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Background: Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer deaths in the US due to lack of effective targeted therapeutics and extremely poor prognosis. Objective: To investigate the role of miR-193b and related signaling mechanism in PDAC cell proliferation, invasion and tumor growth. Methods: Using PDAC cell lines we performed cell viability, colony formation, in vitro wound healing and matrigel invasion assays following transfection with miR-193b mimic or control-miR. To identify potential downstream targets of miR-193b we utilized miRNA-target prediction algorithms and investigate regulation of eukaryotic elongation factor-2 kinase (eEF2K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways and mediators of epithelial mesenchymal transition (EMT) The role of miR-193b in PDAC tumorigenesis was evaluated in in vivo tumor growth of Panc-1 xenograft model in nude mice. Results: We found that miR-193b is under expressed in PDAC cells compared to corresponding normal pancreatic epithelial cells and demonstrated that ectopic expression of miR-193b reduced cell proliferation, migration and invasion and EMT through downregulation of eEF2K signaling in PDAC cells. miR-193b expression led to increased expression of E-Cadherin and Claudin-1, while decreasing Snail and TCF8/ZEB1 expressions via eEF2K and MAPK/ERK axis. In vivo systemic injection of miR-193b using lipid-nanoparticles twice a week reduced tumor growth of Panc-1 xenografts and eEF2K expression in nude mice. Conclusions: Our findings suggest that miR-193b expression suppresses PDAC cell proliferation, migration, invasion and EMT through inhibition of eEF2K/MAPK-ERK oncogenic axis and that miR-193b-based RNA therapy might be an effective therapeutic strategy to control growth of PDAC.

Supplementary Table 1. Composition of the basal diet given to the study hens. Supplementary Table 2. Incidence of serous and mucinous adenocarcinoma in the study animals. Supplementary Table 3. RAS gene family mutation analysis. Supplementary Figure 1. Detection of curcumin in serum of the animals fed with curcumin by HPLC.

We investigated the effect of daily dietary curcumin intake on the development and progression of spontaneous ovarian cancer in a galline (hen) model, as the chicken is the only nonhuman animal in which ovarian cancer spontaneously develops with a high prevalence. At the end of 12 months, ovarian cancer had spontaneously developed in 39% (35/90) of control hens not fed curcumin (n = 90). In comparison, it spontaneously developed in 27% (24/90) and 17% (15/90) of hens given curcumin at 25.8 (n = 90) and 53.0 mg/day (n = 90), respectively (P = 0.004). This represented significant dose-dependent reductions in overall ovarian cancer incidence in the 25.8 and 53.0 mg/day curcumin-fed groups (31% and 57%, respectively). Daily curcumin intake also reduced ovarian tumor sizes (P = 0.04) and number of tumors (P = 0.006). Evaluation of the molecular mechanisms underlying the chemopreventive and antitumor effects of curcumin revealed that NF-κB and STAT3 signaling pathways were significantly inhibited but that the nuclear factor erythroid 2/heme oxygenase 1 antioxidant pathway was induced by curcumin intake in a dose-dependent manner in ovarian tissues (P < 0.05). Sequencing of the Ras family genes (KRAS, NRAS, and HRAS) revealed less frequent KRAS and HRAS mutations in ovarian tumors in the curcumin-fed animals. In conclusion, our results demonstrated for the first time that daily curcumin intake leads to a significant and dose-dependent reduction in spontaneous ovarian cancer incidence and tumor growth, indicating a tremendous role for curcumin as a chemopreventive strategy for ovarian cancer. Cancer Prev Res; 11(1); 59–67. ©2017 AACR.

Blood biochemistry of mice treated with liposomal miR-34a or control mimic.

Supplementary Figure Legends (Figures 1-6)

In vivo nanoliposomal miR-34a treatment is safe and effectively suppresses eEF2K and FOXM1 protein expression in TNBC tumors.

Purpose: Zoledronic acid is being increasingly recognized for its antitumor properties, but the underlying functions are not well understood. In this study, we hypothesized that zoledronic acid inhibits ovarian cancer angiogenesis preventing Rac1 activation.Experimental Design: The biologic effects of zoledronic acid were examined using a series of in vitro [cell invasion, cytokine production, Rac1 activation, reverse-phase protein array, and in vivo (orthotopic mouse models)] experiments.Results: There was significant inhibition of ovarian cancer (HeyA8-MDR and OVCAR-5) cell invasion as well as reduced production of proangiogenic cytokines in response to zoledronic acid treatment. Furthermore, zoledronic acid inactivated Rac1 and decreased the levels of Pak1/p38/matrix metalloproteinase-2 in ovarian cancer cells. In vivo, zoledronic acid reduced tumor growth, angiogenesis, and cell proliferation and inactivated Rac1 in both HeyA8-MDR and OVCAR-5 models. These in vivo antitumor effects were enhanced in both models when zoledronic acid was combined with nab-paclitaxel.Conclusions: Zoledronic acid has robust antitumor and antiangiogenic activity and merits further clinical development as ovarian cancer treatment. Clin Cancer Res; 21(9); 2127–37. ©2015 AACR.

Purpose: Recent studies indicated that dysregulation of noncoding RNAs (ncRNA) such as miRNAs is involved in pathogenesis of various human cancers. However, the molecular mechanisms underlying miR-34a are not fully understood in triple-negative breast cancer (TNBC).Experimental Design: We performed in vitro functional assays on TNBC cell lines to investigate the role of miR-34a in FOXM1/eEF2K signaling axis. TNBC tumor xenograft models were used for in vivo therapeutic delivery of miR-34a.Results: In this study, we investigated the role of p53-driven ncRNA miR-34a and found that miR-34a is associated with significantly longer patient survival in TNBC and inversely correlated with levels of proto-oncogenic eEF2K, which was associated with significantly shorter overall patient survival. We showed that miR-34a directly binds to the 3′-untranslated region of eEF2K and FOXM1 mRNAs and suppresses their expression, leading to inhibition of TNBC cell proliferation, motility, and invasion. Notably, restoring miR-34a expression recapitulated the effects of inhibition of eEF2K and FOXM1, the transcription factor for eEF2K and the direct target of p53, in TNBC cell lines, whereas overexpression of eEF2K and FOXM1 rescued the effects and signaling pathways mediated by miR-34a. Moreover, in vivo therapeutic delivery of miR-34a nanoparticles by systemic intravenous administration delayed tumor growth of two different orthotopic TNBC tumor xenograft models by inhibiting eEF2K and FOXM1, intratumoral proliferation and angiogenesis, and inducing apoptosis.Conclusions: Overall, our findings provide new insights into the tumor suppressor role of miR-34a by dual-targeting of FOXM1/eEF2K signaling axis and suggest that miR-34a–based gene therapy may be a potential therapeutic strategy in TNBC. Clin Cancer Res; 24(17); 4225–41. ©2018 AACR.

Supplementary Figures 1-6. Supplementary Figure S1: In vivo and in vitro effects of ZA on apoptosis. Supplementary Figure S2: Nab-paclitaxel inhibits tube formation.Supplementary Figure S3: RPPA analysis in HeyA8 MDR treated with ZA. Supplementary Figure S4: Functional changes induced by ZA in HeyA8 MDR. Supplementary Figure S5: ZA decreases angiogenic factors. Supplementary Figure S6: ZA inhibits cell invasion

Supplementary Materials and Methods

One-carbon (1C) metabolism has a key role in metabolic programming with both mitochondrial (m1C) and cytoplasmic (c1C) components. Here we show that activating transcription factor 4 (ATF4) exclusively activates gene expression involved in m1C, but not the c1C cycle in prostate cancer cells. This includes activation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) expression, the central player in the m1C cycle. Consistent with the key role of m1C cycle in prostate cancer, MTHFD2 knockdown inhibited prostate cancer cell growth, prostatosphere formation, and growth of patient-derived xenograft organoids. In addition, therapeutic silencing of MTHFD2 by systemically administered nanoliposomal siRNA profoundly inhibited tumor growth in preclinical prostate cancer mouse models. Consistently, MTHFD2 expression is significantly increased in human prostate cancer, and a gene expression signature based on the m1C cycle has significant prognostic value. Furthermore, MTHFD2 expression is coordinately regulated by ATF4 and the oncoprotein c-MYC, which has been implicated in prostate cancer. These data suggest that the m1C cycle is essential for prostate cancer progression and may serve as a novel biomarker and therapeutic target.Significance:These findings demonstrate that the mitochondrial, but not cytoplasmic, one-carbon cycle has a key role in prostate cancer cell growth and survival and may serve as a biomarker and/or therapeutic target.

