Differential gene expression in normal and transformed human mammary epithelial cells in response to oxidative stress

Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMECs), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5) were exposed to increased levels of reactive oxygen species (ROS) by treatment with glucose oxidase. Functional analysis of the metabolic pathways enriched with differentially expressed genes demonstrated that normal and malignant breast epithelial cells diverge substantially in their response to oxidative stress. Whereas normal cells exhibit the up-regulation of antioxidant mechanisms, cancer cells are unresponsive to the ROS insult. However, the gene expression response of normal HMECs under oxidative stress is comparable to that of the malignant cells under normal conditions, indicating that altered redox status is persistent in breast cancer cells, which makes them resistant to increased generation of ROS. We discuss some of the possible adaptation mechanisms of breast cancer cells under persistent oxidative stress that differentiate them from normal mammary epithelial cells as regards the response to acute oxidative stress.     

Phosphorylation of DARPP-32 regulates breast cancer cell migration downstream of the receptor tyrosine kinase DDR1

Cell migration plays a central role in processes such as development, wound healing and cancer metastasis. Here we describe a novel interaction between DDR1, a receptor tyrosine kinase activated by collagen, and the phosphoprotein DARPP-32 in mammary epithelial cells. DARPP-32 expression was readily detected in non-transformed mammary cell lines, but was strongly reduced or even absent in breast tumor cell lines, such as MCF7. Transfection of MCF7 cells with DARPP-32 resulted in severely impaired cell migration, while DARPP-32 transfection into the DDR1-deficient breast cancer cell line MDA-MB-231 did not alter migration. Co-expression of both DDR1 and DARPP-32 in MDA-MB-231 cells inhibited migration, thereby supporting a critical role of the DDR1/DARPP-32 complex in motility. Mutational substitution of the phosphorylation sites Thr-34 or Thr-75 on DARPP-32 revealed that phosphorylation of Thr-34 is necessary for the ability of DARPP-32 to impair breast tumor cell migration. Thus, DARPP-32 signaling downstream of DDR1 is a potential new target for effective anti-metastatic breast cancer therapy.     

Drug-resistant endothelial cells facilitate progression,EMT and chemoresistance in nasopharyngeal carcinoma via exosomes

Recent antitumor drug development has included investigation of a wide variety of anti-angiogenesis therapies. Because cancer cells in tumors require new blood vessels to grow and spread, they stimulate capillary proliferation from existing vessels as well as new vessel formation from endothelial precursor cells. Our previous findings suggested that drug resistance in mouse endothelial cells supported tumor growth, but the relationship between endothelial cells (ECs) and nasopharyngeal carcinoma (NPC) cells remained unclear. Exosomes are small membrane vesicles that are released by several cell types, including human microvascular ECs (HMECs). Exosomes carrying membrane and cytoplasmic constituents have been described as participants in a novel mechanism of cell-to-cell communication. In the present study, we investigated the mechanisms underlying the interactions between HMECs and NPC cells. We found that drug-resistant HMECs secreted small heterogeneous 40–100 nm vesicles, defined as exosomes. Co-incubation of NPC cells with doxorubicin-resistant (R-DOX) HMEC-derived exosomes resulted in promotion of their proliferation, migration, and chemoresistance, as well as changes in the expression of epithelial–mesenchymal transition (EMT) markers. These effects were significantly inhibited by treatment with GW4869 (an exosome inhibitor). We also found that GW4869 inhibited the stimulation of drug-resistant HMECs on NPC progression by modulating EMT in vivo. These data suggest that exosomes participate in a novel mechanism by which drug-resistant ECs enhance NPC progression.     

