Wassim Abou-Kheir

MiR-1 and miR-200 inhibit EMT via Slug-dependent and tumorigenesis via Slug-independent mechanisms.

Epithelial–mesenchymal transition (EMT) is a developmental program of signaling pathways that determine commitment to epithelial and mesenchymal phenotypes. In the prostate, EMT processes have been implicated in benign prostatic hyperplasia and prostate cancer progression. In a model of Pten- and TP53-null prostate adenocarcinoma that progresses via transforming growth factor β-induced EMT, mesenchymal transformation is characterized by plasticity, leading to various mesenchymal lineages and the production of bone. Here we show that SLUG is a major regulator of mesenchymal differentiation. As microRNAs (miRs) are pleiotropic regulators of differentiation and tumorigenesis, we evaluated miR expression associated with tumorigenesis and EMT. Mir-1 and miR-200 were reduced with progression of prostate adenocarcinoma, and we identify Slug as one of the phylogenetically conserved targets of these miRs. We demonstrate that SLUG is a direct repressor of miR-1 and miR-200 transcription. Thus, SLUG and miR-1/miR-200 act in a self-reinforcing regulatory loop, leading to amplification of EMT. Depletion of Slug inhibited EMT during tumorigenesis, whereas forced expression of miR-1 or miR-200 inhibited both EMT and tumorigenesis in human and mouse model systems. Various miR targets were analyzed, and our findings suggest that miR-1 has roles in regulating EMT and mesenchymal differentiation through Slug and functions in tumor-suppressive programs by regulating additional targets.

Critical and reciprocal regulation of KLF4 and SLUG in transforming growth factor β-initiated prostate cancer epithelial-mesenchymal transition.

ABSTRACT Epithelial-mesenchymal transition (EMT) is implicated in various pathological processes within the prostate, including benign prostate hyperplasia (BPH) and prostate cancer progression. However, an ordered sequence of signaling events initiating carcinoma-associated EMT has not been established. In a model of transforming growth factor β (TGFβ)-induced prostatic EMT, SLUG is the dominant regulator of EMT initiation in vitro and in vivo, as demonstrated by the inhibition of EMT following Slug depletion. In contrast, SNAIL depletion was significantly less rate limiting. TGFβ-stimulated KLF4 degradation is required for SLUG induction. Expression of a degradation-resistant KLF4 mutant inhibited EMT, and furthermore, depletion of Klf4 was sufficient to initiate SLUG-dependent EMT. We show that KLF4 and another epithelial determinant, FOXA1, are direct transcriptional inhibitors of SLUG expression in mouse and human prostate cancer cells. Furthermore, self-reinforcing regulatory loops for SLUG-KLF4 and SLUG-FOXA1 lead to SLUG-dependent binding of polycomb repressive complexes to the Klf4 and Foxa1 promoters, silencing transcription and consolidating mesenchymal commitment. Analysis of tissue arrays demonstrated decreased KLF4 and increased SLUG expression in advanced-stage primary prostate cancer, substantiating the involvement of the EMT signaling events described in model systems.

Regulation of podosome dynamics by WASp phosphorylation: implication in matrix degradation and chemotaxis in macrophages

Podosomes, adhesion structures capable of matrix degradation, have been linked with the ability of cells to perform chemotaxis and invade tissues. Wiskott-Aldrich Syndrome protein (WASp), an effector of the RhoGTPase Cdc42 and a Src family kinase substrate, regulates macrophage podosome formation. In this study, we demonstrate that WASp is active in podosomes by using TIRF-FRET microscopy. Pharmacological and RNA interference approaches suggested that continuous WASp activity is required for podosome formation and function. Rescue experiments using point mutations demonstrate an absolute requirement for Cdc42 binding to WASp in podosome formation. Although tyrosine phosphorylation was not absolutely required for podosome formation, phosphorylation did regulate the rate of podosome nucleation and actin filament stability. Importantly, WASp tyrosine phosphorylation does not alter WASp activation, instead phosphorylation appears to be important for the restriction of WASp activity to podosomes. In addition, the matrix-degrading ability of cells requires WASp phosphorylation. Chemotactic responses to CSF-1 were also attenuated in the absence of endogenous WASp, which could not be rescued with either tyrosine mutation. These results suggest a more complex role for tyrosine phosphorylation than simply in the regulation of WASp activity, and suggest a link between podosome dynamics and macrophage migration.