Eukaryotic elongation factor 2 kinase (eEF-2K) is an unusual alpha kinase involved in protein synthesis through phosphorylation of elongation factor 2 (EF2). eEF-2K is highly overexpressed in breast cancer, and its activity is associated with significantly shortened patient survival and proven to be a potential molecular target in breast cancer. The crystal structure of eEF-2K remains unknown, and there is no potent, safe, and effective inhibitor available for clinical applications. We designed and synthesized several generations of potential inhibitors. The effect of the inhibitors at the binding pocket of eEF-2K was analyzed after developing a 3D target model by using a domain of another ?-kinase called myosin heavy-chain kinase A (MHCKA) that closely resembles eEF-2K. In silico studies showed that compounds with a coumarin-chalcone core have high predicted binding affinities for eEF-2K. Using in vitro studies in highly aggressive and invasive (MDA-MB-436, MDA-MB-231, and BT20) and noninvazive (MCF-7) breast cancer cells, we identified a lead compound that was highly effective in inhibiting eEF-2K activity at submicromolar concentrations and at inhibiting cell proliferation by induction of apoptosis with no toxicity in normal breast epithelial cells. In vivo systemic administration of the lead compound encapsulated in single lipid-based liposomal nanoparticles twice a week significantly suppressed growth of MDA-MB-231 tumors in orthotopic breast cancer models in nude mice with no observed toxicity. In conclusion, our study provides a highly potent and in vivo effective novel small-molecule eEF-2K inhibitor that may be used as a molecularly targeted therapy breast cancer or other eEF-2K-dependent tumors. © 2021 American Chemical Society. 1R01CA244344; University of Texas MD Anderson Cancer Center; Türkiye Bilimsel ve Teknolojik Araştirma Kurumu, TÜBITAK: 215S008, TUBITAK-BIDEB 2214A This study was funded by The Scientific and Technological Research Council of Turkey (TUBITAK) (grant number 215S008 and TUBITAK-BIDEB 2214A program, F.C.O.) and The University of Texas-MD Anderson Cancer Center Bridge fund (B.O. and N.K.) and NIH-NCI 1R01CA244344 grants (B.O. and N.K.).

Breast cancer (BC) is the most commonly diagnosed cancer in women and the second leading cause of cancer related deaths in the US. About 70-80% of patients have ER+ BC and initially respond to anti-estrogen (ie, Tamoxifen) or endocrine/homonal treatments. However, about 60% of the patients inevitably develops resistance to anti-estrogen therapies (due to intrinsic (15-20%) and acquire resistance (30-40%), representing a major clinical problem, resulting in relapses and metastasis, leading to significant patient mortality. Therefore, understanding the underlying mechanisms causing resitance to hormonal or anti-estrogen treatments is needed the for developing highly effective threapies and improving patient survival. Cell surface proteases, including serine proteases, are proteolytic modifiers of particular targets, including growth factors and protease-activated receptors, which are critical for the activation of oncogenic signaling pathways. TMPRSS2, a member of type II transmembrane serine protease (TTSP) family, has been shown to be highly expressed in prostate cancer cells and its role in carcinogenesis. We found that TMPRSS2 is highly upregulated well established tamoxifene resistant cell lines derivative of MCF7 (LCC2 and LCC9) compared to MCF7 cells. Performing Kaplan-Meier survival analyses in TCGA-BC database we also found that TMPRSS2 expression is associated with poor prognosis and significantly shorter patient survival in ER+ BC patients (p=0.015, n=221 patients). In addition, we found that TMPRSS2 is regulated by Estrogen/Estrogen Receptor-alpha (ERα) signaling as in vitro inhibition of estrogen/ERα signaling by ERα siRNA, tamoxifen or faslodex reduced TMPRSS2 protein expression ER+ MCF7 BC cells. Moreover, we found that inhibition of TMPRSS2 by 3 different siRNA molecules suppressed cell proliferation of MCF7 cells and reduced expression critical proteins for cell cycle and proliferation, including cyclin D1, c-Myc and pERK/MAPK signaling, suggesting that TMPRSS2 promotes the oncogenic signaling pathways in ER+ BC cells. Currently, we are targeting TMPRSS2 in in vitro and in vivo Tamox-resistant LCC2 and LCC9 models in mice by siRNA alone and in combination with Tamoxifen or faslodex to demostrate inhibition of TMPRSS2 sensitize tumors to anti-estrogen treatments. In conclusion, TMPRSS2 is a potential biomarker for poor patient survival and anti-estrogen resistance, and targeting TMPRSS2 alone as a monothreapy and in combination with anti- Tamoxifen or faslodex may be a promising approach for overcoming resistance to estrogen thereapies in ER+ BC patients. Citation Format: Rumeysa Ozyurt, Nermin Kahraman, Pinar Atalay Dundar, Bulent Ozpolat. TMPRSS2 serin protease is a novel biomarker for ER+ breast cancer patient prognosis and survival and mediates resistance to anti-estrogen treatment in ER+ breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3890.

Deregulation of noncoding RNAs, including microRNAs (miRs), is implicated in the pathogenesis of many human cancers, including breast cancer. Through extensive analysis of The Cancer Genome Atlas, we found that expression of miR-22-3p is markedly lower in triple-negative breast cancer (TNBC) than in normal breast tissue. The restoration of miR-22-3p expression led to significant inhibition of TNBC cell proliferation, colony formation, migration, and invasion. We demonstrated that miR-22-3p reduces eukaryotic elongation factor 2 kinase (eEF2K) expression by directly binding to the 3′ untranslated region of eEF2K mRNA. Inhibition of EF2K expression recapitulated the effects of miR-22-3p on TNBC cell proliferation, motility, invasion, and suppression of phosphatidylinositol 3-kinase/Akt and Src signaling. Systemic administration of miR-22-3p in single-lipid nanoparticles significantly suppressed tumor growth in orthotopic MDA-MB-231 and MDA-MB-436 TNBC models. Evaluation of the tumor response, following miR-22-3p therapy in these models using a novel mathematical model factoring in various in vivo parameters, demonstrated that the therapy is highly effective against TNBC. These findings suggest that miR-22-3p functions as a tumor suppressor by targeting clinically significant oncogenic pathways and that miR-22-3p loss contributes to TNBC growth and progression. The restoration of miR-22-3p expression is a potential novel noncoding RNA-based therapy for TNBC., Graphical Abstract, Our study demonstrates that microRNA-22-3p (miR-22-3p) acts as a tumor suppressor in which its expression is markedly reduced in TNBC tumors. Restoration of its expression by miR-22-3p nanotherapy suppress tumor growth and progression by directly inhibiting of eEF2K-PI3K/AKT and Src signaling. miR-22-3p-based RNA therapy may be novel treatment for TNBC.

Background The TMPRSS2/ERG (TE) fusion gene is present in half of the prostate cancers (PCas). The TMPRSS2 and ERG junction of the fusion messenger RNA (mRNA) constitutes a cancer-specific target. Although docetaxel-based chemotherapy is the second line of therapy following development resistance to androgen ablation therapies, it is not curative. Therefore, the development of nontoxic novel monotherapies for targeting TE mRNA in PCa patients and for increasing the clinical efficacy of docetaxel treatment are needed. Methods We evaluated multiple approaches to enhance the delivery of TE small interfering RNA (siRNA) containing liposomes including PEGylation, topical treatment with nitroglycerin (NG) to increase permeability and retention, and three different PEG modifications: folate, RGD cyclic peptide, and a bFGF fibroblast growth factor receptor-targeting peptide. The efficacy of the optimized TE siRNA liposome in combination with docetaxel was then evaluated in vivo with or without topical NG in vivo using a VCaP xenograft model. TE fusion protein knockdown in residual tumors was assessed using Western blotting and immunohistochemistry. Results In vivo therapeutic targeting of TE fusion gene by systemic delivery of RGD-peptide-coated liposomal siRNA nanovectors led to sustained target silencing, suppressed tumor growth in xenograft models and enhanced the efficacy of docetaxel chemotherapy. Simultaneous application of the vasodilator NG to the skin further increased tissue the delivery of siRNA and enhanced target knockdown. Conclusion TE-targeted gene silencing therapy using liposomal nanovectors is a potential therapeutic strategy as a monotherapy and to enhance the efficacy of chemotherapy in patients with advanced PCa.

