Zakaria Y. Abd Elmageed

Neoplastic Reprogramming of Patient-Derived Adipose Stem Cells by Prostate Cancer Cell-Associated Exosomes

Emerging evidence suggests that mesenchymal stem cells (MSCs) are often recruited to tumor sites but their functional significance in tumor growth and disease progression remains elusive. Herein we report that prostate cancer (PC) cell microenvironment subverts PC patient adipose‐derived stem cells (pASCs) to undergo neoplastic transformation. Unlike normal ASCs, the pASCs primed with PC cell conditioned media (CM) formed prostate‐like neoplastic lesions in vivo and reproduced aggressive tumors in secondary recipients. The pASC tumors acquired cytogenetic aberrations and mesenchymal‐to‐epithelial transition and expressed epithelial, neoplastic, and vasculogenic markers reminiscent of molecular features of PC tumor xenografts. Our mechanistic studies revealed that PC cell‐derived exosomes are sufficient to recapitulate formation of prostate tumorigenic mimicry generated by CM‐primed pASCs in vivo. In addition to downregulation of the large tumor suppressor homolog2 and the programmed cell death protein 4, a neoplastic transformation inhibitor, the tumorigenic reprogramming of pASCs was associated with trafficking by PC cell‐derived exosomes of oncogenic factors, including H‐ras and K‐ras transcripts, oncomiRNAs miR‐125b, miR‐130b, and miR‐155 as well as the Ras superfamily of GTPases Rab1a, Rab1b, and Rab11a. Our findings implicate a new role for PC cell‐derived exosomes in clonal expansion of tumors through neoplastic reprogramming of tumor tropic ASCs in cancer patients. Stem Cells 2014;32:983–997

Modified multipotent stromal cells with epidermal growth factor restore vasculogenesis and blood flow in ischemic hind-limb of type II diabetic mice

Diabetes is increasing in the world and causes severe cardiovascular complications. Diabetes-induced limb ischemia leads to foot amputation and therapeutic remedies are urgently needed. Here we report that local injection of mesenchymal stem cells (MSCs) prestimulated with epidermal growth factor (EGF) restored blood flow and vasculogenesis in the ischemic hind-limb of type II diabetic (db−/db−) mice. Bone marrow cells from db−/db− mice are altered as evidenced by increased oxidative stress and reduced Akt and adhesion molecules when compared with control (db−/db+). Femoral artery ligation-induced ischemia was performed in the hind-limb of db−/db− and db−/db+ mice for 28 days. Enhanced green fluorescent protein (EGFP)-MSCs stimulated±exogenous EGF for 24 h were injected locally into the ischemic muscle. Blood flow measured with MoorLDI-Laser and microangiography assessed with X-ray showed 100% recovery in db−/db+ compared to 50% recovery in db−/db− mice. Interestingly, db−/db− mice had 60 and 96% blood flow recovery and 61 and 98% of vasculogenesis when treated with MSCs alone or MSCs modified with EGF, respectively. Western blot analysis of hind-limb muscles revealed an increase in Akt and vascular endothelial growth factor receptor phosphorylation and hypoxia-inducible factor) expression in db−/db− mice injected with MSCs or MSCs+EGF compared to db−/db− mice. Fluorescent microscopic images show that EGFP-MSCs differentiate into new microvessels. Adhesion and migration of MSCs on cultured endothelial cells were ICAM1-, VCAM1- and Akt-dependent mechanism and elevated when MSCs were prestimulated with EGF compared with nonstimulated MSCs. Our novel study data provide evidence that in type II diabetes, stimulated MSCs with EGF enhance the recovery of blood flow and angiogenesis.

