Fatma Valiyeva

Silencing Survivin Splice Variant 2B Leads to Antitumor Activity in Taxane-Resistant Ovarian Cancer

Purpose: To study the role of survivin and its splice variants in taxane-resistant ovarian cancer. Experimental Design: We assessed the mRNA levels of survivin splice variants in ovarian cancer cell lines and ovarian tumor samples. siRNAs targeting survivin were designed to silence all survivin splice variants (T-siRNA) or survivin 2B (2B-siRNA) in vitro and orthotopic murine models of ovarian cancer. The mechanism of cell death was studied in taxane-resistant ovarian cancer cells and in tumor sections obtained from different mouse tumors. Results: Taxane-resistant ovarian cancer cells express higher survivin mRNA levels than their taxane-sensitive counterparts. Survivin 2B expression was significantly higher in taxane-resistant compared with -sensitive cells. Silencing survivin 2B induced growth inhibitory effects similar to silencing total survivin in vitro. In addition, survivin 2B-siRNA incorporated into DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) nanoliposomes resulted in significant reduction in tumor growth (P < 0.05) in orthotopic murine models of ovarian cancer, and these effects were similar to T-siRNA-DOPC. The antitumor effects were further enhanced in combination with docetaxel chemotherapy (P < 0.01). Finally, we found a significant association between survivin 2B expression and progression-free survival in 117 epithelial ovarian cancers obtained at primary debulking surgery. Conclusions: These data identify survivin 2B as an important target in ovarian cancer and provide a translational path forward for developing new therapies against this target. Clin Cancer Res; 17(11); 3716–26. ©2011 AACR.

Upregulation of miR-21 in Cisplatin Resistant Ovarian Cancer via JNK-1/c-Jun Pathway

Cisplatin has been the most accepted drug for the treatment of ovarian cancer for almost 40 years. Although the majority of patients with ovarian cancer respond to front-line platinum combination chemotherapy, many patients will develop cisplatin-resistance disease, which is extremely rapid and fatal. Although various mechanisms of cisplatin resistance have been postulated, the key molecules involved in such resistance have not been identified. MiRNAs are endogenously expressed small non-coding RNAs, which are evolutionarily conserved and function as post-transcriptional regulators of gene expression. Dysregulation of miRNAs have been associated with cancer initiation, progression and drug resistance. The oncogenic miRNA-21, one of the best-studied miRNAs, is upregulated in almost all human cancers. However, the regulation of miR-21 in cisplatin resistant ovarian cancer cells has not been assessed. In this study, we measured the miR-21 expression by real-time PCR and found upregulation of miR-21 in cisplatin resistant compared with cisplatin sensitive ovarian cancer cells. Chromatin immunoprecipitation studies demonstrated the association of the c-Jun transcription factor to the pri-mir-21 DNA promoter regions. Blocking the JNK-1, the major activator of c-Jun phosphorylation, reduced the expression of pre-mir-21 and increased the expression of its well-known target gene, PDCD4. Overexpression of miR-21 in cisplatin sensitive cells decreased PDCD4 levels and increased cell proliferation. Finally, targeting miR-21 reduced cell growth, proliferation and invasion of cisplatin resistant ovarian cancer cells. These results suggest that the JNK-1/c-Jun/miR-21 pathway contributes to the cisplatin resistance of ovarian cancer cells and demonstrated that miR-21 is a plausible target to overcome cisplatin resistance.

Molecular targeted enhanced ultrasound imaging of flk1 reveals diagnosis and prognosis potential in a genetically engineered mouse prostate cancer model.

Molecular imaging techniques used to detect the initiation of disease have the potential to provide the best opportunity for early treatment and cure. This report aimed at testing the possibility that Flk1+ (vascular endothelial growth factor receptor 2), a crucial angiogenesis factor of most tumor cells, could be a molecular targeted imaging marker for the diagnosis and prognosis of cancer. We performed Flk1-targeted microbubble-enhanced ultrasound (US) imaging of prostate cancer in a genetically engineered mouse model with normal-appearing intact US (negative) prostates and with three different tumor sizes (small, medium, and large). Higher levels of Flk1+ molecular signals were identified in the intact US (negative) prostate group by US-targeted imaging and immunohistochemical analysis. The increase in Flk1+ expression occurred prior to the angiogenesis switch-on phase and vascularity peak. After this peak accumulation stage of Flk1+ molecules, lower and stabilized levels of Flk1+ signals were maintained together with tumor growth from small, to medium, to large size. In a longitudinal observation in a subset (n = 5) of mice with established tumors, elevated Flk1+ signals were observed in tissues surrounding the prostate cancer, for example, the ipsilateral boundary zones between two developing tumor lobes, new tumor blood vessel recruits, the urethra border, and the pelvic node basin. The potential of Flk1-targeted US imaging as a predictive imaging tool was confirmed by correlation studies of three-dimensional US B-mode imaging, gross pathology, and histology analyses. The results of the application in a genetically engineered mouse model with prostate cancer of molecular Flk1-targeted US imaging support the contention that Flk1 can be used as a molecular imaging marker for small tumors undetectable by microimaging and as a molecular diagnostic and prognosis marker for tumor metastasis and progression.

