Radiotherapy Resistance in Prostate Cancer Cells: AKR1C3 Inhibition of Ubiquitinated Degradation of Nrf2 through Interaction with KEAP1

Abstract

Radiotherapy is the most fundamental treatment for prostate cancer (PCa), and although radiotherapy for overall PCa patients is effective, poor prognosis and resistance to multiple treatments regimes in some highly malignant PCa, such as those with high Gleason Scores (GS) (≥9), are important bottlenecks limiting the improvement of treatment outcomes for clinical. AKR1C3 is a key PCa resistance gene that our team identified previously, but the induction of the specific mechanism of radiotherapy resistance has not been fully revealed and understand. To analyze the correlation between its expression level and clinical radiotherapy, we used the gene expression profiles data of PCa patients in TCGA database. We generated a stepwise increase of radiotherapy dose to established PCa radiotherapy resistant cell lines and detect the AKR1C3 expression level. In addition, to explore the molecular mechanism of AKR1C3 induced prostate cancer radiation tolerance through functional enrichment analysis. Then, to treat cells with cycloheximide and the protein stability of the Nrf2 was detected. Last, the protein ubiquitination level was assayed by co-immunoprecipitation (co-IP) after treatment with MG132. Finally, protein-protein interactions were identified using co-IP to mine possible binding molecules. By analyzing the expression profiles data of PCa patients in the TCGA database, we found that in the population of PCa patients treated with radiotherapy, all patients with high AKR1C3 expression died after radiotherapy, suggesting that high AKR1C3 expression may be a biomarker of resistance to radiation. Accordingly, AKR1C3 expression levels showed a positive correlation with GS score, which may be a symbol for patients with highly malignant PCa. A PCa radiotherapy resistant cell line was constructed by a stepwise increase of ionizing radiation (IR) dose, and the total IR dose of radiotherapy was 84Gy, which reached the dose of radical radiotherapy for prostate cancer. The expression of AKR1C3 was further detected by RT-qPCR and WB, and it was found that the expression of AKR1C3 was significantly up-regulated in the resistant cell line, accompanied by milder DNA damage. What's more, by GSEA functional enrichment analysis, we discovered that AKR1C3 overexpression might be related with intracellular oxidative stress damage. After CHX treatment, the protein stability of Nrf2 was significantly enhanced in AKR1C3 overexpression groups than control groups. And the administration of MG132 showed the same results, indicating that the ubiquitinated degradation of Nrf2 was inhibited in AKR1C3 overexpression groups. Further by co-IP, we found that Nrf2 was less ubiquitinated in the cytoplasm after AKR1C3 overexpression, and AKR1C3 could bind Keap1. In sum, we found that AKR1C3 can bind with Keap1 leading to reduced ubiquitination level of Nrf2, causing upregulation of Nrf2 expression and providing new insights into PCa radiotherapy resistance.