed on estrogen receptor α positive or negative (ER+/-) samples, and commonly known differentially expressed proteins related to ER expression in breast cancer were identified. Therefore, we show that our tissue proteomics pipeline is robust and applicable for the identification of breast cancer specific protein markers.
Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Separation; Down-Regulation; Epithelium; Female; Gene Expression Profiling; Humans; Laser Capture Microdissection; Mammary Glands, Human; Neoplasm Proteins; Peptide Mapping; Peptides; Proteomics; Receptors, Estrogen; Receptors, Progesterone; Tumor Cells, Cultured; Up-Regulation
PubMed: 22644111
DOI: 10.1007/s10911-012-9252-6 -
PLoS Biology Feb 2004Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130) (pRb(f), collectively), play a pivotal role in... (Review)
Review
Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130) (pRb(f), collectively), play a pivotal role in regulating eukaryotic cell cycle progression, apoptosis, and terminal differentiation. While aberrations in the pRb-signaling pathway are common in human cancers, the consequence of pRb(f) loss in the mammary gland has not been directly assayed in vivo. We reported previously that inactivating these critical cell cycle regulators in divergent cell types, either brain epithelium or astrocytes, abrogates the cell cycle restriction point, leading to increased cell proliferation and apoptosis, and predisposing to cancer. Here we report that mouse mammary epithelium is similar in its requirements for pRb(f) function; Rb(f) inactivation by T(121), a fragment of SV40 T antigen that binds to and inactivates pRb(f) proteins, increases proliferation and apoptosis. Mammary adenocarcinomas form within 16 mo. Most apoptosis is regulated by p53, which has no impact on proliferation, and heterozygosity for a p53 null allele significantly shortens tumor latency. Most tumors in p53 heterozygous mice undergo loss of the wild-type p53 allele. We show that the mechanism of p53 loss of heterozygosity is not simply the consequence of Chromosome 11 aneuploidy and further that chromosomal instability subsequent to p53 loss is minimal. The mechanisms for pRb and p53 tumor suppression in the epithelia of two distinct tissues, mammary gland and brain, are indistinguishable. Further, this study has produced a highly penetrant breast cancer model based on aberrations commonly observed in the human disease.
Topics: Brain Neoplasms; Breast; Breast Neoplasms; Cell Differentiation; Cell Division; Disease Progression; Epithelial Cells; Female; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes, Retinoblastoma; Humans
PubMed: 14966529
DOI: 10.1371/journal.pbio.0020022 -
Journal of Molecular Endocrinology Sep 2021Epidemiological studies inversely associate BMI with breast cancer risk in premenopausal women, but the pathophysiological linkage remains ill-defined. Despite the...
Epidemiological studies inversely associate BMI with breast cancer risk in premenopausal women, but the pathophysiological linkage remains ill-defined. Despite the documented relevance of the 'local' environment to breast cancer progression and the well-accepted differences in transcriptome and metabolic properties of anatomically distinct fat depots, specific breast adipose contributions to the proliferative potential of non-diseased breast glandular compartment are not fully understood. To address early breast cancer causation in the context of obesity status, we compared the cellular and molecular phenotypes of breast adipose and matched breast glandular tissue from premenopausal non-obese (mean BMI = 27 kg/m2) and obese (mean BMI = 44 kg/m2) women. Breast adipose from obese women showed higher expression levels of adipogenic, pro-inflammatory, and estrogen synthetic genes than from non-obese women. Obese breast glandular tissue displayed lower proliferation and inflammatory status and higher expression of anti-proliferative/pro-senescence biomarkers TP53 and p21 than from non-obese women. Transcript levels for T-cell receptor and co-receptors CD3 and CD4 were higher in breast adipose of obese cohorts, coincident with elevated adipose interleukin 10 (IL10) and FOXP3 gene expression. In human breast epithelial cell lines MCF10A and HMEC, recombinant human IL10 reduced cell viability and CCND1 transcript levels, increased those of TP53 and p21, and promoted (MCF10A) apoptosis. Our findings suggest that breast adipose-associated IL10 may mediate paracrine interactions between non-diseased breast adipose and breast glandular compartments and highlight how breast adipose may program the local inflammatory milieu, partly by recruiting FOXP3+ T regulatory cells, to influence premenopausal breast cancer risk.
Topics: Adipocytes; Adipose Tissue; Adiposity; Adult; Biomarkers; Breast; Breast Neoplasms; Cytokines; Epithelium; Female; Gene Expression; Gonadal Steroid Hormones; Humans; Immunohistochemistry; Inflammation Mediators; Interleukin-10; Middle Aged; Models, Biological; Obesity; Phenotype; Premenopause; T-Lymphocyte Subsets; Telomere; Young Adult
PubMed: 34382943
DOI: 10.1530/JME-21-0100 -
Cell Death and Differentiation Mar 2012Signalling through the janus kinase (JAK)/signal transducer and activator of transcription (Stat) pathway is required at different stages of mammary gland development,...