Targeting miR-21-3p inhibits proliferation and invasion of ovarian cancer cells.

MicroRNA-21 is overexpressed in most cancers and has been implicated in tumorigenesis. Accumulating evidence supports a central role for the miR-21 guide strand (miR-21-5p) in ovarian cancer initiation, progression, and chemoresistance. However, there is limited information regarding the biological role of the miR-21 passenger strand (miR-21-3p) in ovarian cancer cells. The aim of this study was to investigate the role of miR-21-3p and its target genes in cisplatin-resistant ovarian cancer cells. Expression profiling of miR-21-5p and miR-21-3p was performed in a panel of cancer cells by qPCR. Colony formation and invasion assays were carried out on ovarian and prostate cancer cells transfected with miR-21-5p and miR-21-3p inhibitors. Dual luciferase reporter assays were used to identify the miR-21-3p target genes in ovarian cancer cells. Our results show that miR-21-5p had higher expression levels compared to miR-21-3p on a panel of cancer cells. Moreover, inhibition of miR-21-5p or miR-21-3p resulted in a significant decrease in ovarian and prostate cancer cell proliferation and invasion. Luciferase reporter assays identify RNA Binding Protein with Multiple Splicing (RBPMS), Regulator of Chromosome Condensation and POZ Domain Containing Protein 1 (RCBTB1), and Zinc Finger protein 608 (ZNF608) as miR-21-3p target genes. SiRNA-induced RBPMS silencing reduced the sensitivity of ovarian cancer cells to cisplatin treatment. Immunohistochemical analyses of serous ovarian cancer patient samples suggest a significant decrease of RBMPS levels when compared to normal ovarian epithelium. Taken together, the data generated in this study suggests a functional role for miR-21-3p in ovarian cancer and other solid tumors.

Characterization of the Oncogenic Activity of the Novel TRIM59 Gene in Mouse Cancer Models

A novel TRIM family member, TRIM59 gene was characterized to be upregulated in SV40 Tag oncogene–directed transgenic and knockout mouse prostate cancer models as a signaling pathway effector. We identified two phosphorylated forms of TRIM59 (p53 and p55) and characterized them using purified TRIM59 proteins from mouse prostate cancer models at different stages with wild-type mice and NIH3T3 cells as controls. p53/p55-TRIM59 proteins possibly represent Ser/Thr and Tyr phosphorylation modifications, respectively. Quantitative measurements by ELISA showed that the p-Ser/Thr TRIM59 correlated with tumorigenesis, whereas the p-Tyr-TRIM59 protein correlated with advanced cancer of the prostate (CaP). The function of TRIM59 was elucidated using short hairpin RNA (shRNA)-mediated knockdown of the gene in human CaP cells, which caused S-phase cell-cycle arrest and cell growth retardation. A hit-and-run effect of TRIM59 shRNA knockdown was observed 24 hours posttransfection. Differential cDNA microarrray analysis was conducted, which showed that the initial and rapid knockdown occurred early in the Ras signaling pathway. To confirm the proto-oncogenic function of TRIM59 in the Ras signaling pathway, we generated a transgenic mouse model using a prostate tissue–specific gene (PSP94) to direct the upregulation of the TRIM59 gene. Restricted TRIM59 gene upregulation in the prostate revealed the full potential for inducing tumorigenesis, similar to the expression of SV40 Tag, and coincided with the upregulation of genes specific to the Ras signaling pathway and bridging genes for SV40 Tag–mediated oncogenesis. The finding of a possible novel oncogene in animal models will implicate a novel strategy for diagnosis, prognosis, and therapy for cancer. Mol Cancer Ther; 10(7); 1229–40. ©2011 AACR.

