Mechanisms of Intrinsic Radioresistance in Breast Cancer Identify Potential Therapeutic Vulnerabilities

Abstract

Clinical management of breast cancer (BC) includes radiation therapy (RT) for most women, though the molecular mechanisms that underly RT response and intrinsic radioresistance are poorly understood. Both in vitro and in vivo models aid in our understanding of radiobiology, and we hypothesized that transcriptional changes caused by radiation in vitro in BC cell lines would be recapitulated in an in vivo mouse xenograft model and uncover targetable mechanisms of radioresistance in BC. Radiosensitivity was measured with clonogenic survival assays in 16 cell lines. RNA-seq experiments in vitro and in vivo were performed in an RT resistant (SUM-159) and RT sensitive (ZR-75) cell line 24 hrs after 4 Gy or after 2 Gy x 6 fractions, respectively. Differentially expressed genes (DEGs) were identified from RNA-seq data with DeSeq2 followed by pathway analysis with iPathwayGuide. RT sensitivity was subtype independent in 16 BC cell lines, with SUM-159 radioresistant (SF 0.88) and ZR-75-1 radiosensitive (SF 0.29). There were 75 unique pathways that were significantly altered after RT in SUM-159 cells (53 pathways in vivo only, 36 pathways in vitro only, 14 both conditions; adjusted p-value < 0.05) and 85 unique pathways that were significantly altered after RT in ZR-75-1 cells (16 pathways in vivo only, 72 in vivo only, 3 both conditions; adjusted p-value < 0.05). Pathways that were significantly affected in both cell lines exclusively in the in vitro condition include canonical RT response pathways such as cell cycle, cellular senescence, and DNA replication, though the direction of DEGs were opposite in the two cell lines for each of these pathways. The IL-17 signaling pathway was significantly altered for both cell lines in vivo. Of the pathways that were significantly altered in both conditions for SUM-159 cells, inflammation, including chemokine signaling pathway and cytokine-cytokine receptor interaction, were among the most significant. Significantly more cytokines were upregulated following RT in vivo than in vitro. Cytokines were not upregulated in ZR-75-1 cells in vitro or in vivo. Taken together, the significant changes in the IL-17 pathway and the upregulation of cytokines only in vivo indicate a potential of the tumor microenvironment in the in vivo condition that the in vitro condition lacks. Increased heterogeneity in vivo relative to in vitro may also explain the absence of several canonical RT response pathways in the in vivo conditions for each cell line. Notably, the opposite direction of DEG changes in the canonical RT response pathways between the 2 cell lines with disparate radiosensitivity levels may point to important biologic vulnerabilities that may be targeted in the resistant SUM-159 cells. Future studies are underway using additional BC cell lines and single-cell analysis to better understand RT response heterogeneity.