Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial-Mesenchymal Transition and Responds to Oxidative Stress.

Breast cancer cells that have undergone partial epithelial-mesenchymal transition (EMT) are believed to be more invasive than cells that have completed EMT. To study metabolic reprogramming in different mesenchymal states, we analyzed protein expression following EMT in the breast epithelial cell model D492 with single-shot LFQ supported by a SILAC proteomics approach. The D492 EMT cell model contains three cell lines: the epithelial D492 cells, the mesenchymal D492M cells, and a partial mesenchymal, tumorigenic variant of D492 that overexpresses the oncogene HER2. The analysis classified the D492 and D492M cells as basal-like and D492HER2 as claudin-low. Comparative analysis of D492 and D492M to tumorigenic D492HER2 differentiated metabolic markers of migration from those of invasion. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) was one of the top dysregulated enzymes in D492HER2. Gene expression analysis of the cancer genome atlas showed that GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA-mediated knockdown of GFPT2 influenced the EMT marker vimentin and both cell growth and invasion in vitro and was accompanied by lowered metabolic flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway that regulates H2S production and mitochondrial homeostasis. Moreover, GFPT2 was within the regulation network of insulin and EGF, and its expression was regulated by reduced glutathione (GSH) and suppressed by the oxidative stress regulator GSK3-ß. Our results demonstrate that GFPT2 controls growth and invasion in the D492 EMT model, is a marker for oxidative stress, and associated with poor prognosis in claudin-low breast cancer.

Nonantimicrobial Actions of Macrolides: Overview and Perspectives for Future Development.

Macrolides are among the most widely prescribed broad spectrum antibacterials, particularly for respiratory infections. It is now recognized that these drugs, in particular azithromycin, also exert time-dependent immunomodulatory actions that contribute to their therapeutic benefit in both infectious and other chronic inflammatory diseases. Their increased chronic use in airway inflammation and, more recently, of azithromycin in COVID-19, however, has led to a rise in bacterial resistance. An additional crucial aspect of chronic airway inflammation, such as chronic obstructive pulmonary disease, as well as other inflammatory disorders, is the loss of epithelial barrier protection against pathogens and pollutants. In recent years, azithromycin has been shown with time to enhance the barrier properties of airway epithelial cells, an action that makes an important contribution to its therapeutic efficacy. In this article, we review the background and evidence for various immunomodulatory and time-dependent actions of macrolides on inflammatory processes and on the epithelium and highlight novel nonantibacterial macrolides that are being studied for immunomodulatory and barrier-strengthening properties to circumvent the risk of bacterial resistance that occurs with macrolide antibacterials. We also briefly review the clinical effects of macrolides in respiratory and other inflammatory diseases associated with epithelial injury and propose that the beneficial epithelial effects of nonantibacterial azithromycin derivatives in chronic inflammation, even given prophylactically, are likely to gain increasing attention in the future. SIGNIFICANCE STATEMENT: Based on its immunomodulatory properties and ability to enhance the protective role of the lung epithelium against pathogens, azithromycin has proven superior to other macrolides in treating chronic respiratory inflammation. A nonantibiotic azithromycin derivative is likely to offer prophylactic benefits against inflammation and epithelial damage of differing causes while preserving the use of macrolides as antibiotics.

Peroxidasin Enhances Basal Phenotype and Inhibits Branching Morphogenesis in Breast Epithelial Progenitor Cell Line D492.

