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Celastrol Induces Autophagy by Targeting AR/miR-101 in Prostate Cancer Cells.

Guo J, Huang X, Wang H, Yang H - PLoS ONE (2015)

Bottom Line: In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy.Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR.Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Technology, Harbin Institute of Technology, Harbin, 150001, China.

ABSTRACT
Autophagy is an evolutionarily conserved process responsible for the degradation and recycling of cytoplasmic components through autolysosomes. Targeting AR axis is a standard strategy for prostate cancer treatment; however, the role of AR in autophagic processes is still not fully understood. In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy. Knockdown of AR in AR-positive prostate cancer cells resulted in enhanced autophagy. 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. 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. AR binding site was defined in the upstream of miR-101 gene by luciferase reporter and ChIP assays. MiR-101 expression correlated with AR status in prostate cancer cell lines. 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. Finally, autophagy inhibition by miR-101 mimic 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, thus combination of miR-101 mimics with celastrol may represent a promising therapeutic approach for treating prostate cancer.

No MeSH data available.


Related in: MedlinePlus

AR suppression mediates miR-101 reduction after celastrol treatment.A, miR-101 and AR expressions after celastrol (CEL) treatment. **, P<0.01, compared with DMSO. B, Schematic depiction of luciferase reporter constructs as detailed in " Materials and Methods". Wild type and mutant AR binding sites were indicated by italic dash and cross, respectively. C, DU145 cells were transfected with indicated reporter constructs in the presence of pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) plasmid for 24 h, and then luciferase activity was detected. pRLSV40 plasmid was co-transfected for normalization. **, P<0.01, between EGFP-AR and EGFP-V transfections. D, ChIP assay. Cell extracts from DU145 or LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) were immunoprecipitated with AR antibody or normal mouse IgG. Input was 1/100 of the sonicated chromatin prior to immunoprecipitation. PCR was performed as described in the "Materials and Methods". E, LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) were treated with celastrol (CEL, 2.0 μM) or DMSO for 3 h, pri-miR-101 and mature miR-101 levels were determined by qPCR. Asterisks denote significance between EGFP-AR and EGFP-V transfections. *, P <0.05; **, P <0.01.
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pone.0140745.g003: AR suppression mediates miR-101 reduction after celastrol treatment.A, miR-101 and AR expressions after celastrol (CEL) treatment. **, P<0.01, compared with DMSO. B, Schematic depiction of luciferase reporter constructs as detailed in " Materials and Methods". Wild type and mutant AR binding sites were indicated by italic dash and cross, respectively. C, DU145 cells were transfected with indicated reporter constructs in the presence of pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) plasmid for 24 h, and then luciferase activity was detected. pRLSV40 plasmid was co-transfected for normalization. **, P<0.01, between EGFP-AR and EGFP-V transfections. D, ChIP assay. Cell extracts from DU145 or LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) were immunoprecipitated with AR antibody or normal mouse IgG. Input was 1/100 of the sonicated chromatin prior to immunoprecipitation. PCR was performed as described in the "Materials and Methods". E, LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) were treated with celastrol (CEL, 2.0 μM) or DMSO for 3 h, pri-miR-101 and mature miR-101 levels were determined by qPCR. Asterisks denote significance between EGFP-AR and EGFP-V transfections. *, P <0.05; **, P <0.01.

Mentions: To see miRNA involvement in the process of celastrol-induced autophagy, LNCaP cells were treated with celastrol for 12 h to induce autophagy. MiRNA array was performed and showed that the expression of miR-101, an autophagy inhibitor, was decreased by celastrol (data not shown). Suppression of miR-101 expression by celastrol was confirmed by RT-PCR, which revealed a ~3-fold decrease of pri-miR-101 and mature miR-101 in LNCaP cells post celastrol treatment (Fig 3A, left and middle). This was accompanied by an almost complete depletion of AR (Fig 3A, right), suggesting a positive correlation between AR and miR-101 expression after celastrol treatment. AR is a transcriptional factor and its binding site has been predicted in the upstream region of the miR-101 gene [13]. Thus, it is conceivable that AR is the mediator of the suppression of miR-101 expression by celastrol. To test this possibility, we first determined if AR could bind to the predicted AR binding site in the upstream region of the miR-101 gene by luciferase reporter assays. Luciferase reporter constructs were generated as shown in Fig 3B. pGL3-B-miR-101-S (without the predicted AR binding site) showed about 5-fold decreased luciferase activity in comparison with pGL3-B-miR-101-L (with fragments encompassing the predicted AR binding site) (Fig 3C). In addition, pGL3-B-miR-101-MBS in which the AR binding site was mutated showed ~5-fold decreased luciferase activity compared with pGL3-B-miR-101-WBS that contained the wild type binding site (Fig 3C), indicating that this specific sequence is responsible for AR identification. Next, we used ChIP assays to determine if AR could bind to this sequence. DU145 cells were transfected with pEGFP-AR or pEGFP-V. Nuclear proteins were isolated and immunoprecipitated with either AR antibody or control mouse IgG. As expected, miR-101 fragment containing the binding site was specifically amplified in cells transfected with pEGFP-AR (Fig 3D upper), demonstrating that AR could be recruited to this site. In LNCaP cells, miR-101 fragment was amplified after mock transfection (Fig 3D bottom), showing that endogenous AR could be recruited to the ARE of miR-101. Finally, AR overexpression completely rescued pri-miR-101 and mature miR-101 expressions after celastrol treatment, showing AR mediating miR-101 transcription reduction upon celastrol treatment (Fig 3E).