PubMed ID: 31432749 Aim: To investigate the role of EF2K in BRCA1-mutated breast cancer. Materials & methods: We developed silica coated cobalt-ferrite (CoFe) nanoparticles for in vivo delivery of small interfering RNAs (siRNAs) into BRCA1-mutated breast cancer. Results: Expression of EF2K is highly upregulated in the majority (78.5%) of BRCA1-mutated patients and significantly associated with poor patient survival and metastasis. Silencing of EF2K reduced cell proliferation, migration and invasion of the cancer cells. In vivo therapeutic targeting of EF2K by CoFe-siRNA-nanoparticles leads to sustained EF2K gene knockdown and suppressed tumor growth in orthotopic xenograft models of BRCA1-mutated breast cancer. Conclusion: EF2K is a potential novel molecular target in BRCA1-mutated tumors and CoFe-based siRNA nanotherapy may be used as a novel approach to target EF2K. © 2019 © 2019 Future Medicine Ltd. Türkiye Bilimsel ve Teknolojik Araştirma Kurumu --This study has been supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under the 2214-A International Research Fellowship for Doctoral Students. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Background Circulating miRNAs are known to play important roles in intercellular communication. However, the effects of exosomal miRNAs on cells are not fully understood. Methods To investigate the role of exosomal miR-1246 in ovarian cancer (OC) microenvironment, we performed RPPA as well as many other in vitro functional assays in ovarian cancer cells (sensitive; HeyA8, Skov3ip1, A2780 and chemoresistant; HeyA8-MDR, Skov3-TR, A2780-CP20). Therapeutic effect of miR-1246 inhibitor treatment was tested in OC animal model. We showed the effect of OC exosomal miR-1246 uptake on macrophages by co-culture experiments. Findings Substantial expression of oncogenic miR-1246 OC exosomes was found. We showed that Cav1 gene, which is the direct target of miR-1246, is involved in the process of exosomal transfer. A significantly worse overall prognosis were found for OC patients with high miR-1246 and low Cav1 expression based on TCGA data. miR-1246 expression were significantly higher in paclitaxel-resistant OC exosomes than in their sensitive counterparts. Overexpression of Cav1 and anti-miR-1246 treatment significantly sensitized OC cells to paclitaxel. We showed that Cav1 and multi drug resistance (MDR) gene is involved in the process of exosomal transfer. Our proteomic approach also revealed that miR-1246 inhibits Cav1 and acts through PDGFβ receptor at the recipient cells to inhibit cell proliferation. miR-1246 inhibitor treatment in combination with chemotherapy led to reduced tumor burden in vivo. Finally, we demonstrated that when OC cells are co-cultured with macrophages, they are capable of transferring their oncogenic miR-1246 to M2-type macrophages, but not M0-type macrophages. Interpretation Our results suggest that cancer exosomes may contribute to oncogenesis by manipulating neighboring infiltrating immune cells. This study provide a new mechanistic therapeutic approach to overcome chemoresistance and tumor progression through exosomal miR-1246 in OC patients.

KRAS is one of the most frequently mutated proto-oncogenes in pancreatic ductal adenocarcinoma (PDAC) and aberrantly activated in triple-negative breast cancer (TNBC). A profound role of microRNAs (miRNAs) in the pathogenesis of human cancer is being uncovered, including in cancer therapy. Using in silico prediction algorithms, we identified miR-873 as a potential regulator of KRAS, and we investigated its role in PDAC and TNBC. We found that reduced miR-873 expression is associated with shorter patient survival in both cancers. miR-873 expression is significantly repressed in PDAC and TNBC cell lines and inversely correlated with KRAS levels. We demonstrate that miR-873 directly bound to the 3′ UTR of KRAS mRNA and suppressed its expression. Notably, restoring miR-873 expression induced apoptosis; recapitulated the effects of KRAS inhibition on cell proliferation, colony formation, and invasion; and suppressed the activity of ERK and PI3K/AKT, while overexpression of KRAS rescued the effects mediated by miR-873. Moreover, in vivo delivery of miR-873 nanoparticles inhibited KRAS expression and tumor growth in PDAC and TNBC tumor models. In conclusion, we provide the first evidence that miR-873 acts as a tumor suppressor by targeting KRAS and that miR-873-based gene therapy may be a therapeutic strategy in PDAC and TNBC. Keywords: KRAS, oncogene, non-coding RNA, microRNA, ncRNA, miR-873, proliferation, invasion, gene regulation, tumorigenesis, gene silencing, therapy, nanoparticles, pancreatic cancer, liposomes, breast cancer, triple-negative breast cancer

One-carbon (1C) metabolism has a key role in metabolic programming with both mitochondrial (m1C) and cytoplasmic (c1C) components. Here we show that activating transcription factor 4 (ATF4) exclusively activates gene expression involved in m1C, but not the c1C cycle in prostate cancer cells. This includes activation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) expression, the central player in the m1C cycle. Consistent with the key role of m1C cycle in prostate cancer, MTHFD2 knockdown inhibited prostate cancer cell growth, prostatosphere formation, and growth of patient-derived xenograft organoids. In addition, therapeutic silencing of MTHFD2 by systemically administered nanoliposomal siRNA profoundly inhibited tumor growth in preclinical prostate cancer mouse models. Consistently, MTHFD2 expression is significantly increased in human prostate cancer, and a gene expression signature based on the m1C cycle has significant prognostic value. Furthermore, MTHFD2 expression is coordinately regulated by ATF4 and the oncoprotein c-MYC, which has been implicated in prostate cancer. These data suggest that the m1C cycle is essential for prostate cancer progression and may serve as a novel biomarker and therapeutic target. Significance: These findings demonstrate that the mitochondrial, but not cytoplasmic, one-carbon cycle has a key role in prostate cancer cell growth and survival and may serve as a biomarker and/or therapeutic target.

Eukaryotic elongation factor 2 kinase (eEF2K) is an unusual alpha kinase whose expression is highly upregulated in various cancers and contributes to tumor growth, metastasis, and progression. More importantly, expression of eEF2K is associated with poor clinical outcome and shorter patient survival triple negative breast cancer (TNBC). Therefore, eEF2K is an emerging molecular target for development of novel targeted therapeutics and precision medicine in solid cancers. However, currently potent, and specific inhibitors of eEF2K are not available for clinical translation. In the current study, we investigated the effects of various newly designed and synthesized a series of compounds with coumarin scaffold substitutions in inhibiting eEF2K activity using in silico approaches and in vitro studies in TNBC cells. We utilized an amide substitution at 3-position on the coumarin ring with their pharmacologically active groups containing pyrrolidine, piperidine, morpholine and piperazine groups with –(CH2)2– bridged for aliphatic amides. To evaluate substituent effects on coumarin scaffold, boronic acid pinacol ester and boronic acids on phenyl rings were investigated using in silico and in vitro analyses. Due to their ability to form covalent binding to the target enzyme, we investigated the effects of boron containing groups on functionalized coumarin ring (3 compounds) and designed novel aliphatic and aromatic derivatives of coumarin scaffolds (10 compounds) and phenyl ring with boron groups (4 compounds). In silico analysis and molecular docking studies were performed using the Glide/SP module of Maestro molecular modeling package. According to obtained results, structure activity relationship (SAR) was carried out. Among the newly designed, synthesized, and tested compounds, our in vitro findings revealed that several compounds displayed a highly effective eEF2K inhibition at submicromolar concentration in in vitro breast cancer cells. In conclusion, we identified novel eEF2K inhibitors as promising anticancer drug substance candidates which should be further evaluated by in vivo studies, preclinical and clinical studies.

