X. Huang, B. Tian, J. Yu, and D. Chen; Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China
Purpose/Objective(s): Radioresistance is the leading cause of radiotherapy failure in esophageal squamous cell carcinoma (ESCC), significantly affecting the therapeutic efficacy and prognosis of ESCC patients. Hence, it is crucial to understand the molecular mechanisms of radiation resistance in ESCC. Integrin a5 (ITGA5) was involved in various biological processes of malignant tumors. However, few studies have focused on the regulation of ESCC radiosensitivity by ITGA5, and the mechanisms still remain poorly understood. Materials/
Methods: RNA-seq analysis of ESCC radioresistant cell lines was initially performed to screen and determine our research subject. KYSE150 and KYSE520 cells were selected to construct stably transduced cell lines expressing ITGA5 by lentivirus system, and the radioresistant functions of ITGA5 were examined using colony formation assay, comet assay, flow cytometry, western blot and nude mice xenograft tumor model. Mechanism analysis was conducted using mass spectroscopy (MS), chromatin immunoprecipitation (CHIP), luciferase reporter assay, and rescue experiment. Immunohistochemistry (IHC) was used to assess ITGA5 expression in ESCC patients receiving adjuvant radiotherapy. Results: Here, we found that ITGA5 expression was increased in recurrent ESCC tissues of patients receiving radiotherapy compared to those without relapse, and a higher level of ITGA5 was associated with poor prognosis. Also, ITGA5 mRNA and protein expression levels were higher in radioresistant cells than in their parental cells. Moreover, further investigation demonstrated that cells with ITGA5 overexpression had an increased ability for proliferation, survival, DNA damage repair (DDR), and resistance to radiation in ESCC, while knockdown of ITGA5 could sensitize ESCC cells to radiation. Further in vivo assays demonstrated that ITGA5 overexpression group exhibited increased tumor volume and tumor weight in a synergistic manner with radiotherapy, whereas ITGA5 knockdown group produced a synergistic reduction in tumor volume and tumor weight when combined with radiotherapy. To further investigate the molecular mechanism of ESCC radiosensitivity, radiation increased the phosphorylation level of the FAK/AKT/GSK3ß/Myc axis by increasing the expression of ITGA5. Meanwhile, our findings indicated that ITGA5 could promote DDR by upregulating RAD51AP1 expression. Myc, a transcription factor, binds to the promoter region of RAD51AP1 and promotes RAD51AP1 transcription upon radiation. Finally, these results were validated in vitro and in vivo by using ATN-161, an ITGA5 antagonist. Conclusion: Overall, our study shows for the first time that ITGA5 plays a key role in radiation resistance, and that targeting ITGA5 could be a promising strategy to improve radiotherapy efficacy in patients with ESCC.