Molecular characterization of exosome-like vesicles from breast cancer cells

BackgroundMembrane vesicles released by neoplastic cells into extracellular medium contain potential of carrying arrays of oncogenic molecules including proteins and microRNAs (miRNA). Extracellular (exosome-like) vesicles play a major role in cell-to-cell communication. Thus, the characterization of proteins and miRNAs of exosome-like vesicles is imperative in clarifying intercellular signaling as well as identifying disease markers.MethodsExosome-like vesicles were isolated using gradient centrifugation from MCF-7 and MDA-MB 231 cultures. Proteomic profiling of vesicles using liquid chromatography-mass spectrometry (LC-MS/MS) revealed different protein profiles of exosome-like vesicles derived from MCF-7 cells (MCF-Exo) than those from MDA-MB 231 cells (MDA-Exo).ResultsThe protein database search has identified 88 proteins in MDA-Exo and 59 proteins from MCF-Exo. Analysis showed that among all, 27 proteins were common between the two exosome-like vesicle types. Additionally, MDA-Exo contains a higher amount of matrix-metalloproteinases, which might be linked to the enhanced metastatic property of MDA-MB 231 cells. In addition, microarray analysis identified several oncogenic miRNA between the two types vesicles.ConclusionsIdentification of the oncogenic factors in exosome-like vesicles is important since such vesicles could convey signals to non-malignant cells and could have an implication in tumor progression and metastasis.

Requirement for Inducible Nitric Oxide Synthase in Chronic Allergen Exposure-Induced Pulmonary Fibrosis but Not Inflammation

The role of inducible NO synthase (iNOS) in allergic airway inflammation remains elusive. We tested the hypothesis that iNOS plays different roles during acute versus chronic airway inflammation. Acute and chronic mouse models of OVA-induced airway inflammation were used to conduct the study. We showed that iNOS deletion was associated with a reduction in eosinophilia, mucus hypersecretion, and IL-5 and IL-13 production upon the acute protocol. Such protection was completely abolished upon the chronic protocol. Interestingly, pulmonary fibrosis observed in wild-type mice under the chronic protocol was completely absent in iNOS−/− mice despite persistent IL-5 and IL-13 production, suggesting that these cytokines were insufficient for pulmonary fibrosis. Such protection was associated with reduced collagen synthesis and indirect but severe TGF-β modulation as confirmed using primary lung smooth muscle cells. Although activation of matrix metalloproteinase-2/-9 exhibited little change, the large tissue inhibitor of metalloproteinase-2 (TIMP-2) increase detected in wild-type mice was absent in the iNOS−/− counterparts. The regulatory effect of iNOS on TIMP-2 may be mediated by peroxynitrite, as the latter reversed TIMP-2 expression in iNOS−/− lung smooth muscle cells and fibroblasts, suggesting that the iNOS–TIMP-2 link may explain the protective effect of iNOS-knockout against pulmonary fibrosis. Analysis of lung sections from chronically OVA-exposed iNOS−/− mice revealed evidence of residual but significant protein nitration, prevalent oxidative DNA damage, and poly(ADP-ribose) polymerase-1 activation. Such tissue damage, inflammatory cell recruitment, and mucus hypersecretion may be associated with substantial arginase expression and activity. The results in this study exemplify the complexity of the role of iNOS in asthma and the preservation of its potential as a therapeutic a target.

Intratunical Injection of Genetically Modified Adipose Tissue‐Derived Stem Cells with Human Interferon α‐2b for Treatment of Erectile Dysfunction in a Rat Model of Tunica Albugineal Fibrosis