Periostin Contributes to the Acquisition of Multipotent Stem Cell-Like Properties in Human Mammary Epithelial Cells and Breast Cancer Cells

Periostin (POSTN), a recently characterised matricellular protein, is frequently dysregulated in various malignant cancers and promotes tumor metastatic growth. POSTN plays a critical role in the crosstalk between murine breast cancer stem cells (CSCs) and their niche to permit metastatic colonization. However, whether pro-metastatic capability of POSTN is associated with multipotent potentials of mesenchymal stem cells (MSCs) has not been documented. Here we demonstrate that POSTN promotes a stem cell-like trait and a mesenchymal phenotype in human mammary epithelial cells and breast cancer cells. Interestingly, ectopic overexpression of POSTN or recombinant POSTN treatment can induce human mammary epithelial cells and breast cancer cells differentiation into multiple cell lineages that recapitulate part of the multilineage differentiation potentials of MSCs. Moreover, POSTN is highly expressed in bone marrow-derived MSCs and their derived adipocytes, chondrocytes, and osteoblasts in vitro. Furthermore, POSTN promotes the growth of xenograft tumors in vivo. POSTN-overexpressing human mammary epithelial cells enhance breast tumor growth and metastasis. These data thus provide evidence of a new role for POSTN in mammary epithelial neoplasia and metastasis, suggesting that epithelial cancer cells might acquire CSC-like traits and a mesenchymal phenotype, as well as the multipotent potentials of MSCs to promote tumorigenesis and metastasis. Therefore, targeting POSTN and other extracellular matrix components of tumor microenvironment may help to develop new therapeutical strategies to inhibit tumor metastasis.     

Autophagy inhibition in breast cancer cells induces ROS-mediated MIF expression and M1 macrophage polarization

Autophagy can function as a survival mechanism for cancer cells and therefore, its inhibition is currently being explored as a therapy for different cancer types. For breast cancer, triple negative breast cancer (TNBC) is the subtype most sensitive to the inhibition of autophagy; but its inhibition has also been shown to promote ROS-dependent secretion of macrophage migration inhibitory factor (MIF), a pro-tumorigenic cytokine. In this work, we explore the role of MIF in breast cancer, the mechanism by which autophagy inhibition promotes MIF secretion and its effects on neighboring cancer cell signaling and macrophage polarization. We analyzed MIF mRNA expression levels in tumors from breast cancer patients from different subtypes and found that Luminal B, HER2 and Basal subtypes, which are associated to high proliferation, displayed high MIF levels. However, MIF expression had no prognostic relevance in any breast cancer subtype. In addition, we found that autophagy inhibition in 66cl4 TNBC cells increased intracellular Reactive Oxygen Species (ROS) levels, which increased MIF expression and secretion. MIF secreted from 66cl4 TNBC cells induced the activation of MIF-regulated pathways in syngeneic cell lines, increasing Akt phosphorylation in 4T1 cells and ERK phosphorylation in 67NR cells. Regarding MIF/ chemokine receptors, higher levels of CD74 and CXCR2 were found in TNBC tumor cell lines when compared to non-tumorigenic cells and CXCR7 was elevated in the highly metastatic 4T1 cell line. Finally, secreted MIF from autophagy deficient 66cl4 cells induced macrophage polarization towards the M1 subtype. Together, our results indicate an important role for the inhibition of autophagy in the regulation of ROS-mediated MIF gene expression and secretion, with paracrine effects on cancer cell signaling and pro-inflammatory repercussions in macrophage M1 polarization. This data should be considered when considering the inhibition of autophagy as a therapy for different types of cancer.     

Cytoplasmic PELP1 and ERRgamma Protect Human Mammary Epithelial Cells from Tam-Induced Cell Death

Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman''s risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.     

Individual‐specific variation in the respiratory activities of HMECs and their bioenergetic response to IGF1 and TNFα

