Abstract 1246: Driver and signaling cascade in therapy pressure driven cancer cell plasticity
Authors: Poorvi Subramanian, Sreenidhi Mohanvelu, Sivasubramani Narayanan, Afsana Parveen Jahir Hussain, Sabir Salim, Madhi Oli Ramamurthy, Loganayaki Periyasamy, Natarajan Aravindan
Journal: Cancer Research (2025)

Abstract Dynamic adaptations in cancer cell-state due to therapy is a growing concern. Despite multimodal clinical therapy, tumor cells surpass treatment resulting in frequent relapses in rapidly decreasing timeline. We have shown that, in accordance with therapy pressure associated tumor microenvironment, progressive disease (PD) cells exhibit reversible lineage transformation and unparalleled plasticity. However, our understanding of the genetic determinant(s) and/ or mechanism(s) that coordinate cancer cell plasticity is a far reach. Herein, we investigated the crucial cancer cell phenotypic switching in response to therapy pressure. Using a panel of 14 well characterized human neuroblastoma (NB) cells derived during diagnosis (Dx) and PD, we investigated the association of retinal degeneration protein 3 (RD3) status (ELISA) to their migratory and invasive capabilities (transwell assay), clonal expansion (BrDu), EMT (E-CAD, N-CAD, Vimentin, SLUG), metastatic state (Ron-β, MMP-9, MMP-2), stemness maintenance (CD133, CD117, CD114, Oct3/4, Sox2, NANOG, CD54, NOTCH1) and cellular dedifferentiation (β-3 tubulin, NF-L, GFAP, GAP43) (immunoblotting). Further, to investigate the function of RD3 in coordinating cancer cell plasticity, we utilized six select NB cells, three derived during Dx and three derived during PD, representing both primary and metastatic tumors. These patient derived cells were reverse engineered for stable expression of RD3 in RD3- PD cell lines; stable silencing of RD3 in RD3+ Dx cell lines and were subjected to end-point readouts as above. RD3 loss in PD correlated with the increased cellular migration, invasion, metastatic potential, EMT and stemness maintenance. Directed simulation experiments with stable restoration and silencing systems clearly identified the function of RD3-loss in cancer cell plasticity by regulating stemness (ALDH, CD133), EMT (Vimentin, E-CAD, N-CAD), metastasis (Ron-β, MMP-9), pluripotency maintenance (Oct3/4, Sox2, NANOG), dedifferentiation (β-3 tubulin, NF-H, GFAP, GAP43) and clonal expansion (BrDu). Our findings for the first time identified the defined function and relevance of RD3 in regulating cancer cell EMT, stemness and pluripotency maintenance, metastatic state and prompting differentiation. Crucially, our findings recognized therapy pressure directed RD3 loss dictates cancer cell lineage transformation to cancer stem cells. These outcomes together pave way to develop novel and effective molecular-targeted maintenance therapeutic strategy for patients presented with therapy defying progressive disease. Funding: This work was partially or in full, funded by Department of Defense CA-210339; OCAST-HR19-04; NIH-P20GM103639 to Dr. Aravindan; and by the NIH-NCI-Cancer center support Grant P30CA225520, NIGMS P30GM154635, Oklahoma Tobacco Settlement Endowment Trust [R23-03] to the OU Health Stephenson Cancer Center. Citation Format: Poorvi Subramanian, Sreenidhi Mohanvelu, Sivasubramani Narayanan, Afsana Parveen Jahir Hussain, Sabir Salim, Madhi Oli Ramamurthy, Loganayaki Periyasamy, Natarajan Aravindan. Driver and signaling cascade in therapy pressure driven cancer cell plasticity [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 1246.