Targeting c-MYC in Platinum-Resistant Ovarian Cancer

The purpose of this study was to investigate the molecular and therapeutic effects of siRNA-mediated c-MYC silencing in cisplatin-resistant ovarian cancer. Statistical analysis of patients data extracted from The Cancer Genome Atlas (TCGA) portal showed that the disease-free (DFS) and the overall (OS) survival were decreased in ovarian cancer patients with high c-MYC mRNA levels. Furthermore, analysis of a panel of ovarian cancer cell lines showed that c-MYC protein levels were higher in cisplatin-resistant cells when compared with their cisplatin-sensitive counterparts. In vitro cell viability, growth, cell-cycle progression, and apoptosis, as well as in vivo therapeutic effectiveness in murine xenograft models, were also assessed following siRNA-mediated c-MYC silencing in cisplatin-resistant ovarian cancer cells. Significant inhibition of cell growth and viability, cell-cycle arrest, and activation of apoptosis were observed upon siRNA-mediated c-MYC depletion. In addition, single weekly doses of c-MYC–siRNA incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG-2000)-based nanoliposomes resulted in significant reduction in tumor growth. These findings identify c-MYC as a potential therapeutic target for ovarian cancers expressing high levels of this oncoprotein. Mol Cancer Ther; 14(10); 2260–9. ©2015 AACR.

Targeting MicroRNA-143 Leads to Inhibition of Glioblastoma Tumor Progression

Glioblastoma (GBM) is the most common and aggressive of all brain tumors, with a median survival of only 14 months after initial diagnosis. Novel therapeutic approaches are an unmet need for GBM treatment. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. Several dysregulated miRNAs have been identified in all cancer types including GBM. In this study, we aimed to uncover the role of miR-143 in GBM cell lines, patient samples, and mouse models. Quantitative real-time RT-PCR of RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples showed that the relative expression of miR-143 was higher in GBM patients compared to control individuals. Transient transfection of GBM cells with a miR-143 oligonucleotide inhibitor (miR-143-inh) resulted in reduced cell proliferation, increased apoptosis, and cell cycle arrest. SLC30A8, a glucose metabolism-related protein, was identified as a direct target of miR-143 in GBM cells. Moreover, multiple injections of GBM tumor-bearing mice with a miR-143-inh-liposomal formulation significantly reduced tumor growth compared to control mice. The reduced in vitro cell growth and in vivo tumor growth following miRNA-143 inhibition suggests that miR-143 is a potential therapeutic target for GBM therapy.

Abstract 3437: Targeting microRNA-143 in glioblastoma in vivo increases tumor growth

The purpose of this study is to assess the biological role of the microRNA-143 (miR-143) in Glioblastoma multiforme (GBM). GBM is the most common and lethal of all brain tumors. In the United States, the incidence of GBM is about 17% of all primary brain tumors and about 60-75% of all Astrocytomas (American Brain Tumor Association, 2014). The standard therapy is surgical tumor removal followed by chemotherapy and radiotherapy. However, many patients recur after treatment and the median survival rate for GBM has remained 15 months for the past 20 years. Thus, novel therapies for GBM treatment are urgently necessary. MicroRNAs (miRNAs) are a class of small non-coding RNAs (18-22 nucleotides in length) that regulate gene expression at the post-transcriptional level. MiRNAs bind to the 3’-untranslate region (UTR) of messenger RNAs (mRNAs) and regulate protein synthesis. Several deregulated miRNAs have been identified in all cancer types including GBM. In this study we aim to thoroughly uncover the role of miR-143 by using GBM cell lines, mouse models and patient samples. Total RNA was isolated from FFPE samples from brain tumor patients. TaqMan-based Real-time PCR showed that the relative expression of miR-143 was higher in GBM patients compared to control individuals, and with paired surrounding non-cancerous tissue. Furthermore, GBM cells transiently transfected with a miR-143 oligonucleotide inhibitor showed reduced cell proliferation (68.5%) (clonogenicity assay), increased apoptosis and cell cycle arrest of GBM cells in the S phase (Flow cytometry and Western blots). In vivo studies using primary GBM cells injected in the flank of nude mice showed that repeated doses of miR-143-inhibitor liposomal formulation increased the tumor size compared with control mice. These contradictory results could be due to effects of the microenvironment where the tumor is growing. Further studies will be made using intracranial injections in an orthotopic xenograft mouse model to confirm this hypothesis. Western blot analysis and luciferase reporter assays are also underway to identify novel miR-143 target genes in GBM cells. This research project is being supported by: PRCTRC: NCRR (U54 RR 026139-01A1), NIMHD (8U54 MD 007587-03), and RCMI: MBRS-RISE, NCRR (2G12-RR003051) and NIMHD (8G12-MD007600) from the NIH. Citation Format: Eunice Lozada-Delgado, Fatma Valiyeva, Maria Marcos, Pablo Vivas. Targeting microRNA-143 in glioblastoma in vivo increases tumor growth [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 3437. doi:10.1158/1538-7445.AM2017-3437