Eukaryotic elongation factor 2 kinase (eEF-2K), an unusual alpha kinase, is involved in protein synthesis through phosphorylation of elongation factor 2 (EF2). eEF-2K is indicated as one of the critical drivers of breast cancer and associated with poor clinical prognosis, representing a potential molecular target. The crystal structure of eEF-2K is unknown and there is no potent and effective eEF-2K inhibitor reported for clinical applications. To meet this challenge, we designed and synthesized several generations of potential inhibitor compounds and performedin silicostudies. The effect of the inhibitors at the binding pocket of eEF-2K is analyzed after developing a 3D target model by homology modeling approaches using a domain of another α-kinase called myosin heavy-chain kinase A (MHCKA) that is closely resembling eEF-2K. Our results showed that compounds with coumarin-chalcone cores have a high predicted binding affinity for binding to eEF-2K. Followingin vitrostudies, we identified a compound that was highly effective in inhibiting eEF-2K activity at submicromolar concentrations and inhibited proliferation of various breast cancer cells with different features (BT20, MDA-MB-231, MDA-MB-436 and MCF-7) by induction of apoptosis while sparing normal cells.In vivosystemic administration of the the lead inhibitor encapsulated in single lipid-based nanoparticles twice a week significantly supressed growth of MDA-MB-231 tumors in orthotopic breast cancer models in nude mice. In conclusion, our study provides the firstin vivoeffective small molecule eEF-2K inhibitor that may be used for molecularly targeted precison medicine strategies in breast cancer or other eEF-2K-dependent tumors.

Therapeutic options for treatment of basal-like breast cancers remain limited. Here, we demonstrate that bromodomain and extra-terminal (BET) inhibition induces an adaptive response leading to MCL1 protein-driven evasion of apoptosis in breast cancer cells. Consequently, co-targeting MCL1 and BET is highly synergistic in breast cancer models. The mechanism of adaptive response to BET inhibition involves the upregulation of lipid synthesis enzymes including the rate-limiting stearoyl-coenzyme A (CoA) desaturase. Changes in lipid synthesis pathway are associated with increases in cell motility and membrane fluidity as well as re-localization and activation of HER2/EGFR. In turn, the HER2/EGFR signaling results in the accumulation of and vulnerability to the inhibition of MCL1. Drug response and genomics analyses reveal that MCL1 copy-number alterations are associated with effective BET and MCL1 co-targeting. The high frequency of MCL1 chromosomal amplifications (30%) in basal-like breast cancers suggests that BET and MCL1 co-targeting may have therapeutic utility in this aggressive subtype of breast cancer.

Circulating miRNAs are known to play important roles in intercellular communication. However, the effects of exosomal miRNAs on cells are not fully understood.To investigate the role of exosomal miR-1246 in ovarian cancer (OC) microenvironment, we performed RPPA as well as many other in vitro functional assays in ovarian cancer cells (sensitive; HeyA8, Skov3ip1, A2780 and chemoresistant; HeyA8-MDR, Skov3-TR, A2780-CP20). Therapeutic effect of miR-1246 inhibitor treatment was tested in OC animal model. We showed the effect of OC exosomal miR-1246 uptake on macrophages by co-culture experiments.Substantial expression of oncogenic miR-1246 OC exosomes was found. We showed that Cav1 gene, which is the direct target of miR-1246, is involved in the process of exosomal transfer. A significantly worse overall prognosis were found for OC patients with high miR-1246 and low Cav1 expression based on TCGA data. miR-1246 expression were significantly higher in paclitaxel-resistant OC exosomes than in their sensitive counterparts. Overexpression of Cav1 and anti-miR-1246 treatment significantly sensitized OC cells to paclitaxel. We showed that Cav1 and multi drug resistance (MDR) gene is involved in the process of exosomal transfer. Our proteomic approach also revealed that miR-1246 inhibits Cav1 and acts through PDGFβ receptor at the recipient cells to inhibit cell proliferation. miR-1246 inhibitor treatment in combination with chemotherapy led to reduced tumor burden in vivo. Finally, we demonstrated that when OC cells are co-cultured with macrophages, they are capable of transferring their oncogenic miR-1246 to M2-type macrophages, but not M0-type macrophages.Our results suggest that cancer exosomes may contribute to oncogenesis by manipulating neighboring infiltrating immune cells. This study provide a new mechanistic therapeutic approach to overcome chemoresistance and tumor progression through exosomal miR-1246 in OC patients.

The development of effective targeted therapies for the treatment of basal-like breast cancers remains challenging. Here, we demonstrate that cellular stress engendered by BET inhibition induces an adaptive response leading to MCL1 protein-driven evasion of apoptosis in breast cancers. Consequently, co-targeting MCL1 and BET is highly synergistic in in vitro and in vivo breast cancer models. The mechanism of adaptive response to BET inhibition involves upregulation of critical lipid metabolism enzymes including the rate-limiting enzyme stearoyl-CoA desaturase (SCD). Changes in the lipid metabolism are associated with increases in cell motility and membrane fluidity as well as transitions in cell morphology. The structural changes in the cell membrane leads to re-localization and activation of HER2/EGFR which can be interdicted by inhibiting SCD activity. Active HER2/EGFR, in turn, induces accumulation of MCL1 protein and therapeutic vulnerability to MCL1 inhibitors. The BET protein, lipid metabolism and receptor tyrosine kinase activation cascade is observed in patient cohorts of basal-like and HER2-amplified breast cancers. Drug response and genomics analyses revealed that MCL1 copy number alterations, including low-level gains, are selectively enriched in basal-like breast cancers and associated with effective BET and MCL1 co-targeting. The high frequency of MCL1 chromosomal amplifications (>30%) and gains (>50%) in basal-like breast cancers suggests that BET and MCL1 co-inhibition may have therapeutic utility in this aggressive subtype.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer and associated with early metastasis, drug resistance, and poor patient survival. Fork head box M1 (FOXM1) is considered as an emerging molecular target due to its oncogenic role and high overexpression profile in 85% in TNBC. However, molecular mechanisms by which FOXM1 transcription factor mediate its oncogenic effects are not fully understood. Integrin β1 is often upregulated in invasive breast cancers and associated with poor clinical outcome and shorter overall patient survival in TNBC. However, the mechanisms regulating integrin β1 (ITGB1) gene expression have not been well elucidated. Normal breast epithelium (MCF10A) and TNBC cells (i.e., MDA-MB-231, BT-20 MDA-MB436) were used for the study. Small interfering RNA (siRNA)-based knockdown was used to inhibit Integrin β1 gene (mRNA) and protein expressions, which are detected by RT-PCR and Western blot, respectively. Chromatin immunoprecipitation (ChiP) and gene reporter (Luciferase) assays were used to demonstrate that FOXM1 transcription factor binds to the promoter of Integrin β1 gene and drives its expression. We demonstrated that FOXM1 directly binds to the promoter of integrin β1 gene and transcriptionally regulates its expression and activity of focal adhesion kinase (FAK) in TNBC cells. Our study suggests that FOXM1 transcription factor regulates Integrin β1 gene expression and that FOXM1/ Integrin-β1/FAK axis may play an important role in the progression of TNBC.

The development of effective targeted therapies for the treatment of basal-like breast cancers remains challenging. Here, we demonstrate that BET inhibition induces a multi-faceted adaptive response program leading to MCL1 protein-driven evasion of apoptosis in breast cancers. Consequently, co-targeting MCL1 and BET is highly synergistic inin vitroandin vivobreast cancer models. Drug response and genomics analyses revealed that MCL1 copy number alterations, including low-level gains, are selectively enriched in basal-like breast cancers and associated with effective BET and MCL1 co-targeting. The mechanism of adaptive response to BET inhibition involves upregulation of critical lipid metabolism enzymes including the rate-limiting enzyme stearoyl-CoA desaturase (SCD). Changes in the lipid metabolism are associated with increases in cell motility and membrane fluidity as well as transitions in cell morphology and adhesion. The structural changes in the cell membrane leads to re-localization and activation of HER2/EGFR which can be interdicted by inhibiting SCD activity. Active HER2/EGFR, in turn, induces accumulation of MCL1 protein and therapeutic vulnerability to MCL1 inhibitors. The BET protein, lipid metabolism and receptor tyrosine kinase activation cascade is observed in patient cohorts of basal-like and HER2-amplified breast cancers. The high frequency of MCL1 chromosomal amplifications (>30%) and gains (>50%) in basal-like breast cancers suggests that BET and MCL1 co-inhibition may have therapeutic utility in this aggressive subtype.

