Ovarian cancer remains one of the most lethal diseases for the female reproductive system with 22,530 new cases and more than 13,980 deaths predicted for 2019 in the United States alone, according to the American Cancer Society. Currently, there is no effective therapy for ovarian cancer patients, in particular for those who become resistant to chemotherapy (cisplatin). We need to identify novel targets for ovarian cancer therapy. We performed RNA deep-sequencing studies in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. Several RNA (messengers and long noncoding) were differentially abundant in cisplatin-resistant versus cisplatin-sensitive cells. Many of these RNAs have not been previously studied in ovarian cancer. Therefore, we performed rigorous bioinformatic studies that included data filtering and survival rates (using Kaplan-Meier plot analysis) and IPA (Ingenuity Pathway Analysis) to select key candidate genes that can be used as potential targets for ovarian cancer therapy. Additional data filtering was used to select a list of candidate genes whose expression correlates with the overall survival and progression-free survival of ovarian cancer patients. Future experiments will investigate the biologic consequence of targeting each of these candidate genes in ovarian cancer cells and ovarian cancer mouse models. Citation Format: Ricardo Noriega, Jeyshka Reyes, Blanca Quiñones, Joseline Serrano, Pablo Vivas, Josué Pérez. Identification of novel targets for ovarian cancer treatment [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A57.
Background: Breast cancer (BC) is the most diagnosed cancer in women worldwide. MicroRNAs (miRNAs) are involved in different processes of BC and their deregulation can cause them to act as oncogenes or tumor suppressors, participating in cancer progression or also as therapeutic target. Using The Cancer Genome Atlas (TCGA) database, we found that miR-660-5p is significantly overexpressed and associated with poor survival in patients with this pathology. Moreover, it is reported that miR-660-5p can induce BC progression through transcription factor CP2 (TFCP2) and the down regulation of tet-eleven translocation 2 (TET2). In this project, we propose to identify the role of miR-660-5p in proliferation, migration, invasion, angiogenesis, and possible targets involved in these processes in BC cell lines. Methods: Basal levels of miR-660-5p were determined in BC cells MDA-MB-231 and MCF-7, and in human epithelial breast cells MCF-10A by RT-qPCR. The effect of miR-660-5p was evaluated on proliferation, migration, and invasion processes using MDA-MB-231 and MCF-7 cells. HUVEC cells were used to assess angiogenesis. All cell lines were transfected with miR-660-5p inhibitor. Analysis of nine miRNA-target prediction databases was made to identify targets of miR-660-5p. We selected the target genes predicted by at least three of these programs, and their expression was evaluated by RT-qPCR in a customized 384-well plate. Results: We found that miR-660-5p is significantly upregulated in MDA-MB-231 and MCF-7, compared to MCF-10A cells. In addition, we observed a significant decrease in proliferation, migration, and invasion of BC cells transfected with miR-660-5p inhibitor, compared to non-treated cells and miRNA inhibitor negative control treated cells. Similarly, we observed a significant decrease in angiogenesis of HUVEC cells transfected with miR-660-5p inhibitor. Furthermore, of all the miR-660-5p targets identified by prediction databases 21 were selected, and of these, 7 were observed upregulated and 1 downregulated. Conclusions: The results show that miR-660-5p is upregulated and involved in proliferation, migration, invasion, and angiogenesis of BC, which may lead us to suggest that this miRNA act as an onco-miRNA. In addition, seven potential miR-660-5p target genes were identified, but further validation assays are needed to clarify their implication in this disease. Citation Format: Valeria Villarreal-García, José Roberto Estupiñan Jimenez, Ricardo Noriega, Recep Bayraktar, Diana Reséndez-Pérez, Cristina Rodríguez-Padilla, José Manuel Vázquez-Guillén, Gabriel Lopez-Berestein, Pablo E. Vivas-Mejía, Vianey Gonzalez-Villasana. Effect of miR-660-5p in breast cancer progression [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-22-03.
Background: MiRNAs are non-coding RNA molecules and its function is the regulation of gene expression. In cancer, the deregulation of miRNAs allows them to act as oncogenes or tumor suppressors. From an analysis of the expression of miRNAs in breast cancer (BC) in The Cancer Genome Atlas (TCGA), it was identified that miR-1307-3p is significantly overexpressed in the tumor tissue compared to healthy tissue from patients. So far, in BC, it has only been reported that this miRNA inhibits SMYD4 and that it is involved in resistance to cisplatin through its effect on Mdm4. In this project we propose to identify the role of miR-1307-3p in proliferation, migration, invasion, angiogenesis, and possible targets involved in these processes in BC cells. Methods: RT-qPCR was used to evaluate basal levels of miR-1307-3p in the BC cell lines MDA-MB-231 and MCF-7, and the human epithelial breast MCF-10A cells. Later, we determined the effect of miR-1307-3p on proliferation, migration, and invasion in MDA-MB-231 and MCF-7, and angiogenesis in the HUVEC endothelial cells. All assays were carried out using the miR-1307-3p inhibitor. Finally, nine miRNA-target prediction databases were analyzed to identify potential miR-1307-3p target genes, and their expression was analyzed by RT-qPCR in a designed 384-well plate. Results: We found that miR-1307-3p is overexpressed in MDA-MB-231 and MCF-7, compared to MCF-10A cells. We also identified that transfection with the miR-1307-3p inhibitor causes a significant decrease in the processes of proliferation, migration, invasion, and angiogenesis, when compared with untreated or negative control transfected cells. For its part, prediction databases analysis allowed us to identify 19 potential targets of miR-1307-3p. Finally, by RT-qPCR, the overexpression of 3 and the downregulation of 2 genes were confirmed. Conclusions: MiR-1307-3p is overexpressed in BC cells. Furthermore, miR-1307-3p induces the processes of proliferation, migration and invasion in BC cells, and angiogenesis in HUVEC cells. These observations suggest that miR-1307-3p can acts as an onco-miRNA. In addition, the expression of 5 of the predicted target genes were altered by miR-1307-3p inhibitor. Further analysis to validate the implication of this miR-1307-3p targets are needed to understand its importance in BC. Citation Format: José Roberto Estupiñan Jimenez, Valeria Villarreal-García, Ricardo Noriega, Recep Bayraktar, Diana Reséndez-Pérez, Cristina Rodríguez-Padilla, José Manuel Vázquez-Guillén, Fermín Mar-Aguilar, Gabriel Lopez-Berestein, Pablo E. Vivas-Mejía, Vianey Gonzalez-Villasana. Functional effect of miR-1307-3p on breast cancer progression [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-22-04.