Metabolic reprograming is a hallmark of cancer cells. However, the roles of pre‐existing differences in normal cells metabolism toward cancer risk is not known. In order to assess pre‐existing variations in normal cell metabolism, we have quantified the inter‐individual variation in oxidative metabolism of normal primary human mammary epithelial cells (HMECs). We then assessed their response to selected cytokines such as insulin growth factor 1 (IGF1) and tumor necrosis factor alpha (TNFα), which are associated with breast cancer risk. Specifically, we compared the oxidative metabolism of HMECs obtained from women with breast cancer and without cancer. Our data show considerable inter‐individual variation in respiratory activities of HMECs from different women. A bioenergetic parameter called pyruvate‐stimulated respiration (PySR) was identified as a key distinguishing feature of HMECs from women with breast cancer and without cancer. Samples showing PySR over 20% of basal respiration rate were considered PySR^+ve and the rest as PySR^?ve. By this criterion, HMECs from tumor‐affected breasts (AB) and non‐tumor affected breasts (NAB) of cancer patients were mostly PySR^?ve (88% and 89%, respectively), while HMECs from non‐cancer patients were mostly PySR^+ve (57%). This suggests that PySR^?ve/+ve phenotypes are individual‐specific and are not caused by field effects due to the presence of tumor. The effects of IGF1 and TNFα treatments on HMECs revealed that both suppressed respiration and extracellular acidification. In addition, IGF1 altered PySR^?ve/+ve phenotypes. These results reveal individual‐specific differences in pyruvate metabolism of normal breast epithelial cells and its association with breast cancer risk.

     

Sclerotium rolfsii Lectin Induces Stronger Inhibition of Proliferation in Human Breast Cancer Cells than Normal Human Mammary Epithelial Cells by Induction of Cell Apoptosis

Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent.     

Expression Profiling during Mammary Epithelial Cell Three-Dimensional Morphogenesis Identifies PTPRO as a Novel Regulator of Morphogenesis and ErbB2-Mediated Transformation

Identification of genes that are upregulated during mammary epithelial cell morphogenesis may reveal novel regulators of tumorigenesis. We have demonstrated that gene expression programs in mammary epithelial cells grown in monolayer cultures differ significantly from those in three-dimensional (3D) cultures. We identify a protein tyrosine phosphate, PTPRO, that was upregulated in mature MCF-10A mammary epithelial 3D structures but had low to undetectable levels in monolayer cultures. Downregulation of PTPRO by RNA interference inhibited proliferation arrest during morphogenesis. Low levels of PTPRO expression correlated with reduced survival for breast cancer patients, suggesting a tumor suppressor function. Furthermore, we showed that the receptor tyrosine kinase ErbB2/HER2 is a direct substrate of PTPRO and that loss of PTPRO increased ErbB2-induced cell proliferation and transformation, together with tyrosine phosphorylation of ErbB2. Moreover, in patients with ErbB2-positive breast tumors, low PTPRO expression correlated with poor clinical prognosis compared to ErbB2-positive patients with high levels of PTPRO. Thus, PTPRO is a novel regulator of ErbB2 signaling, a potential tumor suppressor, and a novel prognostic marker for patients with ErbB2-positive breast cancers. We have identified the protein tyrosine phosphatase PTPRO as a regulator of three-dimensional epithelial morphogenesis of mammary epithelial cells and as a regulator of ErbB2-mediated transformation. In addition, we demonstrated that ErbB2 is a direct substrate of PTPRO and that decreased expression of PTPRO predicts poor prognosis for ErbB2-positive breast cancer patients. Thus, our results identify PTPRO as a novel regulator of mammary epithelial transformation, a potential tumor suppressor, and a predictive biomarker for breast cancer.     

Mitochondrial biogenesis in epithelial cancer cells promotes breast cancer tumor growth and confers autophagy resistance

Here, we set out to test the novel hypothesis that increased mitochondrial biogenesis in epithelial cancer cells would “fuel” enhanced tumor growth. For this purpose, we generated MDA-MB-231 cells (a triple-negative human breast cancer cell line) overexpressing PGC-1α and MitoNEET, which are established molecules that drive mitochondrial biogenesis and increased mitochondrial oxidative phosphorylation (OXPHOS). Interestingly, both PGC-1α and MitoNEET increased the abundance of OXPHOS protein complexes, conferred autophagy resistance under conditions of starvation and increased tumor growth by up to ~3-fold. However, this increase in tumor growth was independent of neo-angiogenesis, as assessed by immunostaining and quantitation of vessel density using CD31 antibodies. Quantitatively similar increases in tumor growth were also observed by overexpression of PGC-1β and POLRMT in MDA-MB-231 cells, which are also responsible for mediating increased mitochondrial biogenesis. Thus, we propose that increased mitochondrial “power” in epithelial cancer cells oncogenically promotes tumor growth by conferring autophagy resistance. As such, PGC-1α, PGC-1β, mitoNEET and POLRMT should all be considered as tumor promoters or “metabolic oncogenes.” Our results are consistent with numerous previous clinical studies showing that metformin (a weak mitochondrial “poison”) prevents the onset of nearly all types of human cancers in diabetic patients. Therefore, metformin (a complex I inhibitor) and other mitochondrial inhibitors should be developed as novel anticancer therapies, targeting mitochondrial metabolism in cancer cells.     

