Protein Pathway Activation After Ionizing Radiation In Luminal And Basal Breast Cancer Cell Lines

A growing body of data suggests that breast tumor biology (luminal, basal) contributes to the probability of locoregional control in women with breast cancer. We hypothesized that this may, in part, be due to molecular differences in breast cancer radiation response seen through distinct patterns of protein activation in biologically diverse breast cancer cell lines exposed to ionizing radiation. Six human breast cancer cell lines known to display gene expression patterns reminiscent of distinct clinical breast subtypes (luminal - ZR751, T47D, MCF7; basal - SUM159, SUM149, MDA-MB-231) were selected for further study. Cell lines were maintained in appropriate media and grown in triplicate to account for individual sample variability. After reaching confluence, a single dose of 5 Gy was applied to each line. Total protein was harvested pre-radiation and post-radiation (4 hours, 24 hours, and 7 days). Samples were frozen and shipped to Theranostics Health, Inc. (Rockville, MD) for reverse-phase protein microarray analysis. Forty-five proteins with roles in angiogenesis, cell growth, apoptosis, and DNA repair were assessed. Post-processed signal intensities were used to identify distinct signaling activation patterns. Significant (p < 0.05) biological variability is represented by z-score (variation in protein intensity distribution normalized for technical variability) greater than 1.65. Clonogenic cell survival assays were also performed to assess cell line radiosensitivity for correlation with patterns of molecular change. Up to 15 proteins, representing angiogenesis, cell growth/survival and inflammation, from each cell line demonstrated significant biologic variation during the time points measured. Seven proteins demonstrated significant response to radiation in three or more of the evaluated cell lines (Table). T47D and SUM159, two of the more resistant cell lines (surviving fraction [SF] 2 Gy and 5 Gy [Table]), show similar patterns of protein activation and contrast with those of ZR751, the most radiosensitive cell line. These data are the first to link patterns of protein activation to radiation response. Our long-term goal is to further characterize radiation response “pathways” in order to identify biomarkers of radiation resistance and potential targets for clinical trials assessing radiosensitization.TableProtein Activation (z-score)Cell linesAkt S473 XPHIF1αCox2p90RSKE cadherinERK1.2 T202.Y204.p70.S6.Kinase T389.SF 2 GySF 5 GySUM1495.424.694.593.863.423.390.2540.020SUM1592.802.511.691.861.921.770.6600.295MD-MBA-23110.552.070.2460.025T47D4.641.6424.708.280.7400.330ZR7511.744.850.2310.007MCF71.755.125.1923.2015.590.2240.060 Open table in a new tab