Tsion Zewdu Minas

Radiation Induces Diffusible Feeder Cell Factor(s) That Cooperate with ROCK Inhibitor to Conditionally Reprogram and Immortalize Epithelial Cells

Both feeder cells and Rho kinase inhibition are required for the conditional reprogramming and immortalization of human epithelial cells. In the present study, we demonstrated that the Rho kinase inhibitor Y-27632, significantly suppresses keratinocyte differentiation and extends life span in serum-containing medium but does not lead to immortalization in the absence of feeder cells. Using Transwell culture plates, we further demonstrated that physical contact between the feeder cells and keratinocytes is not required for inducing immortalization and, more importantly, that irradiation of the feeder cells is required for this induction. Consistent with these experiments, conditioned medium was shown to induce and maintain conditionally immortalized cells, which was accompanied by increased telomerase expression. The activity of conditioned medium directly correlated with radiation-induced apoptosis of the feeder cells. Thus, the induction of conditionally reprogrammed cells is mediated by a combination of Y-27632 and a diffusible factor (or factors) released by apoptotic feeder cells.

Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model

Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES.

YK-4-279 effectively antagonizes EWS-FLI1 induced leukemia in a transgenic mouse model

Ewing sarcoma is an aggressive tumor of bone and soft tissue affecting predominantly children and young adults. Tumor-specific chromosomal translocations create EWS-FLI1 and similar aberrant ETS fusion proteins that drive sarcoma development in patients. ETS family fusion proteins and over-expressed ETS proteins are also found in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Transgenic expression of EWS-FLI1 in mice promotes high penetrance erythroid leukemia with dense hepatic and splenic infiltrations. We identified a small molecule, YK-4-279, that directly binds to EWS-FLI1 and inhibits its oncogenic activity in Ewing sarcoma cell lines and xenograft mouse models. Herein, we tested in vivo therapeutic efficacy and potential side effects of YK-4-279 in the transgenic mouse model with EWS-FLI1 induced leukemia. A two-week course of treatment with YK-4-279 significantly reduced white blood cell count, nucleated erythroblasts in the peripheral blood, splenomegaly, and hepatomegaly of erythroleukemic mice. YK-4-279 inhibited EWS-FLI1 target gene expression in neoplastic cells. Treated animals showed significantly better overall survival compared to control mice that rapidly succumbed to leukemia. YK-4-279 treated mice did not show overt toxicity in liver, spleen, or bone marrow. In conclusion, this in vivo study highlights the efficacy of YK-4-279 to treat EWS-FLI1 expressing neoplasms and support its therapeutic potential for patients with Ewing sarcoma and other ETS-driven malignancies.

Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level.

ABSTRACT Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane.

Identification of novel ezrin inhibitors targeting metastatic osteosarcoma by screening open access malaria box

Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins and functions as a linker between the plasma membrane and the actin cytoskeleton. Ezrin is a key driver of tumor progression and metastatic spread of osteosarcoma. We discovered a quinoline-based small molecule, NSC305787, that directly binds to ezrin and inhibits its functions in promoting invasive phenotype. NSC305787 possesses a very close structural similarity to commonly used quinoline-containing antimalarial drugs. On the basis of this similarity and of recent findings that ezrin has a likely role in the pathogenesis of malaria infection, we screened antimalarial compounds in an attempt to identify novel ezrin inhibitors with better efficacy and drug properties. Screening of Medicines for Malaria Venture (MMV) Malaria Box compounds for their ability to bind to recombinant ezrin protein yielded 12 primary hits with high selective binding activity. The specificity of the hits on ezrin function was confirmed by inhibition of the ezrin-mediated cell motility of osteosarcoma cells. Compounds were further tested for phenocopying the morphologic defects associated with ezrin suppression in zebrafish embryos as well as for inhibiting the lung metastasis of high ezrin-expressing osteosarcoma cells. The compound MMV667492 exhibited potent anti-ezrin activity in all biologic assays and had better physicochemical properties for drug-likeness than NSC305787. The drug-like compounds MMV020549 and MMV666069 also showed promising activities in functional assays. Thus, our study suggests further evaluation of antimalarial compounds as a novel class of antimetastatic agents for the treatment of metastatic osteosarcoma. Mol Cancer Ther; 14(11); 2497–507. ©2015 AACR.