Celastrol Induces Autophagy by Targeting AR/miR-101 in Prostate Cancer Cells.

Guo J, Huang X, Wang H, Yang H - PLoS ONE (2015)

AR suppression mediates miR-101 reduction after celastrol treatment.A, miR-101 and AR expressions after celastrol (CEL) treatment. **, P<0.01, compared with DMSO. B, Schematic depiction of luciferase reporter constructs as detailed in " Materials and Methods". Wild type and mutant AR binding sites were indicated by italic dash and cross, respectively. C, DU145 cells were transfected with indicated reporter constructs in the presence of pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) plasmid for 24 h, and then luciferase activity was detected. pRLSV40 plasmid was co-transfected for normalization. **, P<0.01, between EGFP-AR and EGFP-V transfections. D, ChIP assay. Cell extracts from DU145 or LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) were immunoprecipitated with AR antibody or normal mouse IgG. Input was 1/100 of the sonicated chromatin prior to immunoprecipitation. PCR was performed as described in the "Materials and Methods". E, LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) were treated with celastrol (CEL, 2.0 μM) or DMSO for 3 h, pri-miR-101 and mature miR-101 levels were determined by qPCR. Asterisks denote significance between EGFP-AR and EGFP-V transfections. *, P <0.05; **, P <0.01.
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pone.0140745.g003: AR suppression mediates miR-101 reduction after celastrol treatment.A, miR-101 and AR expressions after celastrol (CEL) treatment. **, P<0.01, compared with DMSO. B, Schematic depiction of luciferase reporter constructs as detailed in " Materials and Methods". Wild type and mutant AR binding sites were indicated by italic dash and cross, respectively. C, DU145 cells were transfected with indicated reporter constructs in the presence of pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) plasmid for 24 h, and then luciferase activity was detected. pRLSV40 plasmid was co-transfected for normalization. **, P<0.01, between EGFP-AR and EGFP-V transfections. D, ChIP assay. Cell extracts from DU145 or LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) were immunoprecipitated with AR antibody or normal mouse IgG. Input was 1/100 of the sonicated chromatin prior to immunoprecipitation. PCR was performed as described in the "Materials and Methods". E, LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) were treated with celastrol (CEL, 2.0 μM) or DMSO for 3 h, pri-miR-101 and mature miR-101 levels were determined by qPCR. Asterisks denote significance between EGFP-AR and EGFP-V transfections. *, P <0.05; **, P <0.01.
Mentions: To see miRNA involvement in the process of celastrol-induced autophagy, LNCaP cells were treated with celastrol for 12 h to induce autophagy. MiRNA array was performed and showed that the expression of miR-101, an autophagy inhibitor, was decreased by celastrol (data not shown). Suppression of miR-101 expression by celastrol was confirmed by RT-PCR, which revealed a ~3-fold decrease of pri-miR-101 and mature miR-101 in LNCaP cells post celastrol treatment (Fig 3A, left and middle). This was accompanied by an almost complete depletion of AR (Fig 3A, right), suggesting a positive correlation between AR and miR-101 expression after celastrol treatment. AR is a transcriptional factor and its binding site has been predicted in the upstream region of the miR-101 gene [13]. Thus, it is conceivable that AR is the mediator of the suppression of miR-101 expression by celastrol. To test this possibility, we first determined if AR could bind to the predicted AR binding site in the upstream region of the miR-101 gene by luciferase reporter assays. Luciferase reporter constructs were generated as shown in Fig 3B. pGL3-B-miR-101-S (without the predicted AR binding site) showed about 5-fold decreased luciferase activity in comparison with pGL3-B-miR-101-L (with fragments encompassing the predicted AR binding site) (Fig 3C). In addition, pGL3-B-miR-101-MBS in which the AR binding site was mutated showed ~5-fold decreased luciferase activity compared with pGL3-B-miR-101-WBS that contained the wild type binding site (Fig 3C), indicating that this specific sequence is responsible for AR identification. Next, we used ChIP assays to determine if AR could bind to this sequence. DU145 cells were transfected with pEGFP-AR or pEGFP-V. Nuclear proteins were isolated and immunoprecipitated with either AR antibody or control mouse IgG. As expected, miR-101 fragment containing the binding site was specifically amplified in cells transfected with pEGFP-AR (Fig 3D upper), demonstrating that AR could be recruited to this site. In LNCaP cells, miR-101 fragment was amplified after mock transfection (Fig 3D bottom), showing that endogenous AR could be recruited to the ARE of miR-101. Finally, AR overexpression completely rescued pri-miR-101 and mature miR-101 expressions after celastrol treatment, showing AR mediating miR-101 transcription reduction upon celastrol treatment (Fig 3E).

Bottom Line: In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy.Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR.Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Technology, Harbin Institute of Technology, Harbin, 150001, China.

ABSTRACT
Autophagy is an evolutionarily conserved process responsible for the degradation and recycling of cytoplasmic components through autolysosomes. Targeting AR axis is a standard strategy for prostate cancer treatment; however, the role of AR in autophagic processes is still not fully understood. In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy. Knockdown of AR in AR-positive prostate cancer cells resulted in enhanced autophagy. 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. 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. AR binding site was defined in the upstream of miR-101 gene by luciferase reporter and ChIP assays. MiR-101 expression correlated with AR status in prostate cancer cell lines. 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. Finally, autophagy inhibition by miR-101 mimic 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, thus combination of miR-101 mimics with celastrol may represent a promising therapeutic approach for treating prostate cancer.

No MeSH data available.


Related in: MedlinePlus