Pinar Kanlikilicer

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.

Rac1/Pak1/p38/MMP-2 Axis Regulates Angiogenesis in Ovarian Cancer

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.

The ZNF304-integrin axis protects against anoikis in cancer

Ovarian cancer (OC) is a highly metastatic disease, but no effective strategies to target this process are currently available. Here, an integrative computational analysis of the Cancer Genome Atlas OC data set and experimental validation identifies a zinc finger transcription factor ZNF304 associated with OC metastasis. High tumoral ZNF304 expression is associated with poor overall survival in OC patients. Through reverse phase protein array analysis, we demonstrate 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 dual assembly nanoparticle leads to sustained gene silencing for 14 days, increased anoikis and reduced tumour growth in orthotopic mouse models of OC. Taken together, ZNF304 is a transcriptional regulator of β1 integrin, promotes cancer cell survival and protects against anoikis in OC.

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.

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.

Abstract 763: Exosomal miR-6126 as a novel tumor suppressor in ovarian cancer

Tumor microenvironment has been known to play a key role in cancer by the development of drug resistance, epithelial-mesenchymal transition, metastasis and angiogenesis. There is emerging evidence that cancer originated exosomes may contribute to the tumor microenvironment to promote tumorigenesis. Exosomes are nano-sized vesicles that contain non-coding RNAs such as miRNAs or siRNAs. Non-coding RNAs are involved in the pathogenesis of the majority of cancer and reveals a new layer of complexity in the molecular architecture of tumorigenicity. The purpose of this study is to identify miRNA content of both sensitive and resistant ovarian cancer-cell derived exosomes for the identification of exosomal RNA-mediated tumorigenesis in ovarian cancer. Using miRNA microarray profiling, we identified differentially expressed exosomal miRNAs in three sensitive (Heya8, Skov3, A2780) and resistant (Heya8-MDR, Skov3-TR, A2780-CP20) isogenic ovarian cancer cell lines. miR-6126 was found to be only miRNA significantly up-regulated in all cancer cell exosomes released to tumor microenvironment. In vitro functional assays showed that miR-6126 act as a tumor suppressor by inhibiting proliferation, migration, invasion and tube formation. miR-6126 acts as a novel potential tumor suppressor through targeting integrin beta-1, leading to inhibition of PI3K/AKT and Ras/Raf signaling. In vivo experiments also demonstrated that administration of miR-6126 into the nude mice with established tumors inhibited tumor weight. Immunohistochemistry results showed that miR-6126 treatment inhibits many oncogenic functions such as proliferation and angiogenesis of ovarian cancer. We believe that these findings help the identification of exosomal RNA-mediated tumorigenesis in ovarian cancer. Citation Format: Pinar Kanlikilicer, Mohammed Mohammed H. S. Rashed, Recep Bayraktar, Rahul Mitra, Cristina Ivan, Burcu Aslan, Xinna Zhang, Rodriguez-Aguayo Cristian, Emine Bayraktar, Martin Picher, Bulent Ozpolat, George A. Calin, Anil K. Sood, Gabriel Lopez-Berestein. Exosomal miR-6126 as a novel tumor suppressor in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 763.

Abstract LB-169: Ovarian cancer cells selectively release exosomes containing tumor suppressor miRNAs to maintain SRC-mediated oncogenic activities

Exosomes have emerged as an important mediator of diverse biological functions including tumor cell progression, invasion, immune escape, and cell-to-cell communication, through the release of molecules such as mRNAs, microRNAs, and proteins. Ovarian cancer (OC) is one of the lethal cancers with extensive local invasion, metastasis, and poor diagnosis. Thus, finding novel targets for OC therapy are urgently needed. Here, we isolated 30-140 nm exosomes from the supernatant of one normal and six different OC cells. In addition, RNAs were isolated from cells and exosomes, and profiles of two miRNA fractions were obtained using affymetrix microarray analysis. By comparing signal intensities of microarray data and validation using RT-PCR analysis, we found that miR-940 was abundant in exosomes from SKOV3IP1, HeyA8, and HeyA8-MDR. Ectopic expression of miR-940 inhibited proliferation, colony formation, invasion, migration, induced G0/G1 cell cycle arrest, and apoptosis in OC. Overexpression of miR-940 also inhibited tumor cell growth in vivo . The target gene of miRNA-940 and the downstream pathway were further investigated using several target prediction algorithms. We identified specific targeting sites for miR-940 in the 3’-untranslated region (3’-UTR) of SRC. We then experimentally validated miR-940 as a direct regulator of SRC using cell transfection and luciferase assays, and showed that miR-940 inhibited SRC expression at mRNA and protein levels. Following this reduction, the activity of proteins downstream of SRC, such as FAK, paxillin and Akt were also reduced. Collectively, our results suggest that OC cells selectively secreted the tumor suppressive miR-940 into the extracellular environment via exosomes, to maintain their invasiveness and tumorigenic phenotype. Citation Format: Mohammed H. Rashed, Pinar Kanlikilicer, Cristian Rodriguez Aguayo, Nashwa N. Kabil, Martin Pichler, Recep Bayraktar, Emine Bayraktar, Cristina Ivan, Rahul Mitra, George A. Calin, Anil K. Sood, Mohamed F. Abd-Ellah, Gouda K. Helal, Gabriel Lopez Berestein. Ovarian cancer cells selectively release exosomes containing tumor suppressor miRNAs to maintain SRC-mediated oncogenic activities. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-169.

The “EX” and the “SOMA”: How They Communicate

Exosomes are nano-sized vesicles that are capable of transferring intracellular proteins or nucleic acids contents to the microenvironment and throughout the body. Our current knowledge about the communication mechanisms between exosomes and their target cells still remains limited. The interaction between exosomes and their target cells is likely to require exquisite specificity. The pairing between membrane fusion and trafficking proteins such as SNAREs or Rab proteins may be one of the mechanisms associated with specificity. Exosomes may stimulate antitumor response by presenting tumor antigens to dendritic cells; on the other hand, their contribution to angiogenesis, invasion, and metastasis of cancer may counteract these effects. Exosomes are known to transport miRNA; however, our knowledge of the interactions of these large non-coding RNA family and their effects and specificity on close or far away target cells is mostly unknown. As we improve our understanding of the biological fate of exosomal-miRNA, we will most likely obtain insights into the mechanisms involved in these events. Exosome mediated drug delivery is a promising strategy to efficiently deliver antitumor miRNAs or siRNAs to target cancer cells and to deliver chemotherapeutics for the treatment of malignant tumors. Here, we review some of the current knowledge on these communication mechanisms and address the potential roles of exosomes in cancer development, diagnosis, and development of therapeutics.