Signalling through the janus kinase (JAK)/signal transducer and activator of transcription (Stat) pathway is required at different stages of mammary gland development, and this pathway is frequently hyper-activated in cancer, including tumours of the breast. Stats 3, 5 and 6 have important roles in the differentiation and survival of mammary alveolar cells, but somewhat paradoxically, both Stat3 and 5 can have oncogenic activity in the mammary gland. Constitutive activation of JAK2 could be anticipated to result in hyper-activation of Stats 1, 3, 5 and 6 with concomitant cell transformation, although the outcome is difficult to envisage, particularly since Stats 3 and 5 play opposing roles in normal mammary gland development. Here, we show that expression of a constitutively active JAK2 mutant, JAK2 V617F, leads to hyper-activation of Stat5 in mammary epithelial cells (MECs), and transgenic mice expressing JAK2 V617F specifically in the mammary gland exhibit accelerated alveologenesis during pregnancy and delayed post-lactational regression. Overexpressing JAK2 V617F in MECs in vitro results in elevated proliferation and resistance to cell death. Furthermore, constitutively active JAK2 enhances anchorage-independent cell growth in the presence of a co-operating oncogene and accelerates tumourigenesis in a xenograft model. Taken together, our results provide insights into signalling downstream of constitutively active JAK2 and could be important for understanding the molecular mechanisms of breast tumourigenesis.
Topics: Animals; Breast Neoplasms; Cell Death; Cell Line, Tumor; Cell Transformation, Neoplastic; Enzyme Activation; Female; Humans; Janus Kinase 2; Lactation; Male; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Animal; Mice; Mice, Knockout; Mice, Nude; Mutation, Missense; Neoplasm Transplantation; Pregnancy; STAT5 Transcription Factor; Signal Transduction; Transplantation, Heterologous
PubMed: 21941370
DOI: 10.1038/cdd.2011.122 -
Cancer Genomics & Proteomics 2022Until now, little emphasis has been placed on the protein expression profile of male breast cancer (MBC) tumors, due to the rarity of the disease. The present study...
BACKGROUND/AIM
Until now, little emphasis has been placed on the protein expression profile of male breast cancer (MBC) tumors, due to the rarity of the disease. The present study aimed to identify a proteomic pattern that is characteristic for malignant male breast tissue epithelium.
MATERIALS AND METHODS
The protein content of four male breast tumors and corresponding adjacent healthy (control) tissues was analyzed by high-throughput nano-liquid chromatography-MS/MS technology.
RESULTS
A total of 2,352 proteins were identified, that correspond to 1,249 single gene products, with diverse biological roles. Of those, a panel of 119 differentially expressed tissue proteins was identified in MBC samples compared to controls; 90 were found to be over-expressed in MBC tissues, while 29 were down-regulated. Concurrently, 844 proteins were detected only in MBC tumors and 197 were expressed exclusively in control mammary samples.
CONCLUSION
Differential proteomic expression was found in MBC tissue, leading to improved understanding of MBC pathology and highlighting the need for personalized management of male patients.
Topics: Biomarkers, Tumor; Breast; Breast Neoplasms; Breast Neoplasms, Male; Chromatography, Liquid; Humans; Male; Proteomics; Tandem Mass Spectrometry
PubMed: 35181590
DOI: 10.21873/cgp.20316 -
PeerJ 2023The epithelial-mesenchymal transition (EMT) is a multi-step morphogenetic process in which epithelial cells lose their epithelial properties and gain mesenchymal...
BACKGROUND
The epithelial-mesenchymal transition (EMT) is a multi-step morphogenetic process in which epithelial cells lose their epithelial properties and gain mesenchymal characteristics. The process of EMT has been shown to mediate mammary gland fibrosis. Understanding how mesenchymal cells emerge from an epithelial default state will aid in unravelling the mechanisms that control fibrosis and, ultimately, in identifying therapeutic targets to alleviate fibrosis.
METHODS
The effects of EGF and high glucose (HG) on EMT in mammary epithelial cells, MCF10A and GMECs, as well as their pathogenic role, were studied. analysis was used to find interacting partners and protein-chemical/drug molecule interactions.
RESULTS
On treatment with EGF and/or HG, qPCR analysis showed a significant increase in the gene expression of EMT markers and downstream signalling genes. The expression of these genes was reduced on treatment with EGF+HG combination in both cell lines. The protein expression of COL1A1 increased as compared to the control in cells treated with EGF or HG alone, but when the cells were treated with EGF and HG together, the protein expression of COL1A1 decreased. ROS levels and cell death increased in cells treated with EGF and HG alone, whereas cells treated with EGF and HG together showed a decrease in ROS production and apoptosis. analysis of protein-protein interactions suggest the possible role of MAPK1, actin alpha 2 (ACTA2), COL1A1, and NFB1 in regulating TGF1, ubiquitin C (UBC), specificity protein 1 (SP1) and E1A binding protein P300 (EP300). Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment suggests advanced glycation end products-receptor for advanced glycation end products (AGE-RAGE) signalling pathway, relaxin signalling pathway and extra cellular matrix (ECM) receptor interactions underlying fibrosis mechanism.
CONCLUSION
This study demonstrates that EGF and HG induce EMT in mammary epithelial cells and may also have a role in fibrosis.
Topics: Humans; Epithelial-Mesenchymal Transition; Epidermal Growth Factor; Mammary Glands, Human; Reactive Oxygen Species; Fibrosis
PubMed: 37187521
DOI: 10.7717/peerj.15207