The human breast is composed of terminal duct lobular units (TDLUs) that are surrounded by stroma. In the TDLUs, basement membrane separates the stroma from the epithelial compartment, which is divided into an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells. Stem cells and progenitor cells also reside within the epithelium and drive a continuous cycle of gland remodelling that occurs throughout the reproductive period. D492 is an epithelial cell line originally isolated from the stem cell population of the breast and generates both luminal and myoepithelial cells in culture. When D492 cells are embedded into 3D reconstituted basement membrane matrix (3D-rBM) they form branching colonies mimicking the TDLUs of the breast, thereby providing a well-suited in vitro model for studies on branching morphogenesis and breast development. Peroxidasin (PXDN) is a heme-containing peroxidase that crosslinks collagen IV with the formation of sulfilimine bonds. Previous studies indicate that PXDN plays an integral role in basement membrane stabilisation by crosslinking collagen IV and as such contributes to epithelial integrity. Although PXDN has been linked to fibrosis and cancer in some organs there is limited information on its role in development, including in the breast. In this study, we demonstrate expression of PXDN in breast epithelium and stroma and apply the D492 cell line to investigate the role of PXDN in cell differentiation and branching morphogenesis in the human breast. Overexpression of PXDN induced basal phenotype in D492 cells, loss of plasticity and inhibition of epithelial-to-mesenchymal transition as is displayed by complete inhibition of branching morphogenesis in 3D culture. This is supported by results from RNA-sequencing which show significant enrichment in genes involved in epithelial differentiation along with significant negative enrichment of EMT factors. Taken together, we provide evidence for a novel role of PXDN in breast epithelial differentiation and mammary gland development.

The Wittig bioconjugation of maleimide derived, water soluble phosphonium ylides to aldehyde-tagged proteins.

Herein we disclose the transformation of maleimides into water-soluble tris(2-carboxyethyl)phosphonium ylides and their subsequent application in the bioconjugation of protein- and peptide-linked aldehydes. The new entry into Wittig bioconjugate chemistry proceeds under mild conditions and relies on highly water soluble reagents, which are likely already part of most biochemists' inventory.

ECM1 secreted by HER2-overexpressing breast cancer cells promotes formation of a vascular niche accelerating cancer cell migration and invasion.

The tumor microenvironment is increasingly recognized as key player in cancer progression. Investigating heterotypic interactions between cancer cells and their microenvironment is important for understanding how specific cell types support cancer. Forming the vasculature, endothelial cells (ECs) are a prominent cell type in the microenvironment of both normal and neoplastic breast gland. Here, we sought out to analyze epithelial-endothelial cross talk in the breast using isogenic non-tumorigenic vs. tumorigenic breast epithelial cell lines and primary ECs. The cellular model used here consists of D492, a breast epithelial cell line with stem cell properties, and two isogenic D492-derived EMT cell lines, D492M and D492HER2. D492M was generated by endothelial-induced EMT and is non-tumorigenic while D492HER2 is tumorigenic, expressing the ErbB2/HER2 oncogene. To investigate cellular cross talk, we used both conditioned medium (CM) and 2D/3D co-culture systems. Secretome analysis of D492 cell lines was performed using mass spectrometry and candidate knockdown (KD), and overexpression (OE) was done using siRNA and CRISPRi/CRISPRa technology. D492HER2 directly enhances endothelial network formation and activates a molecular axis in ECs promoting D492HER2 migration and invasion, suggesting an endothelial feedback response. Secretome analysis identified extracellular matrix protein 1 (ECM1) as potential angiogenic inducer in D492HER2. Confirming its involvement, KD of ECM1 reduced the ability of D492HER2-CM to increase endothelial network formation and induce the endothelial feedback, while recombinant ECM1 (rECM1) increased both. Interestingly, NOTCH1 and NOTCH3 expression was upregulated in ECs upon treatment with D492HER2-CM or rECM1 but not by CM from D492HER2 with ECM1 KD. Blocking endothelial NOTCH signaling inhibited the increase in network formation and the ability of ECs to promote D492HER2 migration and invasion. In summary, our data demonstrate that cancer-secreted ECM1 induces a NOTCH-mediated endothelial feedback promoting cancer progression by enhancing migration and invasion. Targeting this interaction may provide a novel possibility to improve cancer treatment.

Azithromycin ameliorates sulfur dioxide-induced airway epithelial damage and inflammatory responses.

