Effects of manganese superoxide dismutase silencing on androgen receptor function and gene regulation: implications for castration-resistant prostate cancer

Abstract

Purpose: Advanced prostate cancer is first treated with androgen deprivation therapy. However, tumors become resistant to and grow despite castrate levels of testosterone. Growth and proliferation of CRPC is mediated by gain-of-function changes in the AR and AR reactivation. Expression of manganese superoxide dismutase (SOD2), which regulates cellular ROS, is markedly down-regulated in CRPC when compared with hormone-responsive tumors.

Experimental design: Here, we knocked down SOD2 expression in AR-expressing LNCaP prostate cancer cells and determined gene expression changes, transcription factor binding, and AR transcription activity in SOD2 knockdown cells.

Results: SOD2 knockdown results in an increase in ROS. Gene expression changes induced by SOD2 knockdown results in the up-regulation of genes that are also androgen responsive and 46% of genes up-regulated 2-fold by the androgen ligand R1881 are also up-regulated to the same extent with SOD2 knockdown. The induction of many of these genes with SOD2 knockdown, such as VEGFA and FKBP5, is reversible with the antioxidant N-acetylcysteine, suggesting that this mechanism is directly linked to ROS. Furthermore, an array for transcription factor DNA-binding activity shows that SOD2 knockdown induces DNA binding by several transcription factors, including AR. SOD2 knockdown-induced AR activation was confirmed by electrophoretic mobility shift assay and luciferase activity, and both were readily reversible with N-acetylcysteine.

Conclusions: These findings show that down-regulation of SOD2 induces AR activity in a ROS-dependent manner, and suggest that there may be a role for antioxidant therapy in CRPC.

Figure 1

Expression of shSOD2 decreases SOD2 protein and SOD2 enzymatic activity in LNCaP cells. A. Western blot of vector control and shSOD2-expressing LNCaP cells with actin as a loading control. B. Quantitation of decrease in SOD2 protein normalized to actin. C. Decrease in SOD2 enzymatic activity in shSOD2 expressing cells.

Figure 2

Reactive oxygen species (ROS) are increased in the shSOD2 cell line compared with vector control cells. ROS activity was measured in triplicate with the intensity shifting from a mean of 1497 in the vector sample to a mean of 1681 in the shSOD2 sample (p<0.05) by flow cytometry.

Figure 3

Genes regulated by SOD2 show a large overlap with androgen signaling. A. Venn diagrams showing the number of genes regulated by SOD2 and R1881 as well as the intersection of these genes.

Figure 4

Androgen-responsive genes are regulated by SOD2 downregulation. A. The expression of a sub-set of genes that are regulated concordantly by at least two-fold by both the knockdown of SOD2 (shSOD2) and by treatment with R1881 (R1881). Also shown is the expression of these genes when the shSOD2 cells are treated with NAC (NAC). The functional categories of these genes are shown on the left side. B. A subset of genes found to be highly androgen regulated in another study (21), that is also regulated by both shSOD2 and R1881 that is also reversible in shSOD2 cells that were treated with NAC. C. Quantitative PCR (Q-PCR) shows a similar level of regulation of the genes assayed. SOD2 knockdown-inducible and NAC-reversible expression of PSA and TMPRSS2 by QPCR is also shown.

Figure 5

Changes in transcription factor binding in response to SOD2 knockdown. Binding of transcription factors was measured using a Panomic’s array and the fold change in binding activity is shown. An asterisk represents a low level of binding in the control sample, and the value for quantitation was set to 100 over background.

Figure 6

SOD2 knockdown induces NAC-reversible AR DNA binding and transcription activity. A. An electrophoretic mobility shift assay (EMSA) showing androgen receptor (AR) binding. An increase in AR binding occurs in both cells treated with R1881 (R1881) as well as in cells with SOD2 knockdown (shSOD2). Binding of AR is lost when shSOD2 cells are treated with NAC (shSOD2+NAC). B. AR luciferase activity is increased in SOD2 knockdown cells. Luciferase activity was measured in vector and SOD2 knockdown (shSOD2) cells after the indicated treatments and was normalized to Renilla luciferase. The graph shows the fold-increase in luciferase activity for each of the treatments over vector control cells. An asterisk (*) represent p<0.001 with respect to the control. Two asterisks (**) represent p<0.01 for the comparison of shSOD2+NAC with shSOD2.