Cancer cells exploit many of the cellular adaptive responses to support their survival needs. One such critical pathway in eukaryotic cells is the unfolded protein response (UPR) that is important in normal physiology as well as disease states, including cancer. Since UPR can serve as a lever between survival and death, regulated control of its activity is critical for tumor formation and growth although the underlying mechanisms are poorly understood. Here we show that one of the main transcriptional effectors of UPR, activating transcription factor 4 (ATF4), is essential for prostate cancer (PCa) growth and survival. Using systemic unbiased gene expression and proteomic analyses, we identified a novel direct ATF4 target gene, family with sequence similarity 129 member A (FAM129A), which is critical in mediating ATF4 effects on prostate tumorigenesis. Interestingly, FAM129A regulated both PERK and eIF2a in a feedback loop that differentially channeled the UPR output. ATF4 and FAM129A protein expression is increased in patient PCa samples compared with benign prostate. Importantly, in vivo therapeutic silencing of ATF4-FAM129A axis profoundly inhibited tumor growth in a preclinical PCa model. These data support that one of the canonical UPR branches, through ATF4 and its target gene FAM129A, is required for PCa growth and thus may serve as a novel therapeutic target.

Triple-negative breast cancer (TNBC) is an incurable disease with the highest mortality rates among all breast cancer subtypes. Currently, there is no FDA-approved targeted therapies for TNBC. Significant genetic heterogeneity with six genetically defined sub-groups is considered the major reason for treatment failures and high mortality rates in TNBC patients and has been the major limitation in the identification of common molecular targets and development of targeted therapies for this cancer. Recently, interest in the development of non-coding miRNA based therapeutics has increased significantly. miRNA molecules are short, non-coding RNAs that have emerged as critical regulators of gene expression, affecting a multitude of biological processes, including cell proliferation, survival, motility, and tumorigenesis, by acting as oncogenes or tumor suppressors. Recently, first in human miRNA-targeted therapeutics were found safe and well tolerated in phase I clinical trials, suggesting that miRNA based therapeutic interventions may provide novel therapeutic options, especially for TNBC patients. However, successful clinical application of miRNA-based therapeutics requires development of safe and effective nano-delivery systems. To identify potential molecular targets we extensively analyzed miRNA databases and recently discovered that miR-329 expression is associated with significantly longer survival in patients with TNBC. More important, we found that miR-329 expression is lost or significantly reduced in the majority of TNBC cell lines. We demonstrated that in vitro expression of miR-329 significantly suppressed cell proliferation, invasion and migration. To identify molecular mechanism of miR-329 based therapy we utilized 5 different target prediction algorithms and identified that miR-329 has a binding site at 3'-UTR of EF2K gene which encodes Eukaryotic Elongation factor-2 kinase (EF2K). We found that EF2K is significantly upregulated in TNBC patients and is associated with shorter overall survival in TNBC patients. Remarkably, silencing of EF2K by siRNA recapitulated the effects of ectopic expression of miR-329 mimic and successfully suppressed growth and invasion of various TNBC cells. Furthermore, in vivo therapeutic delivery of miR-329 mimic incorporated albumin-based nanoparticles led to significant tumor suppressive effects in multiple TNBC xenograft models (MDA-MB-231 and MDA-MB-436) in mice. Importantly, albumin-based miR-329 nano-therapy in mice did not lead to any detectable side effects during the therapy, suggesting that miR-329 based therapy is safe and clinically feasible approach. Currently, we are conducting luciferase-based gene reporter assay to demonstrate that miR-329 specifically binds to 3'-UTR of EF2K mRNA and inhibits EF2K expression. We are also analyzing the tumors collected after albumin based nanoparticles-miR-329 treatment for miR-329 expression and EF2K downregulation as well as intra-tumoral proliferation markers (Ki67) and apoptosis by TUNEL. Based on clinical significance and our preliminary data, we conclude that miR-329 acts as a tumor suppressor in TNBC, and its reduced expression leads to expression of EF2K signaling. In addition, miR-329-based nano-therapy provides a safe and significant anti-tumor efficacy and may be a potential therapy for TNBC. Citation Format: Goknur Kara, Nermin Kahraman, Emir Baki Denkbas, George Calin, Bulent Ozpolat. MiR-329 mimic based nano-therapy inhibits growth and progression of triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5985.

Objectives Lung cancer is the leading cause of cancer related deaths in worldwide. Despite recent advances in treatment options, patient survival has not improved substantially due to lack of commonly expressed molecular targets and effective targeted therapeutics. Thus, better understanding of the biology of lung cancer and identification of novel therapeutic targets are urgently needed for development of highly effective molecularly targeted therapies. Materials and methods Viability, proliferation and metastatic ability of lung cancer cells were evaluated using methylthiazoltetrazolium (MTT), colony formation and matrigel invasion assays, respectively. Western blotting, RT-PCR, and gene knockdown by siRNA transfections were carried out to investigate the effects of eEF-2K on lung cancer cells. Athymic Nu/Nu mice were treated with liposomal eEF-2KeEF-2K or control siRNA and tumor growth was evaluated in tumor xenograft models of lung cancer. Results and discussion Here, we report that Eukaryotic Elongation Factor-2 kinase (eEF-2K), a member of an atypical alpha kinases family, is significantly upregulated in lung cancer cell lines and its expression is associated with shorter overall patient survival in lung cancer. Inhibition eEF-2K expression by siRNA or a chemical inhibitorsignificantly suppressed lung cancer cell proliferation, colony formation, survival, migration/invasion and tumorigenesis by inhibiting cyclin D1, Src and Mitogen-Activated Protein Kinases/Extracellular Signal-Regulated Kinase (MAPK/ERK) signaling. In vivo targeting of eEF-2K by systemically injected nanoliposomal eEF-2K siRNA resulted in a significant inhibition of lung cancer tumor xenografts in nude mice. Our results suggest, for the first time, that expression of eEF-2K is associated with poor patient prognosis and involved in regulation of critical pathways, including Src and MAPK/ERK and cyclin D1, promoting tumor growth and progression, and thus may be a novel potential therapeutic target in lung cancer.

Purpose: Recent studies indicated that dysregulation of noncoding KNAs (ncRNA) such as miRNAs is involved in pathogenesis of various human cancers. However, the molecular mechanisms underlying miR-34a are not fully understood in triple-negative breast cancer (TNBC). Experimental Design: We performed in vitro functional assays on TNBC cell lines to investigate the role of mi R-34a in FOLM1/eEF2K signaling axis. TNBC tumor xenograft models were used for in vivo therapeutic delivery of miR-34a. Results: In this study, we investigated the role of p53-driven ncRNA miR-34a and found that miR-34a is associated with significantly longer patient survival in TNBC and inversely correlated with levels of proto-oncogenic eEF2K, which was associated with significantly shorter overall patient survival, We showed that miR-34a directly binds to the 3'-untranslated region of eEF2K and FOXM1 mRNAs and suppresses their expression, leading to inhibition of TNBC cell proliferation, motility, and invasion. Notably, restoring miR-34a expression recapitulated the effects of inhibition of eEF2K and FOXM1, the transcription factor for eEF2K and the direct target of p53, in TNBC cell lines, whereas overexpression of eEF2K and FOXM1 rescued the effects and signaling pathways mediated by miR-34a. Moreover, in vivo therapeutic delivery of miR-34a nanopartides by systemic intravenous administration delayed tumor growth of two different orthotopic TNBC tumor xenograft models by inhibiting eEF2K and FOXM1, intratumoral proliferation and angiogenesis, and inducing apoptosis. Conclusions: Overall, our findings provide new insights into the tumor suppressor role of miR-34a by dual-targeting of FOXM1/eEF2K signaling axis and suggest that miR-34a-based gene therapy may be a potential therapeutic strategy in TNBC. (C)2018 AACR. NIH/NCIUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [R21CA199050, P30CA016672]; noncoding RNA center; NATIONAL CANCER INSTITUTEUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [P30CA016672] Funding Source: NIH RePORTER This work was supported in part by grants from the NIH/NCI (R21CA199050 and P30CA016672) and the funding from noncoding RNA center and used the Functional Proteomics RPPA Core Facility.