BACKGROUND: The airway epithelium (AE) forms the first line of defence against harmful particles and pathogens. Barrier failure of the airway epithelium contributes to exacerbations of a range of lung diseases that are commonly treated with Azithromycin (AZM). In addition to its anti-bacterial function, AZM has immunomodulatory effects which are proposed to contribute to its clinical effectiveness. In vitro studies have shown the AE barrier-enhancing effects of AZM. The aim of this study was to analyze whether AE damage caused by inhalation of sulfur dioxide (SO2) in a murine model could be reduced by pre-treatment with AZM. METHODS: The leakiness of the AE barrier was evaluated after SO2 exposure by measuring levels of human serum albumin (HSA) in bronchoalveolar lavage fluid (BALF). Protein composition in BALF was also assessed and lung tissues were evaluated across treatments using histology and gene expression analysis. RESULTS: AZM pre-treatment (2 mg/kg p.o. 5 times/week for 2 weeks) resulted in reduced glutathione-S-transferases in BALF of SO2 injured mice compared to control (without AZM treatment). AZM treated mice had increased intracellular vacuolization including lamellar bodies and a reduction in epithelial shedding after injury in addition to a dampened SO2-induced inflammatory response. CONCLUSIONS: Using a mouse model of AE barrier dysfunction we provide evidence for the protective effects of AZM in vivo, possibly through stabilizing the intracellular microenvironment and reducing inflammatory responses. Our data provide insight into the mechanisms contributing to the efficacy of AZM in the treatment of airway diseases.

Application of the D492 Cell Lines to Explore Breast Morphogenesis, EMT and Cancer Progression in 3D Culture.

The human female breast gland is composed of branching epithelial ducts that extend from the nipple towards the terminal duct lobular units (TDLUs), which are the functional, milk-producing units of the gland and the site of origin of most breast cancers. The epithelium of ducts and TDLUs is composed of an inner layer of polarized luminal epithelial cells and an outer layer of contractile myoepithelial cells, separated from the vascular-rich stroma by a basement membrane. The luminal- and myoepithelial cells share an origin and in recent years, there has been increasing understanding of how these cell types interact and how they contribute to breast cancer. Accumulating evidence links stem/or progenitor cells in the mammary/breast gland to breast cancer. In that regard, much knowledge has been gained from studies in mice due to specific strains that have allowed for gene knock out/in studies and lineage tracing of cellular fates. However, there is a large histologic difference between the human female breast gland and the mouse mammary gland that necessitates that research needs to be done on human material where primary cultures are important due to their close relation to the tissue of origin. However, due to difficulties of long-term cultures and lack of access to material, human cell lines are of great importance to bridge the gap between studies on mouse mammary gland and human primary breast cells. In this review, we describe D492, a breast epithelial progenitor cell line that can generate both luminal- and myoepithelial cells in culture, and in 3D culture it forms branching ducts similar to TDLUs. We have applied D492 and its daughter cell lines to explore cellular and molecular mechanisms of branching morphogenesis and cellular plasticity including EMT and MET. In addition to discussing the application of D492 in studying normal morphogenesis, we will also discuss how this cell line has been used to study breast cancer progression.

Azithromycin induces epidermal differentiation and multivesicular bodies in airway epithelia.

BACKGROUND: Azithromycin (Azm) is a macrolide recognized for its disease-modifying effects and reduction in exacerbation of chronic airway diseases. It is not clear whether the beneficial effects of Azm are due to its anti-microbial activity or other pharmacological actions. We have shown that Azm affects the integrity of the bronchial epithelial barrier measured by increased transepithelial electrical resistance. To better understand these effects of Azm on bronchial epithelia we have investigated global changes in gene expression. METHODS: VA10 bronchial epithelial cells were treated with Azm and cultivated in air-liquid interface conditions for up to 22 days. RNA was isolated at days 4, 10 and 22 and analyzed using high-throughput RNA sequencing. qPCR and immunostaining were used to confirm key findings from bioinformatic analyses. Detailed assessment of cellular changes was done using microscopy, followed by characterization of the lipidomic profiles of the multivesicular bodies present. RESULTS: Bioinformatic analysis revealed that after 10 days of treatment genes encoding effectors of sterol and cholesterol metabolism were prominent. Interestingly, expression of genes associated with epidermal barrier differentiation, KRT1, CRNN, SPINK5 and DSG1, increased significantly at day 22. Together with immunostaining, these results suggest an epidermal differentiation pattern. We also found that Azm induced the formation of multivesicular and lamellar bodies in two different airway epithelial cell lines. Lipidomic analysis revealed that Azm was entrapped in multivesicular bodies linked to different types of lipids, most notably palmitate and stearate. Furthermore, targeted analysis of lipid species showed accumulation of phosphatidylcholines, as well as ceramide derivatives. CONCLUSIONS: Taken together, we demonstrate how Azm might confer its barrier enhancing effects, via activation of epidermal characteristics and changes to intracellular lipid dynamics. These effects of Azm could explain the unexpected clinical benefit observed during Azm-treatment of patients with various lung diseases affecting barrier function.

EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT.

Epithelial to mesenchymal transition (EMT) is an important event during development and cancer metastasis. There is limited understanding of the metabolic alterations that give rise to and take place during EMT. Dysregulation of signalling pathways that impact metabolism, including epidermal growth factor receptor (EGFR), are however a hallmark of EMT and metastasis. In this study, we report the investigation into EGFR signalling and metabolic crosstalk of EMT through constraint-based modelling and analysis of the breast epithelial EMT cell model D492 and its mesenchymal counterpart D492M. We built an EGFR signalling network for EMT based on stoichiometric coefficients and constrained the network with gene expression data to build epithelial (EGFR_E) and mesenchymal (EGFR_M) networks. Metabolic alterations arising from differential expression of EGFR genes was derived from a literature review of AKT regulated metabolic genes. Signaling flux differences between EGFR_E and EGFR_M models subsequently allowed metabolism in D492 and D492M cells to be assessed. Higher flux within AKT pathway in the D492 cells compared to D492M suggested higher glycolytic activity in D492 that we confirmed experimentally through measurements of glucose uptake and lactate secretion rates. The signaling genes from the AKT, RAS/MAPK and CaM pathways were predicted to revert D492M to D492 phenotype. Follow-up analysis of EGFR signaling metabolic crosstalk in three additional breast epithelial cell lines highlighted variability in in vitro cell models of EMT. This study shows that the metabolic phenotype may be predicted by in silico analyses of gene expression data of EGFR signaling genes, but this phenomenon is cell-specific and does not follow a simple trend.

Epithelial Plasticity During Human Breast Morphogenesis and Cancer Progression.

Understanding the complex events leading to formation of an epithelial-based organ such as the breast requires a detailed insight into the crosstalk between epithelial and stromal compartments. These interactions occur both through heterotypic cellular interactions and between cells and matrix components. While in vivo models may partially capture these complex interactions, there is a need for in- vitro models to study these events. In this review we discuss cell-cell interactions in breast development focusing on the stem cell niche and branching morphogenesis. Given the recent understanding that the basic developmental events underlying branching morphogenesis are closely related to pathways important to cancer progression, i.e. epithelial plasticity and epithelial to mesenchymal transition (EMT), we will also discuss aspects relevant to cancer progression. In cancer, the adoption of mesenchymal phenotype by the malignant cells allows stromal invasion and subsequent intravasation to blood- or lymphatic vessels, a route that is a prerequisite for metastasis. A number of publications have demonstrated that tumor initiating cells, sometimes referred to as cancer stem cells adapt an EMT phenotype that renders them more resistant to apoptosis and drug therapy. The mechanism behind this phenomenon is currently unknown but this may partially explain relapse in breast cancer patients. Increased understanding of branching morphogenesis in the breast gland and the regulation of EMT and its reverse process mesenchymal to epithelial transition (MET) may hold the keys for future development of methods/drugs that neutralize the invading properties of cancer cells.

MicroRNA-200c-141 and ∆Np63 are required for breast epithelial differentiation and branching morphogenesis.

The epithelial compartment of the breast contains two lineages, the luminal- and the myoepithelial cells. D492 is a breast epithelial cell line with stem cell properties that forms branching epithelial structures in 3D culture with both luminal- and myoepithelial differentiation. We have recently shown that D492 undergo epithelial to mesenchymal transition (EMT) when co-cultured with endothelial cells. This 3D co-culture model allows critical analysis of breast epithelial lineage development and EMT. In this study, we compared the microRNA (miR) expression profiles for D492 and its mesenchymal-derivative D492M. Suppression of the miR-200 family in D492M was among the most profound changes observed. Exogenous expression of miR-200c-141 in D492M reversed the EMT phenotype resulting in gain of luminal but not myoepithelial differentiation. In contrast, forced expression of ∆Np63 in D492M restored the myoepithelial phenotype only. Co-expression of miR-200c-141 and ∆Np63 in D492M restored the branching morphogenesis in 3D culture underlining the requirement for both luminal and myoepithelial elements for obtaining full branching morphogenesis in breast epithelium. Introduction of a miR-200c-141 construct in both D492 and D492M resulted in resistance to endothelial induced EMT. In conclusion, our data suggests that expression of miR-200c-141 and ∆Np63 in D492M can reverse EMT resulting in luminal- and myoepithelial differentiation, respectively, demonstrating the importance of these molecules in epithelial integrity in the human breast.

