Radiosensitizing Effects of CDK4/6 Inhibitors in Hormone Receptor-Positive and HER2-Negative Breast Cancer are through Downregulating DNA Repair Mechanism and NF-κB-Signaling Pathway

Abstract

CDK4/6 inhibitors combined with endocrine therapy are demonstrated to prolong survival for hormone receptor (HR)-positive and HER2-negative advanced breast cancer. We sought to assess whether cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors can enhance the radiosensitivity and their underlying mechanisms in this subtype of breast cancer through both in vitro and in vivo approaches. We used MCF-7 and T-47D (HR-positive and HER2-negative) breast cancer cell lines, and different doses of CDK4/6 inhibitors (ribociclib and palbociclib) and radiotherapy (RT) to assess the biological functions and mechanisms through in vitro study. The radiation enhancing effect was assessed by clonogenic assay, numbers of the DNA damage response-related molecules, γH2AX and 53BP1, were assessed by immunofluorescence. The p-ERK, c-Myc, and DNA-double strand break (DNA-DSB)-related molecules, p-DNA-PKcs, and p-ATM, were assessed by western blotting. We used NF-κB p65 Transcription Factor Assay Kit to evaluate the transcriptional activity of NF-κB. We evaluated the antitumor effect of combined RT (2 Gy every other day for total 6 Gy) and ribociclib through the MCF-7 orthotopic xenograft model. The synergistic effects of combining RT with different concentrations of ribociclib and palbociclib pretreatment were demonstrated by colonogenic assay. We revealed that CDK4/6 inhibitors synergistically increased the numbers of RT-induced γH2AX and 53BP1, downregulated the expression of p-DNA-PKcs and p-ATM activated by RT, and diminished the expression of RT-triggering p-ERK expression, and NF-κB activation and its down-streaming gene, c-Myc. When compared with control, ribociclib, and RT group, combined ribociclib and RT significantly shrunk the tumor growth of MCF-7 cell xenograft tumors, and downregulated the immunohistochemical expression of p-ERK, p-NF-κB p65, and c-Myc than control group. Our results demonstrated that combining CDK4/6 inhibitors enhanced the radiosensitivity in HR-positive and HER2-negative breast cancer cells, and conceivably radioenhancing mechanisms of CDK4/6 inhibitors are at least through lessening DNA-DSB repairing processes and weakening the activation of ERK signaling and NF-κB signaling by RT.