Multi-omics Characterization of the Breast Cancer Radiation Response Identifies Apoptosis and Cell Cycle Proteins as Mediators of Radiation Resistance

Abstract

Breast cancer (BC) is the most commonly diagnosed cancer in women and the second most deadly. Trimodality therapy consisting of surgery, chemotherapy, and radiation therapy is used to treat the majority of BC patients. However, despite the use of RT, a significant portion of patients, specifically those with basal-like BC, develop recurrences within 5-years of treatment completion. In an effort to understand why patients with basal-like BC are more likely to recur than patients with luminal or Her2-enriched BC we undertook a series of studies to understand the biology underlying radioresistance in basal-like BC. We first performed transcriptomic and proteomic analysis in cell lines representing the spectrum of BC subtypes treated with radiation (RT). RT induced RNA and protein expression changes in cell cycle, DNA damage, and p53 signaling pathways, which may act as modulators of the RT response in basal-like BC and lead to resistance. RT was unable to induce genes and proteins related to apoptosis after RT in radioresistant, p53-mutant BC cell lines, indicating that an inability to activate this pathway may contribute to radioresistance. Activation of the apoptosis pathway, through inhibition of Bcl-2 family anti-apoptotic proteins, radiosensitized p53-mutant, PIK3CA/PTEN wild-type basal-like BC. Specific inhibition of Bcl-xL, but not Bcl-2, lead to radiosensitization of p53-mutant PIKCA/PTEN wild-type basal-like BC. Radiosensitization was mediated through RT induced Mcl-1 degradation, that in combination with Bcl-xL specific inhibition, increased the percentage of apoptotic cells. Overexpression of Mcl-1 in p53-mutant, PIK3CA/PTEN wild-type basal-like BC rescued this radioresistance. In vivo, pan inhibition of Bcl-2 family proteins or specific inhibition of Bcl-xL in combination with RT delayed tumor growth and increase time to tumor doubling and tripling. These data provide a rationale for using Bcl-xL inhibitors for the radiosensitization of p53-mutant, PIK3CA/PTEN wild-type basal-like BC. To expand upon these studies and further identify proteins related to recurrence we correlated gene expression to early recurrence (< 3 years) in four independent datasets with patient outcomes and found TTK, a cell cycle kinase, was most differentially expressed. Inhibition of TTK (both genetic and pharmacologic) radiosensitized basal-like BC. Reintroduction of wild-type TTK, after endogenous TTK knockdown, rescued radiosensitization, however reintroduction of kinase-dead TTK was unable to do so. TTK inhibition (both genetic and pharmacologic) led to unresolved double stranded DNA (dsDNA) damage over time after RT, indicating that TTK inhibition may compromise dsDNA repair efficiency. Using a homologous recombination (HR) specific reporter system, we found that TTK inhibition (both genetic and pharmacologic) decreased HR efficiency. Furthermore, TTK knockdown decreased Rad51 foci formation, a marker for active HR, after RT. Reintroduction of wild-type TTK, after endogenous TTK knockdown, rescued HR repair efficiency and Rad51 foci formation, however reintroduction of kinase-dead TTK was unable to do so. Using a specific non-homologous end joining (NHEJ) reporter system, we found that TTK inhibition (both genetic and pharmacologic) had no effect on NHEJ repair efficiency. In vivo, TTK inhibition (both genetic and pharmacologic) in combination with RT reduced tumor growth and increase time to tumor tripling in subcutaneous basal-like BC cell line models. In an orthotopic patient derived xenograft model, pharmacologic inhibition of TTK in combination with RT synergistically reduced tumor growth and increased time to tumor tripling. Together our results show that a multi-omics characterization of the RT response led to successful nomination of radiosensitization targets that may be clinically tractable.