Met Receptor Acts Uniquely for Survival and Morphogenesis of EGFR-Dependent Normal Mammary Epithelial and Cancer Cells

Mammary gland development and breast cancer growth require multiple factors both of endocrine and paracrine origin. We analyzed the roles of Epidermal Growth Factor Receptor (EGFR) and Hepatocyte Growth Factor Receptor (Met) in mammary epithelial cells and mammary tumor cells derived from a mutated-ErbB2 transgenic mice. By using highly specific tyrosine kinase inhibitors we found that MCF-10A and NMuMG mammary epithelial cell lines are totally dependent on EGFR activation for their growth and survival. Proliferation and 3D-morphogenesis assays showed that HGF had no role in maintaining mammary cell viability, but was the only cytokine able to rescue EGFR-inhibited mammary cells. Insulin-Like Growth Factor-I (IGF-I), basic-Fibroblast Growth Factor (b-FGF) and Neuregulin, which are well known mammary morphogenic factors, did not rescue proliferation or morphogenesis in these cell lines, following EGFR inhibition. Similarly, ErbB2-driven tumor cells are EGFR-dependent and also display HGF-mediated rescue. Western-blot analysis of the signaling pathways involved in rescue after EGFR inhibition indicated that concomitant ERK1/2 and AKT activation was exclusively driven by Met, but not by IGF-I or b-FGF. These results describe a unique role for EGFR and Met in mammary epithelial cells by showing that similar pathways can be used by tumorigenic cells to sustain growth and resist to EGFR-directed anti-tumorigenic drugs.     

Expression and Localization of GEN1 in Mouse Mammary Epithelial Cells

GEN1, a Holliday junction resolvase, is involved in homologous repair of DNA double strand break and in maintaining centrosome integrity. Although GEN1 mutants have been reported in breast cancer patients and cell lines, little is currently known about the functions of GEN1 in the development and oncogenic transformation of mammary gland. In the present study, we demonstrate that GEN1 expression is correlated with mammary epithelial cell proliferation, differentiation in various physiological stages as well as casein. By immunofluorescence analysis, the centrosomal association of GEN1 is confirmed in mammary epithelial cells. Additionally, GEN1 is likely involved in DNA damage response of breast cancer cell lines. These results suggest that GEN1 may play an important role in the development of mammary gland; its response upon DNA damage indicates that GEN1 gene alteration may contribute to breast cancer formation.     

Understanding the metabolic basis of drug resistance: Therapeutic induction of the Warburg effect kills cancer cells

