Abstract 1508: Therapy resistant cancer cells containing stress granules display signatures of oxidative stress

Abstract Understanding and overcoming therapy resistance in cancer is crucial, as it remains a major driver of lethality. We have shown that following chemotherapy, surviving cancer cells repeatedly skip mitosis, leading to increased genomic content concomitant with an increase in cell size. This survival mechanism has been observed in multiple tumor types and in response to multiple therapy classes, suggesting a convergent phenotype. We hypothesize that this surviving cell phenotype is the actuator of therapy resistance observed in patients with cancer recurrence. It is widely known that chemotherapies elevate levels of reactive oxygen species (ROS) by indirectly disrupting the electron transport chain, leading to the generation of superoxides. When ROS levels exceed cellular antioxidant capacities, cells experience oxidative stress whereby ROS molecules directly damage important macromolecules, potentially leading to cell death. Using live cell imaging with DCFDA and CellROX dyes, we found that the cells surviving even long after therapy release contain elevated levels of ROS as compared to untreated controls across two cell lines of different tumor types (PC3 prostate cancer and MDA-MB-231 breast cancer) and following treatment with two classes of chemotherapy (cisplatin and docetaxel). In single-cell RNA sequencing datasets, we further find an enrichment for downstream targets of NRF2, a major transcription factor that regulates the response to oxidative stress in cells that survive therapy. This suggests that the cells that survive chemotherapy continue to experience oxidative stress even after therapy is removed and respond by initiating defensive mechanisms to promote survival. Stress granule formation is a common cellular response to oxidative stress that form immediately following a translational arrest. Stress granules are cytoplasmic aggregates of RNA bound by proteins, such as G3BP1, which form a stable core and recruit binding partners to form a mature granule. Stress granules are transient and offer cells the capacity to resume translation rapidly following recovery from stress. Alternatively, autophagy of stress granules during recovery offers cells a rich pool of biomolecules upon relief from stress. In both of our cell line models, we find that while stress granules are virtually absent in untreated cells, up to 18% of surviving cells induce stress granule formation, depending on cell type and chemotherapy. We further demonstrated that surviving cells that containing stress granule have enriched nuclear NRF2 levels, suggesting a positive relationship between stress granule formation and cellular response to ROS. Based on these data, we hypothesize that surviving cells experiencing oxidative stress induce stress granule formation. Future work aims to show the pro-survival role of stress granule formation through time-lapse imaging of cells expressing a G3BP1-GFP construct. Citation Format: Louis T. Rolle, Luke V. Loftus, Kenneth J. Pienta, Sarah R. Amend. Therapy resistant cancer cells containing stress granules display signatures of oxidative stress [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 1508.