Emine Bayraktar

Exosomes: From garbage bins to promising therapeutic targets

Intercellular communication via cell-released vesicles is a very important process for both normal and tumor cells. Cell communication may involve exosomes, small vesicles of endocytic origin that are released by all types of cells and are found in abundance in body fluids, including blood, saliva, urine, and breast milk. Exosomes have been shown to carry lipids, proteins, mRNAs, non-coding RNAs, and even DNA out of cells. They are more than simply molecular garbage bins, however, in that the molecules they carry can be taken up by other cells. Thus, exosomes transfer biological information to neighboring cells and through this cell-to-cell communication are involved not only in physiological functions such as cell-to-cell communication, but also in the pathogenesis of some diseases, including tumors and neurodegenerative conditions. Our increasing understanding of why cells release exosomes and their role in intercellular communication has revealed the very complex and sophisticated contribution of exosomes to health and disease. The aim of this review is to reveal the emerging roles of exosomes in normal and pathological conditions and describe the controversial biological role of exosomes, as it is now understood, in carcinogenesis. We also summarize what is known about exosome biogenesis, composition, functions, and pathways and discuss the potential clinical applications of exosomes, especially as biomarkers and novel therapeutic agents.

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.

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.

Regulation of hnRNPA1 by microRNAs controls the miR-18a– K-RAS axis in chemotherapy-resistant ovarian cancer

The regulation of microRNA (miRNA) biogenesis, function and degradation involves a range of mechanisms, including interactions with RNA-binding proteins. The potential contribution of regulatory miRNAs to the expression of these RNA interactor proteins that could control other miRNAs expression is still unclear. Here we demonstrate a regulatory circuit involving oncogenic and tumor-suppressor miRNAs and an RNA-binding protein in a chemotherapy-resistant ovarian cancer model. We identified and characterized miR-15a-5p and miR-25-3p as negative regulators of hnRNPA1 expression, which is required for the processing of miR-18a-3p, an inhibitor of the K-RAS oncogene. The inhibition of miR-25-3p and miR-15a-5p decreased the proliferation, motility, invasiveness and angiogenic potential and increased apoptosis when combined with docetaxel. Alteration of this regulatory circuit causes poor overall survival outcome in ovarian cancer patients. These results highlight miR-15a-5p and miR-25-3p as key regulators of miR-18a-3p expression and its downstream target K-RAS, through direct modulation of hnRNPA1 expression. Our results demonstrate the therapeutic potential of inhibiting miR-25-3p and miR-15a-5p and the use of miR-18a-3p/KRAS ratio as a prominent outcome prognostic factor.

OncoLncs: Long Non-Coding RNAs with Oncogenic Functions

A decade before the discovery of non-coding transcripts, approximately 98% of the human genome was known as “transcriptional noise” or “junk DNA”. However, with the recent findings, non-coding transcripts are continuously turning into functional non-coding RNAs (ncRNA). NcRNAs comprise multiple classes of RNA transcripts that are not transcribed into proteins but have shown to regulate the transcription, stability, or translation of protein-coding genes in the mammalian genome. Nowadays the most studied ncRNAs are called miRNAs. They have been involved in the biogenesis and development of cancer. More recently, long non-coding RNAs (lncRNAs) were discovered and they have been shown not only to play a role in transcriptional and translational regulation, but also be involved in several diseases including cancer. LncRNAs are RNA transcripts longer than 200 nucleotides that do not encode proteins. The accumulating body of evidence suggests that lncRNAs play important roles in a variety of biological processes. They have been reported to be altered in many pathological states including cancer. LncRNAs with oncogenic functions (we may call; OncoLncs) were reported to be overexpressed in cancer cells and involved in the hallmarks of cancer including sustained proliferation, invasion, and metastasis. The field of regulatory RNAs is continuously growing especially the lncRNAs. Thus, in this comprehensive review, we discussed the advances on OncoLncs field in the malignant transformation of cancer and the therapeutics opportunities.

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.

FABP4 as a key determinant of metastatic potential of ovarian cancer

The standard treatment for high-grade serous ovarian cancer is primary debulking surgery followed by chemotherapy. The extent of metastasis and invasive potential of lesions can influence the outcome of these primary surgeries. Here, we explored the underlying mechanisms that could increase metastatic potential in ovarian cancer. We discovered that FABP4 (fatty acid binding protein) can substantially increase the metastatic potential of cancer cells. We also found that miR-409-3p regulates FABP4 in ovarian cancer cells and that hypoxia decreases miR-409-3p levels. Treatment with DOPC nanoliposomes containing either miR-409-3p mimic or FABP4 siRNA inhibited tumor progression in mouse models. With RPPA and metabolite arrays, we found that FABP4 regulates pathways associated with metastasis and affects metabolic pathways in ovarian cancer cells. Collectively, these findings demonstrate that FABP4 is functionally responsible for aggressive patterns of disease that likely contribute to poor prognosis in ovarian cancer.In ovarian cancer, metastatic phenotype may impact surgical outcomes. Here, the authors show miR-409-3p regulates FABP4 which can increase metastatic potential of ovarian cancer, and treatment with DOPC nanoliposomes containing either miR-409--3p mimic or FABP4 siRNA inhibits tumor progression in mice.