Membrane targeting of WAVE2 is not sufficient for WAVE2-dependent actin polymerization: a role for IRSp53 in mediating the interaction between Rac and WAVE2

Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous (WAVE) proteins play a major role in Rac-induced actin dynamics, but Rac does not bind directly to WAVE proteins. It has been proposed that either the insulin receptor substrate protein 53 (IRSp53) or a complex of proteins containing Abelson interactor protein 1 (Abi1) mediates the interaction of WAVE2 and Rac. Depletion of endogenous IRSp53 by RNA-mediated interference (RNAi) in a RAW/LR5 macrophage cell line resulted in a significant reduction of Rac1Q61L-induced surface ruffles and colony-stimulating factor 1 (CSF-1)-induced actin polymerization, protrusion and cell migration. However, IRSp53 was not essential for Fcγ-R-mediated phagocytosis, formation of podosomes or for formation of Cdc42V12-induced filopodia. IRSp53 was found to be present in an immunoprecipitable complex with WAVE2 and Abi1 in a Rac1-activation-dependent manner in RAW/LR5 cells in vivo. Importantly, reduction of endogenous IRSp53 or expression of IRSp53 lacking the WAVE2-binding site (IRSp53ΔSH3) resulted in a significant reduction in the association of Rac1 with WAVE2 and Abi1, indicating that the association of Rac1 with WAVE2 and Abi1 is IRSp53 dependent. While it has been proposed that WAVE2 activity is regulated by membrane recruitment, membrane targeting of WAVE2 in RAW/LR5 and Cos-7 cells did not induce actin polymerization or protrusion, suggesting that membrane recruitment was insufficient for regulation of WAVE2. Combined, these data suggest that IRSp53 links Rac1 to WAVE2 in vivo and its function is crucial for production of CSF-1-induced F-actin-rich protrusions and cell migration in macrophages. This study indicates that Rac1, along with IRSp53 and Abi1, is involved in a more complex and tight regulation of WAVE2 than one operating solely through membrane localization.

Differential Role of Leptin and Adiponectin in Cardiovascular System

Leptin and adiponectin are differentially expressed adipokines in obesity and cardiovascular diseases. Leptin levels are directly associated with adipose tissue mass, while adiponectin levels are downregulated in obesity. Although significantly produced by adipocytes, leptin is also produced by vascular smooth muscle cells and cardiomyocytes. Plasma leptin concentrations are elevated in cases of cardiovascular diseases, such as hypertension, congestive heart failure, and myocardial infarction. As for the event of left ventricular hypertrophy, researchers have been stirring controversy about the role of leptin in this form of cardiac remodeling. In this review, we discuss how leptin has been shown to play an antihypertrophic role in the development of left ventricular hypertrophy through in vitro experiments, population-based cross-sectional studies, and longitudinal cohort studies. Conversely, we also examine how leptin may actually promote left ventricular hypertrophy using in vitro analysis and human-based univariate and multiple linear stepwise regression analysis. On the other hand, as opposed to leptins generally detrimental effects on the cardiovascular system, adiponectin is a cardioprotective hormone that reduces left ventricular and vascular hypertrophy, oxidative stress, and inflammation. In this review, we also highlight adiponectin signaling and its protective actions on the cardiovascular system.