Ezrin Inhibition Up-regulates Stress Response Gene Expression

Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes.

Ezrin Enhances EGFR Signaling and Modulates Erlotinib Sensitivity in Non-Small Cell Lung Cancer Cells.

Ezrin is a scaffolding protein that is involved in oncogenesis by linking cytoskeletal and membrane proteins. Ezrin interacts with epidermal growth factor receptor (EGFR) in the cell membrane, but little is known about the effects of this interaction on EGFR signaling pathway. In this study, we established the biological and functional significance of ezrin-EGFR interaction in non–small cell lung cancer (NSCLC) cells. Endogenous ezrin and EGRF interaction was confirmed by co-immunoprecipitation and immunofluorescent staining. When expression of ezrin was inhibited, EGFR activity and phosphorylation levels of downstream signaling pathway proteins ERK and STAT3 were decreased. Cell fractionation experiments revealed that nuclear EGFR was significantly diminished in ezrin-knockdown cells. Consequently, mRNA levels of EGFR target genes AURKA, COX-2, cyclin D1, and iNOS were decreased in ezrin-depleted cells. A small molecule inhibitor of ezrin, NSC305787, reduced EGF-induced phosphorylation of EGFR and downstream target proteins, EGFR nuclear translocation, and mRNA levels of nuclear EGFR target genes similar to ezrin suppression. NSC305787 showed synergism with erlotinib in wild-type EGFR-expressing NSCLC cells, whereas no synergy was observed in EGFR-null cells. Phosphorylation of ezrin on Y146 was found as an enhancer of ezrin-EGFR interaction and required for increased proliferation, colony formation, and drug resistance to erlotinib. These findings suggest that ezrin-EGFR interaction augments oncogenic functions of EGFR and that targeting ezrin may provide a potential novel approach to overcome erlotinib resistance in NSCLC cells.

Analysis of Tumor Biology to Advance Cancer Health Disparity Research

Cancer mortality rates in the United States continue to decline. Reductions in tobacco use, uptake of preventive measures, adoption of early detection methods, and better treatments have resulted in improved cancer outcomes for men and women. Despite this progress, some population groups continue to experience an excessive cancer burden when compared with other population groups. One of the most prominent cancer health disparities exists in prostate cancer. Prostate cancer mortality rates are highest among men of African ancestry when compared with other men, both in the United States and globally. This disparity and other cancer health disparities are largely explained by differences in access to health care, diet, lifestyle, cultural barriers, and disparate exposures to carcinogens and pathogens. Dietary and lifestyle factors, pathogens, and ancestry-related factors can modify tumor biology and induce a more aggressive disease. There are numerous examples of how environmental exposures, like tobacco, chronic stress, or dietary factors, induce an adverse tumor biology, leading to a more aggressive disease and decreased patient survival. Because of population differences in the exposure to these risk factors, they can be the cause of cancer disparities. In this review, we will summarize recent advances in our understanding of prostate and breast cancer disparities in the United States and discuss how the analysis of tumor biology can advance health disparity research.

Abstract 2446: Ezrin inhibition up-regulates stress response gene expression and blocks osteosarcoma metastasis