Abstract 3761: Nanoliposomal c-MYC-siRNA inhibits in vivo tumor growth of cisplatin-resistant ovarian cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ovarian cancer is the deadliest of gynecological cancers in the United States. With fewer than 15% of cases diagnosed early, ovarian cancer continues to be characterized by late-stage presentation. Treatment for ovarian cancer usually involves surgical cytoreduction followed by platinum-based chemotherapy. Unfortunately, despite initial respond, more than 70% of ovarian cancer patients develop cisplatin resistance, relapse and therapeutic failure. Therefore, there is a need of novel therapies focused on targets within cancer cell survival pathways for advanced stage drug resistant ovarian cancer. Evidence indicates that activation of the oncogenic transcription factor c-MYC is involved in drug resistance. Our previous findings indicate that cisplatin-resistant ovarian cancer cells express higher c-MYC protein levels when compared to their sensitive counterparts. Importantly, targeting c-MYC with small interfering RNA (siRNA) in the cisplatin-resistant ovarian cancer cell line, A2780CP20, induced a significant cell growth arrest and inhibition of cell proliferation. Apoptosis and arrest of cell cycle progression were also observed after siRNA-based silencing of c-MYC. These results were confirmed by Western blot analysis. Furthermore, in vivo delivery of c-MYC-siRNA in a murine xenograft model of cisplatin-resistant ovarian cancer was achieved by using DOPC/PEG-2000-based nanoliposomes. A single weekly injection of nanoliposomal c-MYC-siRNA, during a four week period, decreased tumor weight and number of tumor nodules compared with a liposomal-negative control siRNA. These data propose c-MYC as a potential therapeutic target for cisplatin-resistant ovarian cancer. Citation Format: Jeyshka M. Reyes-Gonzalez, Guillermo N. Armaiz, Lingegowda S. Mangala, Fatma Valiyeva, Sunila Pradeep, Anil K. Sood, Pablo E. Vivas-Mejia. Nanoliposomal c-MYC-siRNA inhibits in vivo tumor growth of cisplatin-resistant ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3761. doi:10.1158/1538-7445.AM2014-3761

Abstract 4376: Upregulation of miR-21 in cisplatin-resistant ovarian cancer via JNK-1/c-Jun pathway

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cisplatin has been the most accepted drug for the treatment of ovarian cancer for almost 40 years. Although the majority of patients with ovarian cancer respond to front-line platinum combination chemotherapy, many patients will develop cisplatin-resistance disease, which is extremely rapid and fatal. Although various mechanisms of cisplatin resistance have been postulated, the key molecules involved in such resistance have not been identified. MiRNAs are endogenously expressed small non-coding RNAs, which are evolutionarily conserved and function as post-transcriptional regulators of gene expression. Dysregulation of miRNAs have been associated with cancer initiation, progression and drug resistance. The oncogenic miRNA-21, one of the best-studied miRNAs, is upregulated in almost all human cancers. However, the regulation of miR-21 in cisplatin resistant ovarian cancer cells has not been assessed. In this study, we measured the miR-21 expression by real-time PCR and found upregulation of miR-21 in cisplatin resistant compared with cisplatin sensitive ovarian cancer cells. Chromatin immunoprecipitation studies demonstrated the association of the c-Jun transcription factor to the mir-21 DNA promoter regions. Blocking the JNK-1, the major activator of c-Jun phosphorylation, reduced the expression of miR-21 and increased the expression of its well-known target gene, PDCD4. Overexpression of miR-21 in cisplatin sensitive cells decreased PDCD4 levels and increased cell proliferation. Finally, targeting miR-21 reduced cell growth, proliferation and invasion of cisplatin resistant ovarian cancer cells. These results suggest that the JNK-1/c-Jun/miR-21 pathway contributes to the cisplatin resistance of ovarian cancer cells and demonstrated that miR-21 is a plausible target to overcome cisplatin resistance. Citation Format: Ileabett M. Echevarria, Joel Encarnacion, Fatma Valiyeva, Pablo Vivas. Upregulation of miR-21 in cisplatin-resistant ovarian cancer via JNK-1/c-Jun pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4376. doi:10.1158/1538-7445.AM2014-4376