Previously, we identified a form of epithelial-stromal metabolic coupling, in which cancer cells induce aerobic glycolysis in adjacent stromal fibroblasts, via oxidative stress, driving autophagy and mitophagy. In turn, these cancer-associated fibroblasts provide recycled nutrients to epithelial cancer cells, “fueling” oxidative mitochondrial metabolism and anabolic growth. An additional consequence is that these glycolytic fibroblasts protect cancer cells against apoptosis, by providing a steady nutrient stream to mitochondria in cancer cells. Here, we investigated whether these interactions might be the basis of tamoxifen-resistance in ER(+) breast cancer cells. We show that MCF7 cells alone are Tamoxifen-sensitive, but become resistant when co-cultured with hTERT-immortalized human fibroblasts. Next, we searched for a drug combination (Tamoxifen + Dasatinib) that could over-come fibroblast-induced Tamoxifen-resistance. Importantly, we show that this drug combination acutely induces the Warburg effect (aerobic glycolysis) in MCF7 cancer cells, abruptly cutting off their ability to use their fuel supply, effectively killing these cancer cells. Thus, we believe that the Warburg effect in tumor cells is not the “root cause” of cancer, but rather it may provide the necessary clues to preventing chemoresistance in cancer cells. Finally, we observed that this drug combination (Tamoxifen + Dasatinib) also had a generalized anti-oxidant effect, on both co-cultured fibroblasts and cancer cells alike, potentially reducing tumor-stroma co-evolution. Our results are consistent with the idea that chemo-resistance may be both a metabolic and stromal phenomenon that can be overcome by targeting mitochondrial function in epithelial cancer cells. Thus, simultaneously targeting both (1) the tumor stroma and (2) the epithelial cancer cells, with combination therapies, may be the most successful approach to anti-cancer therapy. This general strategy of combination therapy for overcoming drug resistance could be applicable to many different types of cancer.Key words: drug resistance, tamoxifen, dasatinib, tumor stroma, microenvironment, Warburg effect, aerobic glycolysis, mitochondrial oxidative phosphorylation, glucose uptake, oxidative stress, reactive oxygen species (ROS), cancer-associated fibroblasts     

Cytokine production and inflammation drive autophagy in the tumor microenvironment: Role of stromal caveolin-1 as a key regulator

Recently, we proposed a new paradigm for understanding the role of the tumor microenvironment in breast cancer onset and progression. In this model, cancer cells induce oxidative stress in adjacent fibroblasts. This, in turn, results in the onset of stromal autophagy, which produces recycled nutrients to “feed” anabolic cancer cells. However, it remains unknown how autophagy in the tumor microenvironment relates to inflammation, another key driver of tumorigenesis. To address this issue, here we employed a well-characterized co-culture system in which cancer cells induce autophagy in adjacent fibroblasts via oxidative stress and NFκB-activation. We show, using this co-culture system, that the same experimental conditions that result in an autophagic microenvironment, also drive in the production of numerous inflammatory mediators (including IL-6, IL-8, IL-10, MIp1α, IFNγ, RANTES (CCL5) and GMCSF). Furthermore, we demonstrate that most of these inflammatory mediators are individually sufficient to directly induce the onset of autophagy in fibroblasts. To further validate the in vivo relevance of these findings, we assessed the inflammatory status of Cav-1 (−/−) null mammary fat pads, which are a model of a bonafide autophagic microenvironment. Notably, we show that Cav-1 (−/−) mammary fat pads undergo infiltration with numerous inflammatory cell types, including lymphocytes, T-cells, macrophages and mast cells. Taken together, our results suggest that cytokine production and inflammation are key drivers of autophagy in the tumor microenvironment. These results may explain why a loss of stromal Cav-1 is a powerful predictor of poor clinical outcome in breast cancer patients, as it is a marker of both (1) autophagy and (2) inflammation in the tumor microenvironment. Lastly, hypoxia in fibroblasts was not sufficient to induce the full-blown inflammatory response that we observed during the co-culture of fibroblasts with cancer cells, indicating that key reciprocal interactions between cancer cells and fibroblasts may be required.Key words: caveolin-1, oxidative stress, cytokine production, inflammation, tumor microenvironment, autophagy, breast cancer     