Basal cells of the human airways acquire mesenchymal traits in idiopathic pulmonary fibrosis and in culture.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with high morbidity and mortality. The cellular source of the fibrotic process is currently under debate with one suggested mechanism being epithelial-to-mesenchymal transition (EMT) in the alveolar region. In this study, we show that airway epithelium overlying fibroblastic foci in IPF contains a layer of p63-positive basal cells while lacking ciliated and goblet cells. This basal epithelium shows increased expression of CK14, Vimentin and N-cadherin while retaining E-cadherin. The underlying fibroblastic foci shows both E- and N-cadherin-positive cells. To determine if p63-positive basal cells were able to undergo EMT in culture, we treated VA10, a p63-positive basal cell line, with the serum replacement UltroserG. A sub-population of treated cells acquired a mesenchymal phenotype, including an E- to N-cadherin switch. After isolation, these cells portrayed a phenotype presenting major hallmarks of EMT (loss of epithelial markers, gain of mesenchymal markers, increased migration and anchorage-independent growth). This phenotypic switch was prevented in p63 knockdown (KD) cells. In conclusion, we show that airway epithelium overlying fibroblastic foci in IPF lacks its characteristic functional identity, shows increased reactivity of basal cells and acquisition of a partial EMT phenotype. This study suggests that some p63-positive basal cells are prone to phenotypic changes and could act as EMT progenitors in IPF.

The influence of physical and spatial substrate characteristics on endothelial cells.

Cardiovascular diseases are a main cause of death worldwide, leading to a growing demand for medical devices to treat this patient group. Central to the engineering of such devices is a good understanding of the biology and physics of cell-surface interactions. In existing blood-contacting devices, such as vascular grafts, the interaction between blood, cells, and material is one of the main limiting factors for their long-term durability. An improved understanding of the material's chemical- and physical properties as well as its structure all play a role in how endothelial cells interact with the material surface. This review provides an overview of how different surface structures influence endothelial cell responses and what is currently known about the underlying mechanisms that guide this behavior. The structures reviewed include decellularized matrices, electrospun fibers, pillars, pits, and grated surfaces.

Endothelial-rich microenvironment supports growth and branching morphogenesis of prostate epithelial cells.

BACKGROUND: Development of epithelial organs depends on interaction between the epithelium and the underlying mesenchyme including the vasculature. The aim of this study was to explore the morphogenic effect of endothelial cells on prostate epithelial cell lines in 3D culture and to establish an in vitro model for prostate branching morphogenesis. METHODS: A panel of eleven cell lines originating in normal or malignant prostate and primary prostate epithelial cells were cultured in reconstituted basement membrane (rBM) matrix with or without non-proliferating but metabolically active endothelial cells. Morphogenesis was evaluated by phase contrast microscopy and further characterized by immunocyto/histocemistry and confocal microscopy. RESULTS: Endothelial cells induced clonogenic potential of most prostate cell lines and formation of branching and mesenchymal-like colonies. One of the normal-derived cell lines in the panel (PZ-HPV-7) displayed unique properties in rBM culture by forming large and complex branching structures resembling the ductal architecture of the prostate. This ability was highly dependent on epithelial seeding density and soluble factors derived from the endothelial cells. High seeding density suppressed branching of PZ-HPV-7 but survival was compromised at low density in the absence of endothelium. CONCLUSIONS: We have generated an endothelial-based clonogenic assay to study prostate epithelial morphogenesis in three-dimensional context. This assay will be important tool to study prostate epithelial-endothelial interactions in 3D context and open up possibilities to study molecular regulation of prostate morphogenesis and cancer progression.

