Recep Bayraktar

Ubiquitous Release of Exosomal Tumor Suppressor miR-6126 from Ovarian Cancer Cells

Cancer cells actively promote their tumorigenic behavior by reprogramming gene expression. Loading intraluminal vesicles with specific miRNAs and releasing them into the tumor microenvironment as exosomes is one mechanism of reprogramming whose regulation remains to be elucidated. Here, we report that miR-6126 is ubiquitously released in high abundance from both chemosensitive and chemoresistant ovarian cancer cells via exosomes. Overexpression of miR-6126 was confirmed in healthy ovarian tissue compared with ovarian cancer patient samples and correlated with better overall survival in patients with high-grade serous ovarian cancer. miR-6126 acted as a tumor suppressor by directly targeting integrin-β1, a key regulator of cancer cell metastasis. miR-6126 mimic treatment of cancer cells resulted in increased miR-6126 and decreased integrin-β1 mRNA levels in the exosome. Functional analysis showed that treatment of endothelial cells with miR-6126 mimic significantly reduced tube formation as well as invasion and migration capacities of ovarian cancer cells in vitro Administration of miR-6126 mimic in an orthotopic mouse model of ovarian cancer elicited a relative reduction in tumor growth, proliferating cells, and microvessel density. miR-6126 inhibition promoted oncogenic behavior by leading ovarian cancer cells to release more exosomes. Our findings provide new insights into the role of exosomal miRNA-mediated tumor progression and suggest a new therapeutic approach to disrupt oncogenic phenotypes in tumors. Cancer Res; 76(24); 7194-207. ©2016 AACR.

Cell-to-cell communication: microRNAs as hormones

Mammalian cells can release different types of extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies. Accumulating evidence suggests that EVs play a role in cell‐to‐cell communication within the tumor microenvironment. EVs’ components, such as proteins, noncoding RNAs [microRNAs (miRNAs), and long noncoding RNAs (lncRNAs)], messenger RNAs (mRNAs), DNA, and lipids, can mediate paracrine signaling in the tumor microenvironment. Recently, miRNAs encapsulated in secreted EVs have been identified in the extracellular space. Mature miRNAs that participate in intercellular communication are released from most cells, often within EVs, and disseminate through the extracellular fluid to reach remote target cells, including tumor cells, whose phenotypes they can influence by regulating mRNA and protein expression either as tumor suppressors or as oncogenes, depending on their targets. In this review, we discuss the roles of miRNAs in intercellular communication, the biological function of extracellular miRNAs, and their potential applications for diagnosis and therapeutics. We will give examples of miRNAs that behave as hormones.

MicroRNA 603 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting elongation factor 2 kinase

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.

Exosomal miR-940 maintains SRC-mediated oncogenic activity in cancer cells: A possible role for exosomal disposal of tumor suppressor miRNAs

Exosomes have emerged as important mediators of diverse biological functions including tumor suppression, tumor progression, invasion, immune escape and cell-to-cell communication, through the release of molecules such as mRNAs, miRNAs, and proteins. Here, we identified differentially expressed exosomal miRNAs between normal epithelial ovarian cell line and both resistant and sensitive ovarian cancer (OC) cell lines. We found miR-940 as abundant in exosomes from SKOV3-IP1, HeyA8, and HeyA8-MDR cells. The high expression of miR-940 is associated with better survival in patients with ovarian serous cystadenocarcinoma. Ectopic expression of miR-940 inhibited proliferation, colony formation, invasion, and migration and triggered G0/G1 cell cycle arrest and apoptosis in OC cells. Overexpression of miR-940 also inhibited tumor cell growth in vivo. We showed that proto-oncogene tyrosine-protein kinase (SRC) is directly targeted by miR-940 and that miR-940 inhibited SRC expression at mRNA and protein levels. Following this inhibition, the expression of proteins downstream of SRC, such as FAK, paxillin and Akt was also reduced. Collectively, our results suggest that OC cells secrete the tumor-suppressive miR-940 into the extracellular environment via exosomes, to maintain their invasiveness and tumorigenic phenotype.

Therapeutic Targeting of AXL Receptor Tyrosine Kinase Inhibits Tumor Growth and Intraperitoneal Metastasis in Ovarian Cancer Models

Despite substantial improvements in the treatment strategies, ovarian cancer is still the most lethal gynecological malignancy. Identification of drug treatable therapeutic targets and their safe and effective targeting is critical to improve patient survival in ovarian cancer. AXL receptor tyrosine kinase (RTK) has been proposed to be an important therapeutic target for metastatic and advanced-stage human ovarian cancer. We found that AXL-RTK expression is associated with significantly shorter patient survival based on the The Cancer Genome Atlas patient database. To target AXL-RTK, we developed a chemically modified serum nuclease-stable AXL aptamer (AXL-APTAMER), and we evaluated its in vitro and in vivo antitumor activity using in vitro assays as well as two intraperitoneal animal models. AXL-aptamer treatment inhibited the phosphorylation and the activity of AXL, impaired the migration and invasion ability of ovarian cancer cells, and led to the inhibition of tumor growth and number of intraperitoneal metastatic nodules, which was associated with the inhibition of AXL activity and angiogenesis in tumors. When combined with paclitaxel, in vivo systemic (intravenous [i.v.]) administration of AXL-aptamer treatment markedly enhanced the antitumor efficacy of paclitaxel in mice. Taken together, our data indicate that AXL-aptamers successfully target in vivo AXL-RTK and inhibit its AXL activity and tumor growth and progression, representing a promising strategy for the treatment of ovarian cancer.

Improving vascular maturation using noncoding RNAs increases antitumor effect of chemotherapy

Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2-targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.

miR-155 in cancer drug resistance and as target for miRNA-based therapeutics

Small non-coding microRNAs (miRNAs) are instrumental in physiological processes, such as proliferation, cell cycle, apoptosis, and differentiation, processes which are often disrupted in diseases like cancer. miR-155 is one of the best conserved and multifunctional miRNAs, which is mainly characterized by overexpression in multiple diseases including malignant tumors. Altered expression of miR-155 is found to be associated with various physiological and pathological processes, including hematopoietic lineage differentiation, immune response, inflammation, and tumorigenesis. Furthermore, miR-155 drives therapy resistance mechanisms in various tumor types. Therefore, miR-155-mediated signaling pathways became a potential target for the molecular treatment of cancer. In this review, we summarize the current findings of miR-155 in hematopoietic lineage differentiation, the immune response, inflammation, and cancer therapy resistance. Furthermore, we discuss the potential of miR-155-based therapeutic approaches for the treatment of cancer.

Dual suppressive effect of microRNA-34a on the FOXM1/eEF2-kinase axis regulates triple-negative breast cancer growth and invasion

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.

Elongation factor-2 kinase (eEF-2K) expression is associated with poor patient survival and promotes proliferation, invasion and tumor growth of lung cancer

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.

Abstract 3249: Thymoquinone inhibits elongation factor 2 kinase signaling axis by inducing tumor suppressor miR-603 in triple negative breast cancer cells

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