Abstract 2801: Functional characterization of long non-coding RNA <i>PAINT</i> and mechanistic insights into its crosstalk with splicing factor hnRNPM in cancer progression

Abstract Long non-coding RNAs (lncRNAs) modulate crucial cellular processes in nexus with miRNAs, transcription factors and RNA-binding proteins (RBPs) whose dysregulation is often linked with cancer progression and drug resistance. In aggressive, recurrent and often fatal invasive cancers, there is an unmet need to identify newer targets and their mechanistic roles in cancer progression for improved therapies. Previously, we reported an association of lncRNA PAINT with aggressive prostate cancer (PCa) and its involvement in dysregulated expression of several cancer hallmark genes including genes in WNT and AKT signaling pathways. In this study, we tested our hypothesis on the general involvement of PAINT in cancer. Our study showed an oncogenic role of lncRNA PAINT in metastatic breast cancer (BCa) cells. GSE database-based expression analysis showed PAINT upregulation in tumor samples compared to normal patient samples. In-situ hybridization (ISH) of tissue microarrays revealed differential upregulation of PAINT in aggressive BCa subtypes. PAINT inhibition decreased proliferation and migration of triple negative BCa (TNBC) cell lines MDA-MB-231 (BRCA+) and MDA-MB-436 (BRCA-), and improved the sensitivity of MDA-MB-231 cells to Doxorubicin, Docetaxel and Carboplatin. PAINT over-expression in an inducible MDA-MB-231-PAINT stable subline augmented cell proliferation and migration and increased S-phase cell population. Mass spectrometry of PAINT-bound proteins using in-vitro transcribed biotinylated PAINT and PC-3 cell extracts revealed hnRNPM as an interactor of PAINT, which was validated by RNA immunoprecipitation in both BCa and PCa cell lines. Ectopic expression of PAINT in an inducible PC-3-PAINT PCa subline showed an increased hnRNPM concentration upon induction, promoting CD44 exon-skipping function of hnRNPM in PC-3-PAINT and C4-2B-PAINT PCa stable sublines. In-silico analysis showed prospective hnRNPM-interacting sites on PAINT, overlapping with its stem-loop structure forming regions. hnRNPM knockdown in induced MDA-MB-231-PAINT cells diminished the effect of PAINT over-expression on cell proliferation and migration. Our study demonstrates that the oncogenic role of PAINT is partly mediated by the stabilization of its interactor hnRNPM, in turn exerting its tumorigenic and epithelial-mesenchymal transition effects on cancer progression. This study establishes the benefit of PAINT-silencing on enhancing the antitumor effects of hnRNPM knockdown, that can be utilized in combination with chemotherapeutics for treating aggressive forms of BCa and PCa. Citation Format: Subhoja Chakraborty, Kavya Ganapathy, Angela Rodio, Manasi Joseph, Mitchell Campbell, Julia Soulakova, Domenico Coppola, Ratna Chakrabarti. Functional characterization of long non-coding RNA PAINT and mechanistic insights into its crosstalk with splicing factor hnRNPM in cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2801.