3167 - INHBA is a Key Mediator of Proton Radiotherapy in Reversing Photon-Induced Radioresistance in Breast Cancer
Presenter(s)
Z. Xu1,2, F. Xu2, Y. Lin1, and J. Chen1; 1Department of Radiation Oncology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China, 2Shanghai Key Laboratory of Proton-therapy, Shanghai, China
Purpose/Objective(s):
Proton radiotherapy is the most advanced radiotherapy technology in contemporary practice with unique physical property, yet its antitumor biological mechanisms remain poorly understood which significantly limited its clinical application. Resistance following photon radiotherapy remains a critical challenge in breast cancer (BC) treatment, often leading to early recurrence. This study focused on exploring the mechanisms of photon-induced radioresistance and proton radiotherapy’s potential to reverse this resistance in BC.Materials/Methods:
The photon-radioresistant T47D cell lines (T47D-R) were established by exposing to a total dose of 60 Gy of 6 MV X-ray irradiation. Subsequently, the transcriptomes of the T47D and T47D-R were sequenced and analyzed. CRISPR/Cas9-based screening was used to identify genes associated with proton/photon radiosensitivity after 10 Gy irradiation. By integrating these two approaches, candidate genes associated with proton radiosensitivity yet photon radioresistance were identified. RT-qPCR was utilized to quantify the mRNA expression levels of relevant genes and siRNA was employed to silence the target genes in T47D-R cells and apoptosis was detected by flow cytometry.
Results:
After photon irradiation, the comet assay, apoptosis rate and expression of apoptosis-related proteins in T47D-R were significantly decreased, while surviving fraction of colony formation and the cell proliferation rate increased, compared to parental T47D.
Compared with the transcriptomes of T47D, 1,052 genes in the T47D-R cell line significantly upregulate (Gene Set A) which are associated with photon-induced radioresistance. Through CRISPR/Cas9-based screening, compared with the non-irradiated group, there were 965 and 968 potential proton-sensitive (Gene Set B) and photon-sensitive genes (Gene Set C) were detected, respectively. By taking the intersection of these gene-sets, 27 candidate genes associated with proton radiosensitivity and photon radioresistance were finally obtained.
RT-qPCR results indicated that the expression level of the INHBA gene was significantly upregulated in T47D-R compared with T47D. Furthermore, the expression of INHBA in both T47D and T47D-R cells increased remarkably after 6 Gy proton radiation. Flow cytometry analysis revealed that the rate of apoptosis in T47D-R cells when knockdown INHBA was significantly higher after photon radiation but decreased after proton radiation. By analysis of the TCGA database, the high expression of INHBA is also showed significant association with poor survival and increased risk of local recurrence in BC which is consistent across different molecular subtypes.
Conclusion:
These findings highlight INHBA as a potential target for personalized proton radiotherapy strategies in photon-radioresistant BC. Further studies are warranted to elucidate INHBA’s mechanistic pathways and optimize proton-based regimens.