Mesenchymal Stem Cells Overexpressing ACE2 Favorably Ameliorate LPS-Induced Inflammatory Injury in Mammary Epithelial Cells

Mesenchymal stem cells (MSCs) are capable of homing injury sites to exert anti-inflammatory as well as anti-damage effects and can be used as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2) plays an important role in numerous inflammatory diseases, but fewer studies have been reported in animal mastitis. We hypothesized that MSCs overexpressing ACE2 is more effective in ameliorating lipopolysaccharide (LPS)-induced inflammatory injury in mammary epithelial cells compared to MSCs alone. The results showed that MSC-ACE2 inhibited the LPS induction by upregulation of TNF-α, IL-Iβ, IL-6, and iNOS mRNA expression levels in EpH4-Ev cells compared with MSCs. Furthermore, results showed that both MSC and MSC-ACE2 were significantly activated IL-10/STAT3/SOCS3 signaling pathway as well as inhibited TLR4/NF-κB and MAPK signaling pathways, but MSC-ACE2 had more significant effects. Meanwhile, MSC-ACE2 promoted the expression of proliferation-associated proteins and inhibited the expression of the apoptosis-associated proteins in EpH4-Ev cells. In addition, MSC and MSC-ACE2 reversed the LPS-induced downregulation expression levels of the tight junction proteins in mammary epithelial cells, indicating that both MSC as well as MSC-ACE2 could promote blood-milk barrier repair, and MSC-ACE2 was more effective. These results suggested that MSCs overexpressing ACE2 were more anti-inflammatory as well as anti-injurious action into LPS-induced inflammatory injury in the EpH4-Ev cells. Thus, MSCs overexpressing ACE2 is expected to serve as a potential strategy for mastitis treatment.

Augmentation of extracellular ATP synergizes with chemotherapy in triple negative breast cancer

Introduction: Breast cancer affects two million women worldwide every year and is the most common cause of cancer-related death among women. The triple-negative breast cancer (TNBC) sub-type is associated with an especially poor prognosis because currently available therapies, fail to induce long-lasting responses. Therefore, there is an urgent need to develop novel therapies that result in durable responses. One universal characteristic of the tumor microenvironment is a markedly elevated concentration of extracellular adenosine triphosphate (eATP). Chemotherapy exposure results in further increases in eATP through its release into the extracellular space of cancer cells via P2RX channels. eATP levels are reduced by eATPases. Given that high concentrations of eATP are cytotoxic, we hypothesized that augmenting the release of eATP through P2RX channels and inhibiting extracellular ATPases would sensitize TNBC cells to chemotherapy. Methods: TNBC cell lines MDA-MB 231, Hs 578t and MDA-MB 468 and non-tumorigenic immortalized mammary epithelial MCF-10A cells were treated with increasing concentrations the chemotherapeutic agent paclitaxel in the presence of eATPase inhibitors, specific agonists or antagonists of P2RXs with cell viability and eATP content being measured. Additionally, the mRNA, protein and cell surface expressions of the purinergic receptors P2RX4 and P2RX7 were evaluated in all examined cell lines via qRT-PCR, western blot, and flow cytometry analyses, respectively. Results: In the present study, we observed dose-dependent declines in cell viability and increases in eATP in paclitaxel-treated TNBC cell lines in the presence of inhibitors of eATPases. These effects were reversed by specific antagonists of P2RXs. Similar results were observed with P2RX activators. All examined cell lines expressed both P2RX4 and P2RX7 at the mRNA, protein and cell surface levels. Conclusion: These results reveal that eATP modulates the chemotherapeutic response in TNBC cell lines which could be exploited to enhance the efficacy of chemotherapy regimens for TNBC.

Deciphering the Role of 3D Genome Organization in Breast Cancer Susceptibility

Cancer risk by environmental exposure is modulated by an individual’s genetics and age at exposure. This age-specific period of susceptibility is referred to as the “Window of Susceptibility” (WOS). Rats have a similar WOS for developing breast cancer. A previous study in rat identified an age-specific long-range regulatory interaction for the cancer gene, Pappa, that is associated with breast cancer susceptibility. However, the global role of three-dimensional genome organization and downstream gene expression programs in the WOS is not known. Therefore, we generated Hi-C and RNA-seq data in rat mammary epithelial cells within and outside the WOS. To systematically identify higher-order changes in 3D genome organization, we developed NE-MVNMF that combines network enhancement followed by multitask non-negative matrix factorization. We examined three-dimensional genome organization dynamics at the level of individual loops as well as higher-order domains. Differential chromatin interactions tend to be associated with differentially up-regulated genes with the WOS and recapitulate several human SNP-gene interactions associated with breast cancer susceptibility. Our approach identified genomic blocks of regions with greater overall differences in contact count between the two time points when the cluster assignments change and identified genes and pathways implicated in early carcinogenesis and cancer treatment. Our results suggest that WOS-specific changes in 3D genome organization are linked to transcriptional changes that may influence susceptibility to breast cancer.

Natural and Synthetic Estrogens in Chronic Inflammation and Breast Cancer

The oncogenic role of estrogen receptor (ER) signaling in breast cancer has long been established. Interaction of estrogen with estrogen receptor (ER) in the nucleus activates genomic pathways of estrogen signaling. In contrast, estrogen interaction with the cell membrane-bound G-protein-coupled estrogen receptor (GPER) activates the rapid receptor-mediated signaling transduction cascades. Aberrant estrogen signaling enhances mammary epithelial cell proliferation, survival, and angiogenesis, hence is an important step towards breast cancer initiation and progression. Meanwhile, a growing number of studies also provide evidence for estrogen’s pro- or anti-inflammatory roles. As other articles in this issue cover classic ER and GPER signaling mediated by estrogen, this review will discuss the crucial mechanisms by which estrogen signaling influences chronic inflammation and how that is involved in breast cancer. Xenoestrogens acquired from plant diet or exposure to industrial products constantly interact with and alter innate estrogen signaling at various levels. As such, they can modulate chronic inflammation and breast cancer development. Natural xenoestrogens generally have anti-inflammatory properties, which is consistent with their chemoprotective role in breast cancer. In contrast, synthetic xenoestrogens are proinflammatory and carcinogenic compounds that can increase the risk of breast cancer. This article also highlights important xenoestrogens with a particular focus on their role in inflammation and breast cancer. Improved understanding of the complex relationship between estrogens, inflammation, and breast cancer will guide clinical research on agents that could advance breast cancer prevention and therapy.

PTHrP Induces STAT5 Activation, Secretory Differentiation and Mammary Tumor Progression

Background: Parathyroid hormone-related protein (PTHrP) is required for embryonic breast development and has important functions during lactation, when it is produced by alveolar epithelial cells and secreted into the maternal circulation to mobilize skeletal calcium used for milk production. PTHrP is also produced by breast cancers and GWAS studies suggest that it influences breast cancer risk. However, the exact functions of PTHrP in breast cancer biology remain unsettled. Methods: We developed a tetracyline-regulated, MMTV (mouse mammary tumor virus)-driven model of PTHrP overexpression in mammary epithelial cells (Tet-PTHrP mice) and bred these mice with the MMTV-PyMT (polyoma middle tumor-antigen) breast cancer model to analyze the impact of PTHrP overexpression on normal mammary gland biology and in breast cancer progression. Results: Overexpression of PTHrP in luminal epithelial cells caused alveolar hyperplasia and secretory differentiation of the mammary epithelium with milk production. This was accompanied by activation of Stat5 and increased expression of E74-like factor-5 (Elf5). In MMTV-PyMT mice, overexpression of PTHrP (Tet-PTHrP;PyMT mice) shortened tumor latency and accelerated tumor growth, ultimately reducing overall survival. Tumors overproducing PTHrP also displayed increased expression of nuclear pSTAT5 and Elf5, increased expression of markers of secretory differentiation and milk constituents, and histologically resembled secretory carcinomas of the breast. Overexpression of PTHrP within cells isolated from tumors, but not PTHrP exogenously added to cell culture media, led to activation of STAT5 and milk protein gene expression. In addition, neither ablating the Type 1 PTH/PTHrP receptor (PTH1R) in epithelial cells or treating Tet-PTHrP;PyMT mice with an anti-PTH1R antibody prevented secretory differentiation or altered tumor latency. These data suggest that PTHrP acts in a cell-autonomous, intracrine manner. Finally, expression of PTHrP in human breast cancers is associated with expression of genes involved in milk production and STAT5 signaling. Conclusions: Our study suggests that PTHrP promotes pathways leading to secretory differentiation and proliferation in both normal mammary epithelial cells and in breast tumor cells.

Cis-9, Trans-11 CLA Alleviates Lipopolysaccharide-Induced Depression of Fatty Acid Synthesis by Inhibiting Oxidative Stress and Autophagy in Bovine Mammary Epithelial Cells

Lipopolysaccharide (LPS) is the dominating endotoxin of Gram-negative bacteria, which can cause mastitis. Bovine mammary epithelial cells (BMECs), as major components of the mammary gland, usually suffer LPS challenge. Cis-9, trans-11 conjugated linoleic acid (CLA) has been reported to have anti-inflammatory characteristics, while its anti-oxidative ability to maintain cellular homeostasis in BMECs under LPS challenge is limited. Therefore, we studied whether cis-9, trans-11 CLA can restore the disturbance of cellular homeostasis indicated by the redox status and autophagy level caused by LPS and have an effect on cellular function- milk fat metabolism. For oxidative stress, LPS challenge promoted the formation of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) and decreased the concentration of glutathione. Anti-oxidative signaling regulated by transcription factor nuclear factor, erythroid 2 like 2 (Nrf2) was also depressed by LPS at the mRNA and protein level. However, cis-9, trans-11 CLA pretreatment downregulated the formation of ROS and TBARS and upregulated the expression of antioxidative enzymes. As a part of innate immunity, autophagy was also motivated by LPS challenge, while CLA decreased the autophagy level. LPS and H2O2 inhibited milk fat synthesis-related transcription factor sterol regulatory element binding protein (SREBP1), peroxisome proliferator activated receptor gamma (PPARG) and their downstream enzymes. Furthermore, 50 uM cis-9, trans-11 CLA promoted the mRNA and protein abundance of milk fat synthesis-related genes and lipid droplet formation in BMECs. In conclusion, LPS challenge disturbed the cellular homeostasis and depressed milk fat synthesis in BMECs; while cis-9, trans-11 CLA alleviated oxidative stress and decreased autophagy level, thus promoting milk fat synthesis, which offers a natural therapeutic strategy for mastitis.