Circulating miRNAs are known to play important roles in intercellular communication.However, the effects of exosomal miRNAs on cells are not fully understood. In this study, substantial expression of oncogenic miR-1246 in sensitive as well as chemoresistant (paclitaxel and multidrug) ovarian cancer (OC) exosomes was found. We showed that Cav1 gene, which is the direct target of miR-1246, is involved in the process of exosomal transfer. A significantly worse overall prognosis were found for OC patients with high miR-1246 and low Cav1 expression based on TCGA data. miR-1246 expression were significantly higher in paclitaxel-resistant OC exosomes than in their sensitive counterparts. Overexpression of Cav1 and anti-miR-1246 treatment significantly sensitized OC cells to paclitaxel. We showed that Cav1 and multi drug resistance (MDR) gene is involved in the process of exosomal transfer. Our proteomic approach also revealed that miR-1246 inhibits Cav1 and acts through PDGFI² receptor at the recipient cells to inhibit cell proliferation. miR-1246 inhibitor treatment in combination with chemotherapy led to reduced tumor burden in vivo. Finally, we demonstrated that when OC cells are co-cultured with macrophages, they are capable of transferring their oncogenic miR-1246 to M2-type macrophages, but not M0-type macrophages. Our results suggest that cancer exosomes may contribute to oncogenesis by manipulating neighboring infiltrating immune cells. This study provide a new mechanistic therapeutic approach to overcome chemosensitization and tumor progression through exosomal miR-1246 in OC patients. Funding: This study was funded by the NIH Common Fund (UH3 TR000943), through the Office of Strategic Coordination/Office of the NIH Director and MD Anderson’s Cancer Center Support Grant (CCSG) (CA016672) to G. Lopez-Berestein, A.K. Sood, G.A. Calin,, the American Cancer Society Research Professor Award to A.K. Sood, and The Center for RNA Interference and Non-Coding RNA to G.A. Calin, A.K. Sood, and G. Lopez-Berestein and CA166228 from the National Cancer Institute of the NIH to MLG. Declaration of Interest: We have no conflicts of interest to disclose. Ethical Approval: All animal work was approved by the Institutional Animal Use and Care Committee of MD Anderson.

Triple-negative breast cancer (TNBC) is the most aggressive and chemoresistant subtype of breast cancer. Therefore, new molecular targets and treatments need to be developed to improve poor patient prognosis and survival. We have previously shown that eukaryotic elongation factor 2 kinase (eEF-2K) is highly expressed in TNBC cells, is associated with poor patient survival and prognosis, and promotes cell proliferation, migration, and invasion. In vivo targeting of eEF-2K significantly reduces the tumor growth of orthotopic TNBC xenograft mouse models, suggesting that eEF-2K may serve as a potential novel therapeutic target. In the current study, we identified thymoquinone (TQ), an active ingredient of Nigella sativa, as a potential safe and effective eEF-2K inhibitor in TNBC. We demonstrated for the first time that TQ inhibits the protein and mRNA expression of eEF-2K, as well as the clinically relevant downstream targets, including Src/FAK and Akt, and induces the tumor suppressor miR-603, in response to NF-kB inhibition. This effect was associated with a significant decrease in the proliferation, colony formation, migration, and invasion of TNBC cells. Furthermore, systemic in vivo injection of TQ (20 and 100 mg/kg) significantly reduced the growth of MDA-MB-231 tumors and inhibited the eEF-2K expression in an orthotopic tumor model in mice. Our study provides first evidence that TQ treatment inhibits cell proliferation, migration/invasion, and tumor growth, in part through the inhibition of eEF-2K signaling in TNBC. Thus, our findings suggest that systemic TQ treatment may be used as a targeted therapeutic strategy for the inhibition of eEF-2K in TNBC tumor growth and progression.

Shahbazi, Reza/0000-0002-0816-0733 WOS: 000406812700005 PubMed: 28745127 Aim: In this study, we aimed to therapeutically target eukaryotic elongation factor 2 kinase (eEF-2K) in an in vivo triple-negative breast cancer (TNBC) tumor model. Materials & methods: We synthesized a highly monodisperse nanoformulation using polyethylenimine-modified gold nanoparticles (AuNP-PEI) as siRNA delivery vehicle and evaluated gene downregulation. Results: We found that AuNP-PEI/eEF-2K nanoformulation was highly effective for in vitro and in vivo gene downregulation and showed remarkable antitumor efficacy that was associated with eEF-2K knockdown, inhibition of Src and MAPK-ERK signaling pathways in a TNBC orthotopic tumor model. Conclusion: Our study suggests that eEF-2K plays an important role in TNBC tumorigenesis and its inhibition by AuNP-PEI/eEF-2K siRNA-based nanotherapeutics may be a potential therapeutic strategy for TNBC. Scientific and Technological Research Council of Turkey (TUBITAK-BIDEB)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [2215] This research was supported by the Scientific and Technological Research Council of Turkey (TUBITAK-BIDEB) 2215 scholarship program. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

We investigated the effect of daily dietary curcumin intake on the development and progression of spontaneous ovarian cancer in a galline (hen) model, as the chicken is the only nonhuman animal in which ovarian cancer spontaneously develops with a high prevalence. At the end of 12 months, ovarian cancer had spontaneously developed in 39% (35/90) of control hens not fed curcumin (n = 90). In comparison, it spontaneously developed in 27% (24/90) and 17% (15/90) of hens given curcumin at 25.8 (n = 90) and 53.0 mg/day (n = 90), respectively (P = 0.004). This represented significant dose-dependent reductions in overall ovarian cancer incidence in the 25.8 and 53.0 mg/day curcumin-fed groups (31% and 57%, respectively). Daily curcumin intake also reduced ovarian tumor sizes (P = 0.04) and number of tumors (P = 0.006). Evaluation of the molecular mechanisms underlying the chemopreventive and antitumor effects of curcumin revealed that NF-κB and STAT3 signaling pathways were significantly inhibited but that the nuclear factor erythroid 2/heme oxygenase 1 antioxidant pathway was induced by curcumin intake in a dose-dependent manner in ovarian tissues (P < 0.05). Sequencing of the Ras family genes (KRAS, NRAS, and HRAS) revealed less frequent KRAS and HRAS mutations in ovarian tumors in the curcumin-fed animals. In conclusion, our results demonstrated for the first time that daily curcumin intake leads to a significant and dose-dependent reduction in spontaneous ovarian cancer incidence and tumor growth, indicating a tremendous role for curcumin as a chemopreventive strategy for ovarian cancer. Cancer Prev Res; 11(1); 59–67. ©2017 AACR.

Adult stem cells have been used as regenerative medicine for treating human diseases through gene therapy approach. These stem cells secrete cytokines, growth factors and extracellular vesicles including exosomes, which may induce antitumor activities. We have demonstrated that muscle progenitor cells (MPCs) from adult (murine and human) skeletal muscle have a high regenerative potential to repair bone, skeletal muscle, and cartilage. However, to date, the potential therapeutic use of MPCs in cancers has not yet been investigated. The objectives of this study were to identify and examine the roles of secreted factors including exosomes from MPCs on the proliferation/survival, motility/migration and invasive potential of prostate cancer (PCa) cells. Here we showed that co-culture of PCa cell lines, PC3 and C42B, with MPCs in trans-well inserts strongly suppressed PCa cell proliferation and survival by activating CDK inhibitors (p16 and p27) and apoptotic BBC3 and NOXA genes. Mass spectrometry analysis of factors secreted by MPCs identified several proteins with known anti-proliferative functions, including IGFBP6/7, pigment epithelium-derived factor (PEDF), HPCAL1, NO, and SPARC. Conditioned media (CM) from MPCs significantly increased docetaxel-induced cytotoxicity of PCa cells by suppressing the expression of key genes including AR, MLL, FOXA1, TWF1, and TGIF. Next, we isolated exosomes from cultured mouse and human MPCs through step-wise ultracentrifugation methods and tested their effect in the proliferation of cancer cells. We observed that treatment of PCa cells with MPC-derived exosomes inhibited cell proliferation as assayed with MTT and down-regulated the expression of AR and its target NKX3.1 genes. We also observed that the migration ability of ovarian cancer cells (OVAC5) and pancreatic cancer (Panc1), was markedly reduced following MPC-derived exosome treatments compared to untreated cells. Taken together, our data suggest that MPCs can suppress the proliferation and survival of cancer cells through secreted factors including exosomes. Thus, adult stem cell-based therapy along with existing therapies may be a promising strategy in treating various solid cancers as well as regenerating cancer-induced bone and muscle tissue lesions. Citation Format: Krishna M. Sinha, Rozita Yarmand, Nermin Kahraman, Fahriye Duzagac, Gabriel Lopez, Bulent Ozpolat, Johnny Huard. Skeletal muscle progenitor cell-derived exosomes have therapeutic potential in inhibition of prostate cancer cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1156.

