Background: Clinical management of BC includes radiation therapy (RT), with most women receiving RT as part of their treatment. Although effective, many women develop locoregional recurrence, including a disproportionate number of women with triple-negative or inflammatory BC. Unfortunately, the molecular mechanisms that underly RT response and intrinsic radioresistance are poorly understood. We hypothesized that transcriptomic and proteomic changes that occur after ionizing radiation in intrinsically radiosensitive and resistant BC models would offer mechanistic insight into mediators of this differential response. Methods: Intrinsic radiosensitivity across all 10 cell lines was measured with clonogenic survival assays as the surviving fraction (SF) after 2 Gy RT. Gene expression changes were assessed by RNA-Seq 24 hours after 4 Gy RT. For long-course RT, cell lines were treated with fractionated RT (2 Gy x 5 fractions). For in vivo mouse xenograft experiments mice received fractionated RT (2 Gy x 6 fractions). Differential gene expression analysis with DeSeq2 was performed on all samples, followed by pathway analysis with Advaita Bioinformatics’ iPathwayGuide. Protein was collected 1, 12, and 24 hours after RT for RPPA analysis evaluating expression changes in 100 proteins and phospho-proteins with SuperCurve. Results: Clonogenic survival identified a wide range of radiation sensitivity in human BC cell lines (SF 83% - 19%) with no significant correlation (r %lt 0.3) to intrinsic BC subtype. The most highly affected pathways in both resistant and sensitive cell lines 24 hours after RT include cell cycle, cellular senescence, and estrogen signaling pathways. For the long-course RT samples, several pathways were significantly altered in fractionated samples only, including MAPK and Hippo signaling and EGFR tyrosine kinase inhibitor resistance. From the in vivo experiments, pathways uniquely affected in the in vivo samples include IL-17 signaling and transcriptional misregulation in cancer. From the proteomic data, we found that proteins including p53, Bcl-2 family proteins, and cell cycle proteins exhibit expression changes after 1 hour. A significant number of pathways (N=69, p %lt 0.01, FDR 0.05) were affected in radioresistant BC models compared to radiosensitive cell lines and these pathways may underlie intrinsic radioresistance. Conclusions: Ionizing radiation induces transcriptomic and proteomic expression changes that differ between intrinsically sensitive and resistant BC models in both single fraction and fractionated studies. Pathways identified in these analyses offer potential insight into the mechanisms underlying intrinsic radioresistance and suggest biologic vulnerabilities that may be targeted to more effectively treat women at a high risk of local BC recurrence. Genome wide CRIPSR-Cas9 screens are currently underway in these breast cancer models to confirm these vulnerability targets. Citation Format: Breanna N. McBean, Anna R. Michmerhuizen, Kari Wilder-Romans, Benjamin C. Chandler, Lynn M. Lerner, Connor Ward, Meilan Liu, Alan P. Boyle, Corey W. Speers. Molecular mechanisms of intrinsic radioresistance in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2401.
Purpose: Expression of the androgen receptor (AR) has been identified as a driver of tumor growth in triple negative breast cancers (TNBC), and previous work has nominated AR inhibition as a strategy for radiosensitization in AR+ TNBC. Despite its role in radioresistance in AR+ TNBC, the mechanistic role of AR and specifically its role in mediating DNA damage repair in response to radiation therapy (RT) remains unknown. Methods: Nuclear fractionation experiments were performed to assess cellular localization of AR protein in AR+ TNBC cell lines (ACC-422, MDA-MB-453). Cells were cultured in media containing hormones (FBS) with treatment of enzalutamide (ENZA), apalutamide (APA), or darolutamide (DARO). Cells were alternatively cultured in media containing charcoal stripped serum (CSS) without hormones with R1881 stimulation. RNA-sequencing was performed to compare AR+ TNBC cells treated with CSS or R1881 stimulation alone or in combination with ionizing radiation. Reverse phase protein arrays were performed in cells treated with ENZA, RT, or combination treatment. Results: While stimulation with R1881 was sufficient to induce nuclear translocation of AR in MDA-MB-453 cells, AR inhibition with ENZA, APA, or DARO blocked AR nuclear translocation under CSS or FBS growth conditions. When cells were treated with R1881+RT, AR nuclear translocation was induced at similar or greater levels compared to R1881 alone in MDA-MB-453 and ACC-422 cells. Combination treatment of RT with ENZA in the presence of hormones reduced AR nuclear localization (39% reduction in MDA-MB-453 cells and 32% reduction in ACC-422 cells) compared to RT alone. These results suggest that decreased promoter region binding, and gene expression upregulation may be a mechanism of radiosensitization with AR inhibition. In addition, transcriptomic analyses demonstrated at least 979 genes differentially expressed in multiple models. Pathway analyses in these models showed common affected pathways included ECM-receptor interaction, PPAR-gamma activation, PI3K-Akt signaling pathway, and the MAPK/ERK signaling pathway. Proteomic analysis in the same cell lines identified apoptosis, DNA damage, and cell cycle pathway changes after RT when AR-signaling was blocked. Common affected pathways in combined analyses identified PI3K-Akt and MAPK/ERK signaling pathway changes that may be responsible for this radiosensitizing phenotype. Conclusions: Our data suggest that AR inhibition in AR+ TNBC is sufficient to inhibit AR nuclear translocation suggesting that AR may play a nuclear role in response to RT to promote DNA repair and radioresistance. We identify potential pathways, including ECM-receptor interaction, PI3K-Akt signaling pathway, and the MAPK/ERK signaling pathway that may be regulated by AR in response to RT and therefore may be responsible for radioresistance. Citation Format: Anna R. Michmerhuizen, Andrea M. Pesch, Benjamin C. Chandler, Lynn M. Lerner, Connor Ward, Leah Moubadder, Stephanie The, Breanna McBean, Caleb Cheng, Lori J. Pierce, Corey W. Speers. Multiomics analysis to uncover the mechanism of radiosensitization of AR-positive triple negative breast cancers with AR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3307.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.