Drug delivery characteristics of the progenitor bronchial epithelial cell line VA10.

PURPOSE: To determine the integrity and permeability properties of the immortalized human VA10 bronchial epithelial cell line for its suitability as an in vitro drug permeation model. METHODS: Cells were grown under liquid-covered culture (LCC) or air-liquid interface (ALI) culture, characterized using electron microscopy and immunostaining. Integrity was measured using transepithelial electrical resistance (TER) and permeability of fluorescein sodium (Flu-Na). General permeability was established with dextrans and model drugs and P-glycoprotein (P-gp) function determined with bidirectional flux of rhodamine-123. RESULTS: ALI culture resulted in 2-3 cell layers with differentiation towards ciliated cells but LCC showed undifferentiated morphology. VA10 cells formed TJ, with higher TER in LCC than ALI (∼2500 vs. ∼1200 Ω*cm(2)) and Flu-Na permeability ∼1-2 × 10(-7) cm/s. ALI cultured cells expressed P-gp and distinguished between compounds depending on lipophilicity and size, consistent with previous data from Calu-3 and 16HBE14o-cell lines. CONCLUSIONS: ALI cultured cell layers capture the in vivo-like phenotype of bronchial epithelium and form functional cell barrier capable of discriminating between compounds depending on physiochemical properties. The VA10 cell line is an important alternative to previously published cell lines and a relevant model to study airway drug delivery in vitro.

Prospects for macrolide therapy of asthma and COPD.

Macrolide compounds, many of which are derived from natural sources, all share a lactone ring structure, but of varying sizes. Their biological activities differ with structure and size but tend to overlap. Marketed macrolide drugs include immunosuppressives and antibiotics. Some of the latter have been shown to exert anti-inflammatory activities, due to direct effects on inflammatory cells and processes when used for respiratory infections. Consequently, azithromycin is included in clinical guidelines for COPD and asthma treatment, though it has the disadvantage, as an antibiotic, of increasing bacterial resistance. COPD and asthma, however, like several chronic inflammatory diseases involving other organs, are driven to a large extent by epithelial barrier dysfunction. Recently, azithromycin was shown to directly enhance epithelial barrier function and a new class of derivatives, barriolides, is under development with the lead indication COPD. It is thus likely that by circumventing antibiosis and acting on a crucial etiological disease process, this type of agent will open up a new, safer approach to COPD and asthma therapy with macrolides.

Identification of a core EMT signature that separates basal-like breast cancers into partial- and post-EMT subtypes.

Epithelial-mesenchymal transition (EMT) is a cellular plasticity program critical for embryonic development and tissue regeneration, and aberrant EMT is associated with disease including cancer. The high degree of plasticity in the mammary epithelium is reflected in extensive heterogeneity among breast cancers. Here, we have analyzed RNA-sequencing data from three different mammary epithelial cell line-derived EMT models and identified a robust mammary EMT gene expression signature that separates breast cancers into distinct subgroups. Most strikingly, the basal-like breast cancers form two subgroups displaying partial-EMT and post-EMT gene expression patterns. We present evidence that key EMT-associated transcription factors play distinct roles at different stages of EMT in mammary epithelial cells.

Evidence for a stem cell hierarchy in the adult human breast.

Cellular pathways that contribute to adult human mammary gland architecture and lineages have not been previously described. In this study, we identify a candidate stem cell niche in ducts and zones containing progenitor cells in lobules. Putative stem cells residing in ducts were essentially quiescent, whereas the progenitor cells in the lobules were more likely to be actively dividing. Cells from ducts and lobules collected under the microscope were functionally characterized by colony formation on tissue culture plastic, mammosphere formation in suspension culture, and morphogenesis in laminin-rich extracellular matrix gels. Staining for the lineage markers keratins K14 and K19 further revealed multipotent cells in the stem cell zone and three lineage-restricted cell types outside this zone. Multiparameter cell sorting and functional characterization with reference to anatomical sites in situ confirmed this pattern. The proposal that the four cell types are indeed constituents of an as of yet undescribed stem cell hierarchy was assessed in long-term cultures in which senescence was bypassed. These findings identify an adult human breast ductal stem cell activity and its earliest descendants.