A Studay On The Molecular Mechanisms Of Celastrol Targetting AR Induced Autophagy In Prostate Cancer Treatment

Prostate cancer(PC)is a serious threat to the health of men. Androgen receptor(AR) signaling pathway plays an important role in the development and progression of prostate cancer, thus targeting AR signaling pathway is a standard method for the treatment of early prostate cancer. However, androgen deprivation therapy only can prolong the lifetime of patients for a period of time, and most patients will experience cancer recurrence due to the development of invasive castration resistant prostate cancer(CRPC). It is believed that the reactivation of AR signaling pathway is the main reason for CRPC. Celastrol, a triterpenoid isolated from the traditional Chinese medicine ‘Thunder of God Vine’ has shown potential effectiveness against-PC, because it decreases AR by promoting AR degradation through inhibition of Hsp90 or activation of calpain.Blocking AR signaling pathway has been shown to trigger autophagy in AR positive prostate cancer cell lines. Whether celastrol, as a AR degrader, could induce autophagy in human prostate cancer cells was determined. Our q PCR results showed induction of autophagy-related genes ATG5 and ATG7 after celastrol treatment in LNCa P prostate cancer cells. In accordance with ATG induction, the levels of autophagic marker, LC3 II, were found increased upon celastrol treatment as shown by GFP-LC3 puncta and Western blotting. In addition, p62 protein levels were decreased after celastrol treatment. These results demonstrate that celastrol can induce autophagy and promote the proceed of atophagic flux.Along with autophagy induction, AR protein was decreased by celastrol, suggesting a reverse correlation between AR and celastrol-induced autophagy. To further explore the relationship between AR and autophagy induced by celastrol, AR was knocked down by siRNA to analyze the changes of autophagy. The results showed that knockdown AR could induce autophagy. Inversely, ectopic expression of AR in AR-negative prostate cancer cells, or gain of function of the AR signaling in AR-positive cells, led to suppression of autophagy. These results demonstrated the negative role of AR in celastrol-induced autophagy in prostate cancer cells. Inhibition of AR signaling pathway can induce apoptosis, which is believed to trigger autophagy. To analyze the effect of apoptosis on autophagy induction, we used AR antagonist MDV3100 to inhibit AR signaling pathway, and determined apoptosis and autophagy. Our results demonstrate that targeting AR can induce autophagy under non-apoptotic condition. Furthermore, celastol as a AR degrader also induced autophagy under non-apoptotic condition. Inhibition of apoptosis could not affect celastrol-induced autophagy, while inhibition of celastrol-induced autophagy could promote apoptosis.As a transcription factor, AR not only regulates m RNA, but also regulates the non-coding RNA. Our microarray data showed that miR-101 and miR-17-92 a cluster expressions were down-regulated in the process of autophagy induced by celastrol. Through bioinformatics prediction, we found potential androgen response element in the promoter regions of miR-101 and miR-17-92 a cluster, thus we hypothesize that AR regulates miR-101 and miR-17-92 a cluster at transcription level. We verified that AR could bind to the predicted AREs in the promoter regions of the miR-101 and miR-17-92 a cluster by luciferase reporter assays and Ch IP assays. The expressions of miR-101 and miR-17-92 a cluster were found to correlate with AR status in prostate cancer cell lines. Knockdown of AR led to down regulation of miR-101 and miR-17-92 a cluster in AR positive cell lines. Ectopic expression of AR in AR-negative prostate cancer cells, or gain of function of the AR signaling in AR-positive cells, led to up regulation of miR-101 and miR-17-92 a cluster. These results demonstrate that AR transactivates the expression of miR-101 and miR-17-92 a cluster.Since miR-101 is an inhibitor of autophagy and its expression was decreased along with AR in the process of celastrol-induced autophagy, we hypothesize that AR inhibits autophagy through transactivation of miR-101. The inhibition of celastrol-induced autophagy by AR was compromised by blocking miR-101; while transfection of miR-101 led to inhibition of celastrol-induced autophagy in spite of AR depletion. Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR.In addition, miR-17-92 a cluster was found to inhibit celastrol-induced autophagy. Dissection of miR-17-92 a cluster revealed that miR-17 and miR-20 a were responsible for autophagy inhibition of miR-17-92 a cluster, while miR-18 a could activate celastrol-induced autophagy. Target gene analysis demonstrates miR-17-92 a inhibits autophagy related gene ATG7. Transfection of ATG7 expression vector lack of 3￠UTR demonstrated that miR-17-92 a could inhibit autophagy by regulating ATG7.Finally, autophagy inhibition by bafilomycin or miR-101/miR-17/miR-20 a was found to enhance the cytotoxic effect of celastrol in prostate cancer cells. Our results demonstrate that AR inhibits autophagy via transactivation of miR-101 or miR-17-92 a cluster, thus combination of these autophagy regulated miRNAs with celastrol may represent a promising therapeutic approach for treating prostate cancer.