The Role of the Microenvironment in Mammary Gland Development and Cancer

The mammary gland is composed of a diverse array of cell types that form intricate interaction networks essential for its normal development and physiologic function. Abnormalities in these interactions play an important role throughout different stages of tumorigenesis. Branching ducts and alveoli are lined by an inner layer of secretory luminal epithelial cells that produce milk during lactation and are surrounded by contractile myoepithelial cells and basement membrane. The surrounding stroma comprised of extracellular matrix and various cell types including fibroblasts, endothelial cells, and infiltrating leukocytes not only provides a scaffold for the organ, but also regulates mammary epithelial cell function via paracrine, physical, and hormonal interactions. With rare exceptions breast tumors initiate in the epithelial compartment and in their initial phases are confined to the ducts but this barrier brakes down with invasive progression because of a combination of signals emitted by tumor epithelial and various stromal cells. In this article, we overview the importance of cellular interactions and microenvironmental signals in mammary gland development and cancer.The mammary gland is composed of a combination of multiple cell types that together form complex interaction networks required for the proper development and functioning of the organ. The branching milk ducts are formed by an outer myoepithelial cell layer producing the basement membrane (BM) and an inner luminal epithelial cell layer producing milk during lactation. The ducts are surrounded by the microenvironment composed of extracellular matrix (ECM) and various stromal cell types (e.g., endothelial cells, fibroblasts, myofibroblasts, and leukocytes). Large amount of data suggest that cell-cell and cell-microenvironment interactions modify the proliferation, survival, polarity, differentiation, and invasive capacity of mammary epithelial cells. However, the molecular mechanisms underlying these effects are poorly understood. The purification and comprehensive characterization of each cell type comprising normal and neoplastic human breast tissue combined with hypothesis testing in cell culture and animal models are likely to improve our understanding of the role these cells play in the normal functioning of the mammary gland and in breast tumorigenesis. In this article, we overview cellular and microenvironmental interactions that play important roles in the normal functioning of the mammary gland and their abnormalities in breast cancer.     

Exosomes Derived from Mesenchymal Stem Cells Suppress Angiogenesis by Down-Regulating VEGF Expression in Breast Cancer Cells

Exosomes are small membrane vesicles released by a variety of cell types. Exosomes contain genetic materials, such as mRNAs and microRNAs (miRNAs), implying that they may play a pivotal role in cell-to-cell communication. Mesenchymal stem cells (MSCs), which potentially differentiate into multiple cell types, can migrate to the tumor sites and have been reported to exert complex effects on tumor progression. To elucidate the role of MSCs within the tumor microenvironment, previous studies have suggested various mechanisms such as immune modulation and secreted factors of MSCs. However, the paracrine effects of MSC-derived exosomes on the tumor microenvironment remain to be explored. The hypothesis of this study was that MSC-derived exosomes might reprogram tumor behavior by transferring their molecular contents. To test this hypothesis, exosomes from MSCs were isolated and characterized. MSC-derived exosomes exhibited different protein and RNA profiles compared with their donor cells and these vesicles could be internalized by breast cancer cells. The results demonstrated that MSC-derived exosomes significantly down-regulated the expression of vascular endothelial growth factor (VEGF) in tumor cells, which lead to inhibition of angiogenesis in vitro and in vivo. Additionally, miR-16, a miRNA known to target VEGF, was enriched in MSC-derived exosomes and it was partially responsible for the anti-angiogenic effect of MSC-derived exosomes. The collective results suggest that MSC-derived exosomes may serve as a significant mediator of cell-to-cell communication within the tumor microenvironment and suppress angiogenesis by transferring anti-angiogenic molecules.     

Differential Subcellular Localization Renders HAI-2 a Matriptase Inhibitor in Breast Cancer Cells but Not in Mammary Epithelial Cells

The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells.     

Increased protein stability causes DNA methyltransferase 1 dysregulation in breast cancer

We report that DNA methyltransferase 1 (DNMT1) expression is dysregulated in breast cancer. The elevated protein levels are not a result of increased mRNA levels, but rather an increase in protein half-life. We found that DNMT1 protein levels were elevated in breast cancer tissues and in MCF-7 breast cancer cells relative to normal human mammary epithelial cells (HMECs) without a concomitant increase in DNMT1 mRNA or proliferative fraction. Although DNMT1 mRNA levels were properly S-phase-regulated in both cell types, DNMT1 protein levels did not follow S-phase fraction in MCF-7 cells. Rather, an increase in DNMT1 protein stability was found for MCF-7 cells relative to HMECs, and a destruction domain was mapped to the N-terminal 120 amino acids of DNMT1, which was required for its proper ubiquitination and degradation in HMECs. Furthermore, overexpression of DNMT1 with this deleted destruction domain in HMECs resulted in significantly increased genomic 5-methylcytosine levels relative to overexpression of the full-length protein. The regulation of DNMT1 destruction via this domain may be dysfunctional in cancer cells leading to subsequent cytosine hypermethylation in the genome.