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Background. Posttranscriptional regulation is a critical control point for the expression of genes that promote or retard tumor growth. The mRNA-binding protein hnRNP A1 is required for the processing of miR-18a, an inhibitor of k-ras expression. In this study we hypothesized that downmodulation of hnRNP A1 by miR-25 and miR-15a controls the expression of miR-18a and k-ras by modulating nuclear and cytoplasmic miRNA processing at the posttranscriptional level. Methods. In this study, we determined the expression of hnRNP A1 at mRNA and protein levels in three parental ovarian cancer cells lines (Skov3ip1, HeyA8, and A2780) and their chemotherapy-resistant derivatives (Skov3-TR, HeyA8 MDR, and A2780 cp20). We also predicted in silico which miRNAS can target hnRNP A1 and confirmed the miRNA expression by real-time PCR. Moreover, we inhibited miR-25 and mir-15a to determine their tumorigenic potential in vitro. We also correlated the expression of miR-25 and miR-15a and/or miR-18a and k-ras expression in the TCGA data. Results. Downregulation of hnRNP A1 at the RNA and protein levels was observed in the taxane-resistant cell lines but not in the cisplatin-resistant cell line. Furthermore, we predicted in silico that miR-25and miR-15a can target hnRNP A1 and confirmed the overexpression of these microRNAs in SKOV3-TR (5- and 3-fold, respectively) and HeyA8 MDR (3-fold) by real-time PCR. We also analyzed overall survival, trying to find a correlation between k-ras and miR-15a (high 23.77, low 33.97 months), k-ras and miR-25 (high 16.95, low 35.21 months), and the combination of k-ras, miR-25, and miR18a (k-ras high, miR25 high, mir18a low, 20.73 months; k-ras low, miR25 low, mir18a high, 31.49 months). Comparison of these data with a validation set yielded similar results. Conclusion. These results underscore the importance of a previously uncharacterized circuit of posttranscriptional regulation between miR-25 or -15a and RNA-binding protein hnRNP A1 that regulates miR-18a and k-ras expression. This circuit represents a unique opportunity for novel therapeutics approaches. Citation Format: Cristian Rodriguez-Aguayo, Paloma Monroig, Maria I. Almeida, Cristina Ivan, Vianey Gonzalez-Villasana, Maitri Y. Shah, Anil K. Sood, George Calin, Gabriel Lopez-Berestein. Downregulation of hnRNP A1 by miRNAs as a new mechanism of survival in 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 539. doi:10.1158/1538-7445.AM2014-539

EphA2 has emerged as an important target for ovarian cancer therapy in recent years. In the present study, we identified miR-520d-3p as an independent favorable prognostic marker for epithelial ovarian cancer patients. We demonstrate that dual targeting of EphA2 in vitro using EphA2-siRNA and miR-520d-3p exhibits synergistic downregulation of EphA2 and inhibits cell proliferation, migration and invasion. In vivo treatment using DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) nano-liposomes conjugated with miR-520d-3p and EphA2-siRNA showed synergistic therapeutic efficacy and superior inhibition of tumor proliferation and angiogenesis than either monotherapy alone. We also identified that EphA2(low)/EphB2(low)/miR-520d-3p(high) gene signature predicts favorable clinical prognosis for ovarian cancer patients. This study addresses a new concept of RNA inhibition therapy by combining miRNA and siRNA to target oncogenic pathways altered in ovarian cancer. Citation Format: Maitri Shah, Gabriel Berestein Lopez, Anil Sood, George Calin. Therapeutic synergy between novel tumor suppressor miR-520d-3p and EphA2-targeting siRNA in 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 1455. doi:10.1158/1538-7445.AM2014-1455

Selective ER modulators (SERMs) have shown significant efficacy for the prevention of ER-positive breast cancer, however, SERMs are not effective for patients whose tumors do not express ER. Therefore, novel agents are needed for prevention and intervention of high risk ER-breast cancers. The c-Src plays a pivotal role in the progression of EGFR- and HER2-dependent ER negative breast cancer. Thus, targeting Src using agents with minimal toxicity or in combination with other novel agents may prevent/intervene progression of early stage ER- breast cancer. In this study we used Saracatinib (AztraZeneca), a Src inhibitor well-tolerated in phase II clinical trials. We examined the effect of Src inhibition using Saracatinib alone or with other agents for cancer prevention using human mammary epithelial pre-malignant cell lines (MECs) and MMTV-neu mouse model. The MCF10A MECs stably overexpressing ErbB2 (10A.ErbB2 cells) form irregular (grape-like) acini in 3D culture condition. Saracatinib treatment prevents the progression of abnormal 3D acini growth as well reverses the abnormal 3D acini growth of 10A.ErbB2 cells to normal phenotype. Our reverse phase protein array data showed that Saracatinib induces the expression of cell cycle inhibitors and apoptosis related molecules, e.g. P27kip1, RAR beta, and PDCD4. Our 3D acini confocal microscopy experiment showed that it induces luminal apoptosis and suppresses cell proliferation in 10A.ErbB2 cells as detected by activated caspase-3 and ki-67 staining respectively. Next, we used MMTV-neu mouse model which develop ER negative lesions at early stage, to examine correlation between Src activation and ER negative mammary cancer progression. MMTV-neu mice mammary lesions exhibited super-activated Src which were negatively associated with estrogen receptor (ER) expression. Our preliminary data showed that Saracatinib-containing regimen inhibited MMTV-neu mice mammary lesions. Our study demonstrated that targeting Src by Saracatinib may be effective for the chemoprevention of early stage ER negative breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1857. doi:10.1158/1538-7445.AM2011-1857