EMP2 Is a Novel Regulator of Stemness in Breast Cancer Cells.

Little is known about the role of epithelial membrane protein-2 (EMP2) in breast cancer development or progression. In this study, we tested the hypothesis that EMP2 may regulate the formation or self-renewal of breast cancer stem cells (BCSC) in the tumor microenvironment. In silico analysis of gene expression data demonstrated a correlation of EMP2 expression with known metastasis-related genes and markers of cancer stem cells (CSC) including aldehyde dehydrogenase (ALDH). In breast cancer cell lines, EMP2 overexpression increased and EMP2 knockdown decreased the proportion of stem-like cells as assessed by the expression of the CSC markers CD44+/CD24-, ALDH activity, or by tumor sphere formation. In vivo, upregulation of EMP2 promoted tumor growth, whereas knockdown reduced the ALDHhigh CSC population as well as retarded tumor growth. Mechanistically, EMP2 functionally regulated the response to hypoxia through the upregulation of HIF-1α, a transcription factor previously shown to regulate the self-renewal of ALDHhigh CSCs. Furthermore, in syngeneic mouse models and primary human tumor xenografts, mAbs directed against EMP2 effectively targeted CSCs, reducing the ALDH+ population and blocking their tumor-initiating capacity when implanted into secondary untreated mice. Collectively, our results show that EMP2 increases the proportion of tumor-initiating cells, providing a rationale for the continued development of EMP2-targeting agents.

Connexin 43 Loss Triggers Cell Cycle Entry and Invasion in Non-Neoplastic Breast Epithelium: A Role for Noncanonical Wnt Signaling.

(1) Background: The expression of connexin 43 (Cx43) is disrupted in breast cancer, and re-expression of this protein in human breast cancer cell lines leads to decreased proliferation and invasiveness, suggesting a tumor suppressive role. This study aims to investigate the role of Cx43 in proliferation and invasion starting from non-neoplastic breast epithelium. (2) Methods: Nontumorigenic human mammary epithelial HMT-3522 S1 cells and Cx43 shRNA-transfected counterparts were cultured under 2-dimensional (2-D) and 3-D conditions. (3) Results: Silencing Cx43 induced mislocalization of ß-catenin and Scrib from apicolateral membrane domains in glandular structures or acini formed in 3-D culture, suggesting the loss of apical polarity. Cell cycle entry and proliferation were enhanced, concomitantly with c-Myc and cyclin D1 upregulation, while no detectable activation of Wnt/ß-catenin signaling was observed. Motility and invasion were also triggered and were associated with altered acinar morphology and activation of ERK1/2 and Rho GTPase signaling, which acts downstream of the noncanonical Wnt pathway. The invasion of Cx43-shRNA S1 cells was observed only under permissive stiffness of the extracellular matrix (ECM). (4) Conclusion: Our results suggest that Cx43 controls proliferation and invasion in the normal mammary epithelium in part by regulating noncanonical Wnt signaling.

Molecular typing of Staphylococcus aureus collected from a Major Hospital in Amman, Jordan.

INTRODUCTION: Over the past decade methicillin-resistant Staphylococcus aureus (MRSA) has been recognized as a major cause of healthcare associated infections. Recently, however, epidemiology of this pathogen has changed drastically with the emergence of new clones in the community. Efficient epidemiological typing methods are essential to monitor and limit the occurrence and spread of epidemic clones. METHODOLOGY: A total of sixty S. aureus isolates were collected from the Jordan University hospital in Amman-Jordan. All isolates were characterized using Staphylococcus protein A (spa) typing and pulsed-field gel electrophoresis (PFGE). Samples were tested for their susceptibility patterns against seven antimicrobial agents and for their potential to form biofilms. RESULTS: spa typing showed that spa type t044 was the most common representing 28% of the isolates studied and 38% of the MRSA population. PFGE revealed fourty-six pulsotypes among the sixty tested isolates clustering similar spa types together. The predominant resistance was detected against levofloxacin, chloramphenicol and clindamycin. One MSSA isolate typed as spa t955 showed biofilm formation potential through protein deposition.. CONCLUSION: The study results are based on one hospital, but the findings of this and other studies conducted in the region indicate that there is an urgent need for standardized surveillances combined with the application of well-validated typing methods to assess the occurrence of MRSA and to control its spread.

Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: role of ß-catenin/connexin43 association.

Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of ß-catenin, α-catenin and ZO-2, it decreased nuclear levels of ß-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, ß-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, ß-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering ß-catenin away from nucleus.

Polarity proteins as regulators of cell junction complexes: implications for breast cancer.

The epithelium of multicellular organisms possesses a well-defined architecture, referred to as polarity that coordinates the regulation of essential cell features. Polarity proteins are intimately linked to the protein complexes that make the tight, adherens and gap junctions; they contribute to the proper localization and assembly of these cell-cell junctions within cells and consequently to functional tissue organization. The establishment of cell-cell junctions and polarity are both implicated in the regulation of epithelial modifications in normal and cancer situations. Uncovering the mechanisms through which cell-cell junctions and epithelial polarization are established and how their interaction with the microenvironment directs cell and tissue organization has opened new venues for the development of cancer therapies. In this review, we focus on the breast epithelium to highlight how polarity and cell-cell junction proteins interact together in normal and cancerous contexts to regulate major cellular mechanisms such as migration. The impact of these proteins on epigenetic mechanisms responsible for resetting cells toward oncogenesis is discussed in light of increasing evidence that tissue polarity modulates chromatin function. Finally, we give an overview of recent breast cancer therapies that target proteins involved in cell-cell junctions.

Modulation of Cx43 and Gap Junctional Intercellular Communication by Androstenedione in Rat Polycystic Ovary and Granulosa Cells in vitro.

BACKGROUND: Gap-junctional intercellular communication (GJIC) is implicated in physicological processes and it is vitally important for granulosa cell (GC) differentiation and oocyte growth. We investigated the expression of connexin 43 (Cx43), a gap junctional protein, in normal and androstenedione-induced polycystic ovary (PCO), the effects of androstenedione on Cx43 expression, GJIC and progesterone production in granulosa cells in vitro. METHODS: Isolated GCs from rat ovary were supplemented with FSH and dripped with EHS-matrix (EHS-drip) in culture media, were treated with physiological (10(-7) M) or pathological (10(-5) M) androstenedione concentrations to induce differentiation. Cx43 protein levels were assessed by Western blotting. Immunohistochemistry was also used to determine the localization of Cx43 in GCs and corpus luteum (CL) of controls and PCOs. Differentiation of GCs was determined by progesterone assay and Lucifer yellow dye transfer for GJIC status. The degree of significance of variations between the results was analyzed by ANOVA using SPSS (version 11.5; 2002). RESULTS: Cx43 localized in the GC layer of both the control and PCOs. Its protein levels were upregulated in PCO rat ovaries. GCs in culture with or without androstenedione had a punctate membranous distribution of Cx43. However, androstenedione increased GJIC and upregulated progesterone and Cx43 protein levels. Inhibiting GJIC by 18-α GA in androstenedione-treated GCs caused partial inhibition of progesterone production, suggesting a possible role of GJIC in mediating the action of androstenedione on GC differentiation. CONCLUSION: This study presented a suitable culture model for polycystic ovary syndrome and showed that Cx43 and GJIC might contribute to the pathogenesis of polycystic ovary syndrome.