Prostate epithelial PTEN/TP53 loss leads to transformation of multipotential progenitors and epithelial to mesenchymal transition

Loss of PTEN and loss of TP53 are common genetic aberrations occurring in prostate cancer. PTEN and TP53 contribute to the regulation of self-renewal and differentiation in prostate progenitors, presumptive tumor initiating cells for prostate cancer. Here we characterize the transformed phenotypes resulting from deletion of the Pten and TP53 tumor suppressors in prostate epithelium. Using the PB-Cre4(+)Pten(fl/fl)TP53(fl/fl) model of prostate cancer, we describe the histological and metastatic properties of primary tumors, transplanted primary tumor cells, and clonal cell lines established from tumors. Adenocarcinoma was the major primary tumor type that developed, which progressed to lethal sarcomatoid carcinoma at approximately 6 months of age. In addition, basal carcinomas and prostatic urothelial carcinomas were observed. We show that tumor heterogeneity resulted, at least in part, from the transformation of multipotential progenitors. CK8+ luminal epithelial cells were capable of undergoing epithelial to mesenchymal transition in vivo to sarcomatoid carcinomas containing osseous metaplasia. Metastasis rarely was observed from primary tumors, but metastasis to lung and lymph nodes occurred frequently from orthotopic tumors initiated from a biphenotypic clonal cell line. Androgen deprivation influenced the differentiated phenotypes of metastases. These data show that one functional consequence of Pten/TP53 loss in prostate epithelium is lineage plasticity of transformed cells.

TMPRSS2- Driven ERG Expression In Vivo Increases Self-Renewal and Maintains Expression in a Castration Resistant Subpopulation

Genomic rearrangements commonly occur in many types of cancers and often initiate or alter the progression of disease. Here we describe an in vivo mouse model that recapitulates the most frequent rearrangement in prostate cancer, the fusion of the promoter region of TMPRSS2 with the coding region of the transcription factor, ERG. A recombinant bacterial artificial chromosome including an extended TMPRSS2 promoter driving genomic ERG was constructed and used for transgenesis in mice. TMPRSS2-ERG expression was evaluated in tissue sections and FACS-fractionated prostate cell populations. In addition to the anticipated expression in luminal cells, TMPRSS2-ERG was similarly expressed in the Sca-1hi/EpCAM+ basal/progenitor fraction, where expanded numbers of clonogenic self-renewing progenitors were found, as assayed by in vitro sphere formation. These clonogenic cells increased intrinsic self renewal in subsequent generations. In addition, ERG dependent self-renewal and invasion in vitro was demonstrated in prostate cell lines derived from the model. Clinical studies have suggested that the TMPRSS2-ERG translocation occurs early in prostate cancer development. In the model described here, the presence of the TMPRSS2-ERG fusion alone was not transforming but synergized with heterozygous Pten deletion to promote PIN. Taken together, these data suggest that one function of TMPRSS2-ERG is the expansion of self-renewing cells, which may serve as targets for subsequent mutations. Primary prostate epithelial cells demonstrated increased post transcriptional turnover of ERG compared to the TMPRSS2-ERG positive VCaP cell line, originally isolated from a prostate cancer metastasis. Finally, we determined that TMPRSS2-ERG expression occurred in both castration-sensitive and resistant prostate epithelial subpopulations, suggesting the existence of androgen-independent mechanisms of TMPRSS2 expression in prostate epithelium.

Characterizing the Contribution of Stem/Progenitor Cells to Tumorigenesis in the Pten-/-TP53-/- Prostate Cancer Model