Autophagy is a highly complex lysosomal degradation process. Recently we demonstrated that autophagy genes encoding Beclin1, ATG7, LC3 promotes cell proliferation, survival, and invasion by promoting Cyclin D1 and Integrin β1/ Src signaling in triple negative breast cancer (TNBC). Studies showed that increased basal autophagy is linked to development of chemoresistance, relapses and metastasis and poor prognosis in TNBC. However, the major molecular mechanisms and the integrated global regulators controlling the autophagic machinery still remains unknown. Here, we identified miRNA-873 as a p53-driven tumor suppressor that is associated with favorable patient survival (TCGA database) and the key factor for regulating the major autophagy genes, including BCN1, LC3, ATG7, ATG16L1 and ATG13 in TNBC. Using in silico prediction algorithms we demonstrated that miR-873 has binding sites on the 3’-untranslated region (3’-UTR) of these genes and directly binds and suppresses their expression using by gene reporter assays. Introduction of mutations to the miR-873 binding sites on 3-UTR of these genes reversed the inhibitory effect of miR-873on these genes. Furthermore, knockdown of Beclin1, LC3 and ATG7 genes significantly suppressed Eukaryotic Elongation Factor-2 kinase (eEF2K), an unusual alpha kinase, which is highly overexpressed in TNBC patients and associated with poor prognosis. We also found that miR-873 also binds to the 3’-UTR of eEF2K mRNA and regulates its expression and inhibits starvation induced autophagy. Through knockdown and overexpression studies we also demonstrated that eEF2K regulates expression of abovementioned autophagic proteins. Interestingly, we found that BCN1, LC3, and ATG7 also regulates expression of EF2K, suggesting an existence of a novel negative feed-back loop. Lastly, we demonstrated that miR-873 expression is suppressed in TNBC patients and cell lines and restoration of its expression in vivo in MDA-MB-231 and MDA-MB-436 orthotopic xenograft models by systemic injection (I.V, tail vein once a week, 0.15 mg/kg) of miR-873 mimic molecules incorporated in novel single lipid (SLNP)-nanoparticles suppressed tumor growth. Furthermore, in vivo treatment of mice with SLNP-Beclin1, ATG7 or ATG8 siRNAs also completely suppressed TNBC tumor growth. In conclusion, our data suggest that p53/miR-873/eEF2K axis is a novel post-transcriptional regulator of autophagy and miR-873 functions as a master regulator of the post-transcriptional regulation of the major autophagy genes directly and indirectly through eEF2K dependent dual-suppressor mechanism and modulation of this axis could be used as a potential therapy for TNBC. Citation Format: Hamada Ahmed Mokhlis, Nermin Kahraman, Seyda Baydogan, Abdel-Aziz Hamed Abdel-Aziz, Ahmed Ashour, Cristina Ivan, Gabriel Lopez-Berestein, Bulent Ozpolat. MiR-873 is the master regulator of autophagy genes through a novel negative feedback mechanism mediated by Elongation factor 2 kinase (eEF-2K) and suppresses tumor growth and progression of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4262.

Ovarian cancer, especially high-grade serous ovarian cancer (HGSOC), is the deadliest gynecological cancer with 50-70% 5-year mortality rates. Every year more than 22,000 new cases of ovarian cancer and 15,000 deaths are anticipated within the United States only. Recent clinical and experimental evidence indicates that tumor-associated macrophages (TAMs), the most abundant cells in the tumor microenvironment, play a significant role in tumor growth and progression by contributing to angiogenesis, invasion, metastasis, and drug resistance, leading to poor clinical outcomes and significantly shorter patient survival in HGSOC. More than 50% of cells in the peritoneal tumor microenvironment and malign ascites consist of TAMs in ovarian cancer (OC) patients. Especially, M2 macrophages have been shown to support tumor proliferation and promote tumor progression, angiogenesis, and drug resistance. But the mechanisms of these oncogenic effects are still not clear. The goal of our study to investigate the role of TAM-derived exosomes, which are 30-100nm microvesicles released from cells and are key factors in communication between cancer cells and the tumor microenvironment. To this end, we evaluated differentially expressed miRNAs in high-grade ovarian cancer cells (OVCAR3, OVCAR 432 and OVCAR5) after treatment with exosomes-derived from TAMs (M2 phenotype) using the Affymetrix Gene Chip miRNA 4.0 microarrays. We identified several miRNAs, including miR-6068 that we validated by qPCR and found it to be significantly upregulated in both HGSOC cells and their exosomes. We demonstrated that transfection of HGSOC cells with miR-6068 significantly increased proliferation, migration and invasion capacities of the cells in vitro, suggesting that this miR-6068 act as an oncogenic miR (oncomiR). Using in silico prediction algorithms we found that miR-6068 has binding sites on the 3’-untranslated region (3’-UTR) of PTPN4 gene encoding a phosphatase and demonstrated that it miR-6068 suppresses PTPN4 expression by Western blot and qPCR. Inhibition of PTPN4 by siRNA significantly induced cell proliferation in OC cells, suggesting that PTPN4 acts as a tumor suppressor. In conclusion, our results suggest that miR-6068 has an oncogenic role in ovarian cancer progression by targeting PTPN4 and may be a novel effective therapeutic target for ovarian cancer. Citation Format: Seyda Baydogan, Jianting Sheng, Nermin Kahraman, Pinar Kanlikilicer, Hamada Ahmed Mokhlis, Sayra Dilmac, Stephen T. C. Wong, Bulent Ozpolat. Exosomal transfer of tumor-associated macrophage derived miR-6068 promote ovarian cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3563.

Triple negative breast cancer (TNBC) is an aggressive type of breast cancer characterized by the absence of defined molecular targets, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and is associated with high rates of relapse and distant metastasis despite surgery and adjuvant chemotherapy. The lack of effective targeted therapies for TNBC represents an unmet therapeutic challenge. Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical calcium/calmodulin-dependent serine/threonine kinase that promotes TNBC tumorigenesis, progression, and drug resistance, representing a potential novel molecular target. However, the mechanisms regulating eEF2K expression are unknown. Here, we report that eEF2K protein expression is highly up-regulated in TNBC cells and patient tumors and it is associated with poor patient survival and clinical outcome. We found that loss/reduced expression of miR-603 leads to eEF2K overexpression in TNBC cell lines. Its expression results in inhibition of eEF2K by directly targeting the 3-UTR and the inhibition of tumor cell growth, migration and invasion in TNBC. In vivo therapeutic gene delivery of miR-603 into TNBC xenograft mouse models by systemic administration of miR-603-nanoparticles led to a significant inhibition of eEF2K expression and tumor growth, which was associated with decreased activity of the downstream targets of eEF2K, including Src, Akt, cyclin D1 and c-myc. Our findings suggest that miR-603 functions as a tumor suppressor and loss of miR-603 expression leads to increase in eEF2K expression and contributes to the growth, invasion, and progression of TNBC. Taken together, our data suggest that miR-603-based gene therapy is a potential strategy against TNBC.

Breast cancer (BC) is the most common malignancy and the second leading cause of cancer-related deaths in women worldwide. Triple negative breast cancer (TNBC) is the most aggressive and chemo-resistant subtype of BC that represents an unmet therapeutic challenge. Lack of therapeutic targets and significant tumor heterogeneity are the major reasons contributing to poor prognosis and high mortality rates. To meet these challenges, we extensively analyzed BC patient databases and discovered that the high expression of a novel non coding RNA- microRNA 484- was correlated with poor survival and prognosis of BC patients. Moreover, miR-484 expression was elevated in all molecular subtypes of BC patients, especially in TNBC, compared to healthy controls. Thus, our preliminary data suggests that miR-484 could be a possible oncogenic driver, with clinical and prognostic significance in BC patients, and may thus serve as a potential molecular target. However, the role and mechanism of action of miR-484 in TNBC has not been previously elucidated. Thus, the aim of this study is to elucidate the functional role and mechanism of action of miR-484 in TNBC. To identify potential targets for miR-484, we used four different algorithms that predict the mRNA targets of miRNAs: TargetScan, miRDB, microrna.org, and Dianna microT, and based on their target prediction strategy, HOXA5 was the common target for miR-484 in all four databases. The homeobox protein (HOXA5) is a transcription factor that has been shown to play important roles in cell differentiation, hematopoiesis, and tumorigenesis. Additionally, previous studies have shown that HOXA5 may function as tumor suppressor in BC. Our results showed that ectopic inhibition of miR-484 reduced cell proliferation, colony formation, invasion/migration, and induced apoptosis in TNBC cells. Furthermore, ectopic overexpression of miR-484 reduced HOXA5 mRNA and protein expression levels in TNBC cells, suggesting that miR-484 may function in TNBC cells by inhibition of HOXA5. We further confirmed that HOXA5 is a direct target of miR-484 by luciferase reporter assay, and showed reduced luciferase activity in TNBC and HEK293 cells expressing the 3'UTR region of HOXA5 and treated with miR-484 mimic. Furthermore, in vivo therapeutic targeting of miR-484 significantly reduced tumor growth and progression in orthotopic xenograft TNBC mouse models. Overall, our findings suggest that miR-484 promotes tumor growth and progression by targeting HOXA5 in TNBC. Citation Format: Nashwa N. Kabil, Recep Bayraktar, Cristina Ivan, Nermin Kahraman, Hamada A. Mokhlis, George A. Calin, Gabriel Lopez-Berestein, Bulent Ozpolat. miR-484 acts as an “oncomiR” in triple negative breast cancer cells to promote tumor growth and progression by targeting HOXA5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 504.