Ezrin is a member of the ezrin, radixin, moesin (ERM) protein family of membrane-cytoskeleton linkers. Ezrin has been implicated in many essential cellular functions including cell adhesion, motility, maintenance and determination of cell shape, cell proliferation and apoptosis, regulation of ion channels, morphogenesis and signal transduction. Ezrin promotes invasive and migratory capabilities of cancer cells. A high level of ezrin expression is associated with poor clinical outcome and metastatic behavior of pediatric solid tumors including osteosarcoma and rhabdomyosarcoma as well as multiple other tumor types. Ezrin, therefore, could be a promising molecular target for the prevention and treatment of cancer metastasis. We previously discovered two small molecule inhibitors, NSC305787 and NSC668394, which bind directly to ezrin and inhibit its activity in mediating the invasive phenotype of osteosarcoma cells in multiple in vitro and in vivo assays. In this study, we expand on our previous findings by demonstrating that NSC305787-treatment but not NSC668394 significantly reduces pulmonary metastasis in a genetically engineered mouse model of osteosarcoma. We assessed the pharmacokinetics of compounds in mice and demonstrated that NSC305787 has a more favorable pharmacokinetic profile compared with NSC668394. In order to uncover ezrin-mediated biological pathways that can be used for a specific pharmacodynamic marker(s) of response to ezrin inhibition, we profiled global gene expression in osteosarcoma cells after treatment with inhibitors. We identified several commonly up-regulated genes with functional relevance to integrated stress response, implicating that a common underlying mechanism may be shared by these compounds. We further validated the microarray data through extensive testing using real-time qPCR and verified the specificity of the transcriptional response using another novel ezrin inhibitor MMV667492 that we have identified recently from the MMV400 “Malaria Box” library. The effect of ezrin inhibitors on the expression of stress genes was recapitulated by siRNA-mediated depletion of ezrin. The up-regulation of stress genes was much weaker in cells with reduced ezrin levels compared to wild-type cells, indicating the specificity of the compounds on ezrin-mediated cellular responses. Analysis of the expression of stress genes in white blood cells and skin of NSC305787-treated mice demonstrated up-regulation of the DDIT4/REDD1, suggesting that DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of response to ezrin inhibition. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive, may have important functions regulating gene expression and inhibition of ezrin activity by NSC305787 in osteosarcoma could be an attractive therapy to prevent clinically significant metastasis. Citation Format: Haydar Celik, Gulay Bulut, Jenny Han, Garrett T. Graham, Tsion Z. Minas, Erin J. Conn, Sung-Hyeok Hong, Gary T. Pauly, Mutlu Hayran, Xin Li, Metin Ozdemirli, Ayse Ayhan, Michelle A. Rudek, Jeffrey A. Toretsky, Aykut Uren. Ezrin inhibition up-regulates stress response gene expression and blocks osteosarcoma metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2446.

MP66-15 INHIBITION OF ERG ACTIVITY IN PATIENT DERIVED PROSTATE CANCER XENOGRAFTS USING THE SMALL MOLECULE INHIBITOR YK-4-279

RESULTS: Previously, cFLIP was identified as an androgenresponsive gene. However, we show that cFLIP expression is increased in androgen-independent CaP cells. We provide evidence that (i) cFLIP-expressing CRPC cells exhibit higher proliferative index than cFLIP-deficient counterparts and (ii) cFLIP expression is high in prostatic tissues of castrated (ARR2.IkB-Myc) transgenic mice. The significance of cFLIP as an upstream of AR could be ascertained from the finding that suppressing cFLIP could decrease PSA levels and transcriptional activities of ARFL and ARv7 in CRPC cells. These data prompted to further investigate the correlation between Cflip, ARFL and ARv7. We show that cFLIP forms a complex with ARv-7 and ARFL in CRPC cells. We observed higher levels of cFLIP/ARv7 complex in nuclei than in cytoplasm suggesting the possibility of cFLIP as a protein-carrier. Using transfections with cFLIP-Long, cFLIP-short-variant and mutants (cFLIPL435mt/472mt/439mt), two sequences on C-terminus were identified for nuclear translocation that confirmed its protein-transporter property. These data were validated in PC3 cells (in which ARv7 was ectopically expressed). Next, we observed that b-catenin is required for complex formation and subsequent nuclear translocation of cFLIP/ARv7 in cells. Finally, using a xenograft mouse model, we established the significance of cFLIP/ARv7 complex as a therapeutic target and show that simultaneous targeting (by using nanoparticle-loaded cFLIP-siRNA þ ARv7siRNA) significantly inhibits CRPC-type tumor growth and improves the outcome of docetaxel therapy. We also identified a novel inhibitor of cFLIP/ARv. CONCLUSIONS: We identified a novel therapeutic approach of treating CRPC.