Loss of PTEN is one of the most common mutations in prostate cancer, and loss of wild‐type TP53 is associated with prostate cancer progression and castrate resistance. Modeling prostate cancer in the mouse has shown that while Pten deletion in prostate epithelial cells leads to adenocarcinoma, combined loss of Pten and TP53 results in rapidly developing disease with greater tumor burden and early death. TP53 contributes significantly to the regulation of stem cell self‐renewal, and we hypothesized that loss of Pten/TP53 would result in measurable changes in prostate cancer stem/progenitor cell properties. Clonogenic assays that isolate progenitor function in primary prostate epithelial cells were used to measure self‐renewal, differentiation, and tumorigenic potential. Pten/TP53 null as compared with wild‐type protospheres showed increased self‐renewal activity and modified lineage commitment. Orthotopic transplantation of Pten/TP53 null cells derived from protospheres produced invasive Prostatic Intraepithelial Neoplasia (PIN)/adenocarcinoma, recapitulating the pathology seen in primary tumors. Pten/TP53 null progenitors relative to wild type also demonstrated increased dependence on the AKT/mammalian target of rapamycin complex 1 (mTORC1) and androgen receptor (AR) pathways for clonogenic and tumorigenic growth. These data demonstrate roles for Pten/TP53 in prostate epithelial stem/progenitor cell function, and moreover, as seen in patients with castrate‐resistant prostate cancer, suggest for the involvement of an AR‐dependent axis in the clonogenic expansion of prostate cancer stem cells. STEM CELLS 2010;28:2129–2140

HTR-8/SVneo cell line contains a mixed population of cells

INTRODUCTION The placenta, a transient organ in humans, is essential for pregnancy maintenance and fetal development. Trophoblast and stromal cells are the main cell types present in human placenta. Trophoblast cells are derivatives of the trophectoderm layer and fulfill the endocrine, exchange, invasion and implantation processes of the placenta, whereas stromal cells are of extraembryonic mesenchymal origin and are important for villous formation and maintenance. Different cell lines were developed to study trophoblast functions including BeWo, JEG-3 and JAR from chorioncarcinoma while HTR-8/SVneo was developed using first trimester extravillous trophoblast infected with simian virus 40 large T antigen (SV40). These cell lines are largely used to study trophoblast functions including cell fusion, migration and invasion. Therefore, the purity of each cell lines is crucial in order to be able to use them as a model recapitulating trophoblast cells. METHODS HTR-8/SVneo, BeWo, JEG-3 and JAR were analyzed for epithelial and mesenchymal markers using immunofluorescence, real time PCR and Western blot. RESULTS Our results showed that HTR-8/SVneo cell line contains two populations of cells suggesting the presence of trophoblast and stromal/mesenchymal cells. While all cells in BeWo, JEG-3 and Jar are positive for the trophoblast/epithelial marker CK7, HTR-8/SVneo cells contained few clusters of CK7 positive cells. Interestingly, vimentin expression was detected in a subset of HTR-8/SVneo cells and was completely absent from all other tested placental cell lines. DISCUSSION Our results unveil the presence of a heterogeneous population of trophoblast and stromal cells within HTR-8/SVneo cell line. This mixed population of cells should be taken into consideration when using this cell line to study trophoblast functions.

Modeling Human Neurological and Neurodegenerative Diseases: From Induced Pluripotent Stem Cells to Neuronal Differentiation and Its Applications in Neurotrauma

With the help of several inducing factors, somatic cells can be reprogrammed to become induced pluripotent stem cell (iPSCs) lines. The success is in obtaining iPSCs almost identical to embryonic stem cells (ESCs), therefore various approaches have been tested and ultimately several ones have succeeded. The importance of these cells is in how they serve as models to unveil the molecular pathways and mechanisms underlying several human diseases, and also in its potential roles in the development of regenerative medicine. They further aid in the development of regenerative medicine, autologous cell therapy and drug or toxicity screening. Here, we provide a comprehensive overview of the recent development in the field of iPSCs research, specifically for modeling human neurological and neurodegenerative diseases, and its applications in neurotrauma. These are mainly characterized by progressive functional or structural neuronal loss rendering them extremely challenging to manage. Many of these diseases, including Parkinsons disease (PD), Huntingtons disease (HD), Amyotrophic lateral sclerosis (ALS) and Alzheimers disease (AD) have been explored in vitro. The main purpose is to generate patient-specific iPS cell lines from the somatic cells that carry mutations or genetic instabilities for the aim of studying their differentiation potential and behavior. This new technology will pave the way for future development in the field of stem cell research anticipating its use in clinical settings and in regenerative medicine in order to treat various human diseases, including neurological and neurodegenerative diseases.