INTRODUCTION Peyronies disease (PD) has frequently been associated with erectile dysfunction (ED) and may further compromise coitus. AIM To investigate the efficacy of intratunical injection of genetically modified rat adipose tissue-derived stem cells (ADSCs) expressing human interferon α-2b (ADSCs-IFN) in decreasing fibrosis and restoring erectile function in a rat model of tunica albugineal fibrosis (TAF). METHODS A total of 36 Sprague-Dawley rats (12 weeks old; 300-350 g) were randomly divided in six equal groups: (i) sham group (50 μL saline-injected into the tunica albuginea [TA]); (ii) TAF group (transforming growth factor [TGF]-β1 [0.5 μg/50 μL] injected into the TA); (iii) TGF-β1 plus 5 × 10(5) control ADSCs injected same day; (iv) TGF-β1 plus 5 × 10(5) ADSCs-IFN injected same day; (v) TGF-β1 plus 5 × 10(5) control ADSCs injected after 30 days; and (vi) TGF-β1 plus 5 × 10(5) ADSCs-IFN injected after 30 days. Rat allogeneic ADSCs were harvested from inguinal fat tissue. MAIN OUTCOME MEASURES Forty-five days following the TGF-β1 injection, erectile function was assessed, and penile tissues were harvested for further evaluations. RESULTS In the same-day injection groups, intratunical injection of ADSCs and ADSC-IFN improved erectile response observed upon stimulation of cavernous nerve compared with TAF group. Intratunical ADSC-IFN injection at day 30 improved erectile responses 3.1, 1.8, and 1.3 fold at voltages of 2.5, 5.0, and 7.0, respectively, when compared with TAF group. Furthermore, at voltages of 2.5 and 5.0, treatment on day 30 with ADSCs-IFN improved erectile responses 1.6- and 1.3-fold over treatment with ADSCs alone. Local injection of ADSCs or ADSCs-IFN reduced Peyronies-like manifestations, and these effects might be associated with a decrease in the expression of tissue inhibitors of metalloproteinases. CONCLUSION This study documents that transplantation of genetically modified ADSCs, with or without human IFN α-2b, attenuated Peyronies-like changes and enhanced erectile function in a rat model of TAF.

Meeting report: Discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium

Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.

Synergistic inhibition of thyroid cancer by suppressing MAPK/PI3K/AKT pathways.

BACKGROUND Although a wide spectrum of inhibitors of the MEK/ERK and PI3K/AKT pathways have been discovered and entered clinical trials, the effects of their individual use in thyroid cancer were often disappointing. We hypothesized that dual targeting of these two pathways would be a safe and effective strategy against aggressive thyroid cancers. METHODS We examined the antiproliferative effects of the MEK/ERK inhibitor AZD6244 and the PI3K/AKT inhibitor GDC0941, individually or in combination, on thyroid cancer cells harboring both the BRAF(V600E) and PIK3CA mutations. The effects of drug exposure on both total and phosphorylated (p-) forms of AKT and ERK were monitored by Western blotting analysis. Effects of these inhibitors on cell-cycle progression and apoptosis were measured by flow cytometry and DNA-fragmentation analyses, respectively. RESULTS We observed significant toxicities to viability of cells with low concentrations of AZD6244 or GDC0941, which were synergistic when the two inhibitors were used in combination (P < 0.01). AZD6244 abrogated p-ERK and GDC0941 abrogated p-AKT levels, confirming their expected target effects. Unexpectedly, monotherapy with AZD6244 resulted in activation of the PI3K signaling pathway in some cancer cell lines and co-exposure to AZD6244 and GDC0941 was necessary to suppress both pathways. Flow cytometry showed G1 arrest. DNA fragmentation analysis showed an increased apoptosis of cells dually treated with the two inhibitors. CONCLUSION Concomitant suppression of MEK/ERK and PI3K/AKT pathways by AZD6244 and GDC0941 abrogates compensatory mechanisms of tumor survival and causes synergistic cytotoxicity in thyroid cancer cells.

Subverting ER-Stress towards Apoptosis by Nelfinavir and Curcumin Coexposure Augments Docetaxel Efficacy in Castration Resistant Prostate Cancer Cells