Worldwide, the number of cancer‐related deaths continues to increase due to the ability of cancer cells to become chemotherapy‐resistant and metastasize. For women with ovarian cancer, a staggering 70% will become resistant to the front‐line therapy, cisplatin. RNA binding protein with multiple splicing (RBPMS) is a member of family proteins that bind to the nascent RNA transcripts regulate their processing, which includes splicing, transport, localization and stability. Evidence indicates that RBPMS interact with c‐Fos, a member of the AP‐1 family that dimerizes with c‐Jun to stimulate AP‐1 transcriptional activity. Furthermore, RBPMS interacted with transcription factor Smad3, which bind to c‐Jun increasing AP‐1 transcriptional activity. Three major RBPMS isoforms have been described, RBPMS‐A (21.8 kDa), RBPMS‐B (22.4 kDa) and RBPMS‐C (24.2 kDa). Reports have shown that each RBPMS isoform interacts with proteins and/or RNAs responsible for cell growth, proliferation, and tumor progression. Previous reports of our laboratory have found that RBPMS expression decrease in cisplatin resistance compared with cisplatin sensitive cells. However, the specific RBPMS isoform responsible of this observation has not been elucidated and less is known about its role on invasiveness ability and angiogenesis capacity. We overexpressed each RBPMS variants (A, B and C) in the cisplatin resistant ovarian cancer cells, A2780CP20, and performed cell viability, cell proliferation and invasion assays. Significant reduction in the number of colonies and their area were observed in both isoform A and isoform C clones, compared with isoform B and control empty vector clones. However, clones of isoform A were more sensitive to cisplatin treatment in comparation with isoform B and C clones. In vivo experiments showed a significant reduction in tumor growth between RBPMS A and C when compared with the controls. Tumor tissues were subjected to immunohistochemical analysis for proliferation (Ki67) and angiogenesis (CD31). Elevated signals of both antibodies were detected in tissues from control groups when compared with RBPMS A and C overexpressing tumor tissues. These studies along with the RNAseq suggest that RBPMS, regulate transcripts involved in cell proliferation and invasion capacity of ovarian cancer cells. Moreover, RBPMS A and C represses uncontrolled proliferation, invasiveness, and angiogenesis in ovarian cancer cells. However, only the isoform A contributed to the cisplatin sensitivity of ovarian cancer cells. Future efforts on this project will be focused on determining the RBPMS isoform that represses AP‐1 (c‐Fos and c‐Jun) dependent gene regulation in cisplatin sensitive ovarian cancer cells.
Ovarian cancer ranks 5th in cancer‐related deaths among women around the world. According to the American Cancer Society, it is estimated that for 2020, there will be 21,750 newly diagnosed ovarian cancer women and 13,940 related deaths in the US alone. Around 94% of ovarian cancer patients survive five years after diagnosis when the disease is detected at an early stage. However, less than 45% of women patients survive when this disease is diagnosed at advanced stages. Currently, there is no effective therapy for ovarian cancer patients, in particular for those that become resistant to chemotherapy (cisplatin). Thus, we need to identify novel targets for ovarian cancer therapy. We performed RNA deep sequencing studies in cisplatin‐sensitive and cisplatin‐resistant ovarian cancer cells. Several RNA (messengers and long non‐coding) were differentially abundant in cisplatin‐resistant versus cisplatin sensitive cells. Many of these RNAs have not been previously studied in ovarian cancer. Therefore, we performed rigorous bioinformatic studies that included data filtering and survival rates (using Kaplan‐Meier Plot Analysis) and IPA (Ingenuity Pathway Analysis) to select essential candidate genes that can be used as potential targets for ovarian cancer therapy. Additional data filtering was used to choose a list of candidate genes whose expression correlates with the overall survival and progression‐free survival of ovarian cancer patients. Twenty‐seven resulting genes from our bioinformatics analysis were further evaluated by performing a small‐interfering RNA (siRNA)‐mediated silencing and cell growth/proliferation assays in cisplatin‐resistant ovarian cancer cell lines. Future experiments will investigate the biological consequences of targeting each of these candidate genes in ovarian cancer cells and ovarian cancer mouse models.
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