Abstract 2401: Molecular mechanisms of intrinsic radioresistance in breast cancer
Authors: McBean Breanna N. , Michmerhuizen Anna R. , Wilder-Romans Kari , Chandler Benjamin C. , Lerner Lynn M. , Ward Connor , Liu Meilan , Boyle Alan P. , Speers Corey W. 
Published: April 2023

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.