Despite its side-effects, docetaxel (DTX) remains a first-line treatment against castration resistant prostate cancer (CRPC). Therefore, strategies to increase its anti-tumor efficacy and decrease its side effects are critically needed. Targeting of the constitutive endoplasmic reticulum (ER) stress in cancer cells is being investigated as a chemosensitization approach. We hypothesized that the simultaneous induction of ER-stress and suppression of PI3K/AKT survival pathway will be a more effective approach. In a CRPC cell line, C4-2B, we observed significant (p<0.005) enhancement of DTX-induced cytotoxicity following coexposure to thapsigargin and an AKT-inhibitor. However, since these two agents are not clinically approved, we investigated whether a combination of nelfinavir (NFR) and curcumin (CUR), known to target both these metabolic pathways, can similarly increase DTX cytotoxicity in CRPC cells. Within 24 hrs post-exposure to physiologic concentrations of NFR (5 µM) and CUR (5 µM) a significantly (p<0.005) enhanced cytotoxicity was evident with low concentration of DTX (10 nM). This 3-drug combination rapidly increased apoptosis in aggressive C4-2B cells, but not in RWPE-1 cells or in primary prostate epithelial cells (PrEC). Comparative molecular studies revealed that this 3-drug combination caused a more pronounced suppression of phosphorylated-AKT and higher induction in phosphorylated-eIF2α in C4-2B cells, as compared to RWPE-1 cells. Acute exposure (3–9 hrs) to this 3-drug combination intensified ER-stress induced pro-apoptotic markers, i.e. ATF4, CHOP, and TRIB3. At much lower concentrations, chronic (3 wks) exposures to these three agents drastically reduced colony forming units (CFU) by C4-2B cells. In vivo studies using mice containing C4-2B tumor xenografts showed significant (p<0.05) enhancement of DTX’s (10 mg/kg) anti-tumor efficacy following coexposure to NFR (20 mg/kg) & CUR (100 mg/kg). Immunohistochemical (IHC) analyses of tumor sections indicated decreased Ki-67 staining and increased TUNEL intensity in mice exposed to the 3-drug combination. Therefore, subverting ER-stress towards apoptosis using adjuvant therapy with NFR and CUR can chemosensitize the CRPC cells to DTX therapy.

Biochemical characterization of riboflavin carrier protein (RCP) in prostate cancer

Riboflavin carrier protein (RCP) is a growth- and development-specific protein. Here, we characterized the expression of this protein in prostate cancer by polyclonal and monoclonal antibodies against chicken RCP. RCP was localized to both androgen-dependent and independent prostate cancer cell lines. Compared to controls, RCP was over-expressed in all 45 prostate adenocarcinomas, irrespective of the Gleasons score or the stage of the disease. The identified RCP had a molecular weight of 38 kDa, similar to RCP purified from chicken. Presence of this protein was also confirmed by siRNA inhibition analysis. Antibodies to chicken RCP inhibited incorporation of tritiated thymidine into DNA and prevented riboflavin uptake in PC3 prostate cancer cells, suggesting a critical function of this protein in prostate cancer cell growth. These data suggest that RCP can be used as a tumor biomarker in prostate cancer.

Dysregulation of miR-212 Promotes Castration Resistance through hnRNPH1-Mediated Regulation of AR and AR-V7: Implications for Racial Disparity of Prostate Cancer

Purpose: The causes of disproportionate incidence and mortality of prostate cancer among African Americans (AA) remain elusive. The purpose of this study was to investigate the mechanistic role and assess clinical utility of the splicing factor heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) in prostate cancer progression among AA men. Experimental Design: We employed an unbiased functional genomics approach coupled with suppressive subtractive hybridization (SSH) and custom cDNA microarrays to identify differentially expressed genes in microdissected tumors procured from age- and tumor grade–matched AA and Caucasian American (CA) men. Validation analysis was performed in independent cohorts and tissue microarrays. The underlying mechanisms of hnRNPH1 regulation and its impact on androgen receptor (AR) expression and tumor progression were explored. Results: Aberrant coexpression of AR and hnRNPH1 and downregulation of miR-212 were detected in prostate tumors and correlate with disease progression in AA men compared with CA men. Ectopic expression of miR-212 mimics downregulated hnRNPH1 transcripts, which in turn reduced expression of AR and its splice variant AR-V7 (or AR3) in prostate cancer cells. hnRNPH1 physically interacts with AR and steroid receptor coactivator-3 (SRC-3) and primes activation of androgen-regulated genes in a ligand-dependent and independent manner. siRNA silencing of hnRNPH1 sensitized prostate cancer cells to bicalutamide and inhibited prostate tumorigenesis in vivo. Conclusions: Our findings define novel roles for hnRNPH1 as a putative oncogene, splicing factor, and an auxiliary AR coregulator. Targeted disruption of the hnRNPH1-AR axis may have therapeutic implications to improve clinical outcomes in patients with advanced prostate cancer, especially among AA men. Clin Cancer Res; 22(7); 1744–56. ©2015 AACR.