Ovarian cancer is a highly metastatic disease, but no effective strategies to target this metastatic process currently are known. Here, an integrative computational analysis of The Cancer Genome Atlas ovarian cancer dataset coupled with experimental validation identified a novel zinc finger transcription factor ZNF304 as one of the key factors for ovarian cancer metastasis. High tumoral ZNF304 expression was associated with poor overall survival in ovarian cancer patients. Through reverse phase protein array analysis, we demonstrated that ZNF304 promotes multiple proto-oncogenic pathways important for cell survival, migration, and invasion. ZNF304 transcriptionally regulates β1 integrin, which subsequently regulates Src/focal adhesion kinase and paxillin and prevents anoikis. In vivo delivery of ZNF304 siRNA by a novel dual assembly nanoparticle led to sustained gene silencing for 14 days, increased anoikis, and reduced tumor growth in orthotopic mouse models of ovarian cancer. Taken together, ZNF304 is a novel transcriptional regulator of β1 integrin, promotes cancer cell survival, and protects against anoikis in ovarian cancer.

Eukaryotic elongation factor 2 kinase (eEF2K), an emerging molecular target for cancer therapy, contributes to cancer proliferation, cell survival, tumorigenesis, and invasion, disease progression and drug resistance. Although eEF2K is highly up-regulated in various cancers, the mechanism of gene regulation has not been elucidated. In this study, we examined the role of Forkhead Box M1 (FOXM1) proto-oncogenic transcription factor in triple negative breast cancer (TNBC) cells and the regulation of eEF2K. We found that FOXM1 is highly upregulated in TNBC and its knockdown by RNA interference (siRNA) significantly inhibited eEF2K expression and suppressed cell proliferation, colony formation, migration, invasion and induced apoptotic cell death, recapitulating the effects of eEF2K inhibition. Knockdown of FOXM1 inhibited regulators of cell cycle, migration/invasion and survival, including cyclin D1, Src and MAPK-ERK signaling pathways, respectively. We also demonstrated that FOXM1 (1B and 1C isoforms) directly binds to and transcriptionally regulates eEF2K gene expression by chromatin immunoprecipitation (ChIP) and luciferase gene reporter assays. Furthermore, in vivo inhibition of FOXM1 by liposomal siRNA-nanoparticles suppressed growth of MDA-MB-231 TNBC tumor xenografts in orthotopic models. In conclusion, our study provides the first evidence about the transcriptional regulation of eEF2K in TNBC and the role of FOXM1 in mediating breast cancer cell proliferation, survival, migration/invasion, progression and tumorgenesis and highlighting the potential of FOXM1/eEF2K axis as a molecular target in breast and other cancers.

Triple negative breast cancer (TNBC), is a highly heterogeneous and aggressive subtype of breast cancer (BC), which poses a significant clinical challenge. TNBC constitutes about 15-20 % of BC cases, and is characterized by lack of estrogen (ER), progesterone (PR) and human epidermal growth factor 2 (HER2) receptors. Thus, patients cannot benefit from targeted therapies such as anti-estrogens (eg.Tamoxifen) and anti-HER2 (eg.trastuzumab) treatments. Therefore, identification of new molecular targets and treatment strategies are highly warranted to improve patient outcome. We previously reported that elongation factor 2 kinase (EF-2K) is highly expressed in TNBC cell lines and is associated with poor patient survival and prognosis. Furthermore, in vivo targeting of EF-2K by siRNA nano-therapeutics inhibited cell proliferation, migration/invasion, and significantly decreased tumor growth in 2 different orthotopic TNBC mouse models (MDA MB-231 and MDA MB-436). Collectively, our work suggests that EF-2K is an excellent novel therapeutic target in TNBC. In search of a potential safe and effective EF-2K inhibitor, we identified Thymoquinone (TQ), a dietary natural compound, known to have diverse anti-cancerous properties in several in vitro and in vivo models, including TNBC. However, the mechanism by which TQ mediates its effects are not well elucidated. Our current study is the first to demonstrate that TQ inhibits protein and mRNA expression of EF-2K, as well as its clinically significant downstream targets such as Src/FAK, PI3K/AKT, and CyclinD1; resulting in a significant decrease in proliferation, migration/invasion of TNBC cells. To determine the molecular mechanism by which TQ inhibits EF-2K expression, we investigated if TQ induces tumor suppressor microRNAs that we identified to bind to the 3’-UTR of EF-2K. We found that TQ induces miR-603 expression, which we had reported to directly bind and inhibit EF-2K expression; resulting in decreased TNBC growth and progression, both in vitro and in vivo. Furthermore, we showed that inhibition of nuclear factor kappa B (NF-κB) (a well established target for TQ), also induces miR-603 and inhibits EF-2K expression in TNBC cells. In conclusion, our study is the first to show that TQ treatment decreases EF-2K expression through modulating the NF-κB/miR-603 axis; ultimately resulting in decreased cell proliferation, migration/invasion of TNBC. Our data suggests a novel molecular mechanism for TQ that represents a potential therapeutic strategy in inhibiting TNBC tumor growth and progression. Citation Format: Nashwa N. Kabil, Recep Bayraktar, Nermin Kahraman, Bulent Ozpolat. Thymoquinone inhibits elongation factor 2 kinase signaling axis by inducing tumor suppressor miR-603 in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3249. doi:10.1158/1538-7445.AM2017-3249

BReast CAncer 1 and 2 (BRCA1/2) tumor suppressor genes are frequently mutated in familial breast and ovarian cancers. Recent clinical trials in breast cancers with BRCA1/2 mutations indicate that 50-75% of patients may not have response to poly (ADP-ribose) polymerase inhibitors (PARP) inhibitors. Thus, identification of novel molecular targets and therapeutic interventions are needed to improve poor clical outcome and patient survival. Here we report that Eukaryotic Elongation Factor-2 Kinase (EF2K), an atypical kinase, is highly upregulated in BRCA1+ mutated cell lines and involved in colony formation, migration/ invasion and enhances the effect of PARP inhibitors. Furthermore, in vivo therapeutic targeting of EF2K by once a week systemic injections with magnetic nanoparticles (MNPs) incorporating EF2K siRNA significantly inhibits growth of BRCA1+ tumors in orthotopic xenograft models in mice by inhibition of tumor cell proliferation, angiogenesis and induction of robust apoptosis, which were associated with in vivo inhibition of clinically relevant molecular pathways including, proliferation, survival/drug resistance (PI3K/Akt, c-Myc), cell cycle (Cyclin D1), motility/invasion (Src/FAK/Paxillin), translation (4E-BP1), angiogenesis (VEGF) and stem-cell markers. No toxicity was detected during the study and by postmortem analysis of major organs, indicating that targeting EF2K is safe. The combination of PARP inhibitor (AZD2281, Oleparib) with MNP-EF2K siRNA enhanced significantly growth inhibition in MDA-MB-436 cells indicting EF2K siRNA sensitize BRCA1 + mutation breast cancer cells to PARP inhibitors. Collectively, our results suggest, for the first time, that EF2K is involved in tumorigenesis and progression and may serve as a potential therapeutic target in BRCA1+ mutated breast cancers. Citation Format: Elif Asik, Nermin Kahraman, Tulin Guray, Murvet Volkan, Gabriel Lopez-Berestein, Bulent Ozpolat. Eukaryotic Elongation Factor 2 Kinase (eEF-2K) is a novel therapeutic target in BRCA1+ mutated breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1125. doi:10.1158/1538-7445.AM2017-1125