Systemic influences of mammary cancer on monocytes in mice

There is a growing body of evidence that cancer causes systemic changes. These influences are most evident in the bone marrow and blood, particularly the myeloid compartment. Here we show using mouse models of breast cancer caused by the mammary epithelial expression of the Polyoma middle T antigen that there is an increase in the number of circulating and splenic monocytes. In the circulation, cancer does not affect ratios of classical to non-classical populations monocytes nor their half-lives. Single cell RNA sequencing also indicates that cancer does not induce any new monocyte populations. In the bone marrow cancer does not change monocytic progenitor number is unaffected but the proliferation rate of monocytes is higher thus providing an explanation for expansion in the circulating number. Deep RNA sequencing of these monocytic populations reveals cancer causes changes in the classical monocyte compartment with changes evident in bone marrow monocytes but more in the blood suggesting influences in both compartments. Down regulation of interferon type 1 signalling and antigen presentation were the most prominent. Consistent with this analysis down regulated genes are enriched with STAT1/STAT2 binding sites in their promoter, transcription factors required for type 1 interferon signalling. However, these transcriptome changes in mice did not replicate those found in patients with breast cancer. Consequently, mouse models of cancer may be insufficient to study the systemic influences of human cancer.

Distinct Oncogenic Transcriptomes in Human Mammary Epithelial Cells Infected With Cytomegalovirus

Human cytomegalovirus is being recognized as a potential oncovirus beside its oncomodulation role. We previously isolated two clinical isolates, HCMV-DB (KT959235) and HCMV-BL (MW980585), which in primary human mammary epithelial cells promoted oncogenic molecular pathways, established anchorage-independent growth in vitro, and produced tumorigenicity in mice models, therefore named high-risk oncogenic strains. In contrast, other clinical HCMV strains such as HCMV-FS, KM, and SC did not trigger such traits, therefore named low-risk oncogenic strains. In this study, we compared high-risk oncogenic HCMV-DB and BL strains (high-risk) with low-risk oncogenic strains HCMV-FS, KM, and SC (low-risk) additionally to the prototypic HCMV-TB40/E, knowing that all strains infect HMECs in vitro. Numerous pro-oncogenic features including enhanced expression of oncogenes, cell survival, proliferation, and epithelial-mesenchymal transition genes were observed with HCMV-BL. In vitro, mammosphere formation was observed only in high-risk strains. HCMV-TB40/E showed an intermediate transcriptome landscape with limited mammosphere formation. Since we observed that Ki67 gene expression allows us to discriminate between high and low-risk HCMV strains in vitro, we further tested its expression in vivo. Among HCMV-positive breast cancer biopsies, we only detected high expression of the Ki67 gene in basal tumors which may correspond to the presence of high-risk HCMV strains within tumors. Altogether, the transcriptome of HMECs infected with HCMV clinical isolates displays an “oncogenic gradient” where high-risk strains specifically induce a prooncogenic environment which might participate in breast cancer development.

Nfatc1’s Role in Mammary Epithelial Morphogenesis and Basal Stem/progenitor Cell Self-renewal

Mammary gland is an outstanding system to study the regulatory mechanisms governing adult epithelial stem cell activity. Stem cells in the basal layer of the mammary gland fuel the morphogenesis and regeneration of a complex epithelial network during development and upon transplantation. The self-renewal of basal stem/progenitor cells is subjected to regulation by both cell-intrinsic and extrinsic mechanisms. Nfatc1 is a transcription factor that regulates breast tumorigenesis and metastasis, but its role in mammary epithelial development and stem cell function has not been investigated. Here we show that Nfatc1 is expressed in a small subset of mammary basal epithelial cells and its epithelial-specific deletion results in mild defects in side branching and basal-luminal cell balance. Moreover, Nfatc1-deficient basal cells exhibit reduced colony forming ability in vitro and somewhat compromised regenerative potential upon transplantation. Thus, our study provides evidence for a detectable yet non-essential role of Nfatc1 in mammary epithelial morphogenesis and basal stem/progenitor cell self-renewal.