Abstract Despite advances in therapeutic strategies, epithelial ovarian cancer (EOC) is still the most lethal gynecological malignancy due to late diagnosis, drug resistance, and high relapse rate (∼80%). Although PARP inhibitors (PARPi) like Olaparib have shown significant efficacy against BRCA-mutated tumors, many EOC cases are BRCA wild-type and are intrinsically resistant to these. Moreover, BRCA-mutant tumors also develop resistance to PARPi through BRCA reversion mutations or activation of ATR/CHK1/WEE1 axis. Therefore, novel therapeutic strategies are urgently needed to enhance therapeutic efficacy and overcome resistance mechanisms. Epigenetic alterations of microRNA (miRNA) also contribute to resistance mechanisms. miR-15a (hsa-miR-15a) is a tumor suppressor miRNA that downregulates several oncogenic proteins like WEE1, CHK1, YAP1, and BCL2. miR-15a is frequently downregulated in EOC patients, and restoring the level of miR-15a offers a promising approach for targeted therapy. To enhance therapeutic efficacy, stability, and tumor cell specificity, we engineered MTX-5FU-Gem-miR-15a, combining the therapeutic power of tumor suppressor miR-15a with methotrexate (MTX), 5-fluorouracil (5FU), and gemcitabine (Gem). We hypothesize that this modified miRNA will be able to overcome both intrinsic and acquired PARPi resistance in EOC. To determine its efficacy, Olaparib-resistant EOC cell lines (OVCAR-3 and UWB1. 289) were established through stepwise dose escalation. Both parental and resistant cells were treated with MTX-5FU-Gem-miR-15a or respective controls and evaluated for cell viability (WST-1 assay), induction of apoptosis (Annexin V/PI staining), and cell cycle progression (PI staining). MTX-5FU-Gem-miR-15a significantly reduced cell viability in both parental (IC50: 0. 5-5 nM) and resistant (IC50: 5-15 nM) OC cells without any delivery vehicle, induced S-phase arrest, and significantly increased apoptosis. The therapeutic efficacy of MTX-5FU-Gem-miR-15a was improved by over 1000-fold than that of Olaparib. Western blotting confirmed downregulation of miR-15a target genes. Notably, co-treatment of MTX-5-FU-Gem-miR-15a with olaparib exhibited additional synergistic anti-tumor effects. All experiments were conducted in biological triplicate. Statistical significance was determined using Student’s t-test (p 0. 05). To determine therapeutic efficacy in vivo, a metastatic model of EOC was established by injecting tumor cells via tail vein into female NOD/SCID mice. The mice were then treated with MTX-5FU-Gem-miR-15a (3. 75 mg/kg) or vehicle control. Our results show that MTX-5FU-Gem-miR-15a treatment reduced EOC tumor volume by 5. 5-fold (∼80%) compared to the control group and increased survival without any observable toxicity. Taken together, these results show that MTX-5FU-Gem-miR-15a inhibits tumor growth in both BRCA-mutant and BRCA-wild-type ovarian cancer, and it presents a promising novel miRNA-based therapeutic strategy with strong potential for overcoming drug resistance and enhancing treatment outcomes in both naïve and refractory EOC. Citation Format: Amartya Pal, Anushka Ojha, Jingfang Ju. Development of MTX-5FU-Gem-miR-15a as a novel miRNA-based therapeutic strategy for epithelial ovarian cancer (EOC) abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl): Abstract nr A041.
Pal et al. (Fri,) studied this question.
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