Background: Radioresistance is a major problem in prostate cancer (CaP) radiotherapy (RT). The mechanisms of CaP radioresistance are still unclear. We have recently developed CaP-RR (radioresistant) cell lines which display more aggressive characteristics including increased colony formation, invasion ability, sphere formation capability, and enhanced epithelial mesenchymal transition (EMT) and cancer stem cell (CSC) phenotypes. In addition, we found the PI3K/Akt/mTOR pathway is closely linked with EMT and CSCs expression. Therefore, these CaP-RR cells, representative of the source of recurrence after RT, provide a very good model to mimic the clinical radioresistance condition to find biomarkers and signaling pathways for CaP radiotherapy.
Aim: The objective of this study was to identify candidate proteins and the main signaling pathways involved in CaP radioresistance, validate the identified potential biomarkers in CaP-radioresistant (RR) cell lines and animal xenografts, and perform the functional study from a selected candidate.
Methods: The differential proteins from CaP parental cell lines (PC-3, DU145 and LNCaP) and CaP-RR sublines (PC-3RR, DU145RR and LNCaPRR) were analyzed using LC-MS/MS and identified by a label-free ion count approach. Pathways enriched as a result of radioresistance were assessed. Identified potential markers were validated in CaP-RR cell lines and subcutaneous (s.c) animal xenografts by Western blotting and immunohistochemistry. In addition, the protein fructose-bisphosphate aldolase A (ALDOA) was identified as a key protein in radioresistance and was selected for radiosensitivity study.
Results: A total of 309 signaling pathway proteins were identified to be significantly different between CaP and CaP-RR cells (P ≤ 0.05, fold differences > 1.5, > 80% power). Among these proteins, nineteen are common among three paired CaP cell lines and associated with metastasis, progression, signaling pathways and radioresistance. The PI3K/Akt, VEGF, metabolism and ERK pathways were identified to be associated with CaP radioresistance. The expression of key proteins from the identified pathways was found to be significantly increased in CaP-RR cells and s.c animal xenografts compared to controls. Furthermore, the downregulation of ALDOA combined with RT effectively reduced colony capability, induced more apoptosis and increased radiosensitivity in CaP-RR cells.
Conclusion: CaP radioresistance is associated with EMT and enhanced CSC phenotypes via activation of the PI3K/Akt/mTOR signaling pathway. CaP radioresistance is caused by multifactorial traits and several signaling pathways. Downregulation of ALDOA increases radiosensitivity in CaP-RR cells. Our findings indicate that interfering EMT/CSCs, ALDOA and signaling pathways, in combination with RT is promising for CaP radiotherapy.