2840 - Unraveling the Molecular Mechanisms of Radioresistance in Breast Cancer: A Transcriptomic and Proteomic Analysis

S. Lichtman-Mikol¹, R. Abou Zeidane¹, B. McBean², B. Chandler³, B. Hauk¹, D. Gurdak¹, M. Tao¹, V. Mercer¹, and C. Speers¹; ¹Department of Radiation Oncology, University Hospitals Seidman Cancer Center and Case Western Reserve University, Cleveland, OH, ²Department of Human Genetics, University of Michigan, Ann Arbor, MI, ³Department of Radiation Oncology, University of Michigan, Ann Arbor, MI

Purpose/Objective(s): Clinical management of breast cancer (BC) includes radiation therapy (RT), with most women receiving RT after surgery. Although effective, many women develop locoregional recurrence. The molecular underpinnings of RT response and intrinsic radioresistance remain elusive. We hypothesized that transcriptomic and proteomic changes post-RT in radiosensitive and resistant BC models would offer mechanistic insight into mediators of this differential response that might prove clinically actionable. Materials/

Methods: Radiosensitivity across 10 BC cell lines was assessed via clonogenic survival assays, measuring the surviving fraction (SF) after 2 Gy of RT. Gene expression (GE) and pathway analysis were quantified by RNA-Seq 24 hours after 4 Gy RT. Long-course RT involved treating BC lines with fractionated RT (2 Gy x 5 fractions). For in vivo mouse xenograft experiments, mice received fractionated RT (2 Gy x 6 fractions). Protein was collected 1, 12, and 24 hours after RT for quantitative Reverse Phase Protein Array (RPPA) analysis of 293 proteins and phospho-proteins.
Results: A wide range of RT sensitivity was noted in human BC cell lines (SF 83%-19%) with no significant correlation (r < 0.3) to the intrinsic BC subtype. Key pathways enriched in GE changes 24 hours post-RT included cell cycle, cellular senescence, and estrogen signaling. For the fractionated in vitro samples, several pathways were significantly altered in fractionated samples only compared to single fraction, including MAPK signaling, EGFR tyrosine kinase inhibitor resistance, and Hippo signaling. In vivo, pathways uniquely differentially expressed in the animal xenograft samples for both cell lines include IL-17 signaling and transcriptional misregulation in cancer. Protein and phospho-protein changes were most pronounced in p53, Bcl-2 family, HER family, and cell cycle pathway proteins at 1 hour. There were significant differences in protein and phospho-protein changes in luminal vs. basal models, most notably in p53, apoptotic, and ATM/ATR pathways. A significant number of pathways (N=69, p<0.01, FDR 0.05) were affected in radioresistant BC models compared to radiosensitive cell lines and these pathways may underlie intrinsic radioresistance.
Conclusion: RT induces transcriptomic and proteomic expression changes that differ between intrinsically sensitive and resistant and luminal vs. basal 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, including the EGFR, Bcl-xL, and mitotic checkpoint pathways. Genome wide CRIPSR/Cas9 screens are currently underway to confirm these targets in BC models.