ESTRO 2024 - Abstract Book

S5403

Radiobiology - Tumour biology

ESTRO 2024

Xue Yang 1,2 , Fanbiao Meng 3 , Bo Xu 1,2,3

1 Chongqing University Cancer Hospital, Institute of Intelligent Oncology, Chongqing Key Laboratory of Intelligent Oncology for Breast Cancer, Chongqing, China. 2 Chongqing University, School of Medicine, Chongqing, China. 3 Tianjin Medical University Cancer Institute and Hospital, Department of Biochemistry and Molecular Biology, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin, China

Purpose/Objective:

To investigate the mechanism of ROCK1 in response to irradiation(IR) in cancer cell lines and find potentialstrategies for cancer therapy.

Material/Methods:

Immunofluorescence microscopy, and single cell electrophoresis assay were used to detect the effect of ROCK1 in response to IR. Colony formation assay were used to detect the radiosensitivity of HeLa cells lacking ROCK1 or its enzyme activity. NHEJ and HR reporter assays were used to detect which pathway is ROCK1 involved to take part in the DNA damage response(DDR). Subcellular fractionation and immunofluorescence microscopy were used to analyze the different protein expression of ROCK1 in nuclei and cytoplasm. Endogenous and exogenous co immunoprecipitation(co-IP) assay were used to detected the interaction between ROCK1 and two major components, XRCC6/Ku70 and XRCC5/Ku80, of non-homologous end jointing(NHEJ) pathway. Western blot assays were used to detect the activation of DNA-PKcs and NHEJpathway. Proteome mass spectrometry was used to identify the specific serine/ threonine(S/T) phosphorate sites of Ku70 and Ku80 mediated by ROCK1. Generating a Ku70 S477A inactive phospho-mutant to detect the effect of Ku70 in response to IR and the activation of NHEJ pathway. Immunofluorescence microscopy and single cell electrophoresis assay indicated that lack of ROCK1 in cells increase DNA double strand breaks(DSBs) after IR. Colony formation assay showed that ROCK1 shRNA stably knocked-down cells as well as cells exposed to ROCK1 inhibitor Fasudil were much more sensitive to IR than that of control cells. NHEJ and HR reporter assays suggested that ROCK1 participates in the IR-induced NHEJ repair and radiosensitivity. Subcellular fractionation and immunofluorescence microscopy indicated that ROCK1 translocate from cytoplasm into nuclei following IR. Co-IP and western blot assays showed that ROCK1 interacts with Ku70/Ku80 complex and is required for DNA-PKcs activity in response to IR. Proteome mass spectrometry found that ROCK1 specifically phosphorylates Ku70 at S477 in response to IR. Furthermore, S477 motif of Ku70 is well conserved in the mammalian species. Immunofluorescence microscopy, single cell electrophoresis assay, NHEJ and HR reporter assays and western blots performed in cells transfected with Ku70 S477A construct showed that ROCK1-mediated Ku70 S477 phosphorylation is essential for DSBs repair and the activation of NHEJ. Cell viability assay also indicated that the phosphorylation of Ku70 S477 limits the responsiveness to IR. Results:

Conclusion: