In vitro and in vivo studies show that blocking ARD1 prevents growth of xenografts in mice.
Blocking the activity of a protein that regulates androgen receptor (AR) activation could provide a therapeutic approach against prostate cancer, researchers claim. A team led by scientists at Louisiana State University Health Sciences Center identified N-acetyltransferase arrest-defect 1 (ARD1) as an androgen-induced regulator of AR in prostate cancer.
Their in vitro and in vivo experiments demonstrated that ARD1 is crucial for AR-mediated gene transcription, prostate cancer cell proliferation, and xenograft tumor growth in SCID mice. Studies in addition showed that the ARD1 protein is upregulated in human prostate cancer cell lines and primary tumor biopsies.
Wanguo Liu, Ph.D., and colleagues report their research in PNAS in a paper titled “Inactivation of androgen-induced regulator ARD1 inhibits androgen receptor acetylation and prostate tumorigenesis.”
Androgen receptor activity plays a key role in prostate cancer development and is believed to regulate the expression of genes that are essential for prostate tumorigenesis. AR coactivators that effect phosphorylation, sumoylation, and stabilization of AR act to enhance AR activity and AR-mediated transcriptional regulation.
In particular, the LSU team notes, AR acetylation has recently been shown to play a key role in AR-mediated transactivation and prostate tumorigenesis. Their own work had in addition found that the ARD1 protein, which induces acetylation in a range of proteins, is upregulated in prostate cancer.
These two observations led the researchers to investigate whether ARD1 plays a role in prostate cancer tumorigenesis or progression. Initial studies demonstrated that levels of ARD1 were up to five times as high in prostate cancer cell lines than they were in normal prostatic epithelial cell lines and were highest in cell lines with an intact AR. Immunohistochemical tests on a prostate cancer tissue microarray showed that levels of ARD1 were raised in about 97% of tumor tissue but in only about 6% of normal prostate tissues.
The team found that in fact ARD1 wasn’t induced by androgen at the transcriptional level but rather was induced at the protein level. Experiments using the synthetic androgen R1881 in prostate cancer cell lines demonstrated that levels of ARD1 were dependent on the dose of R1881 administered and that R1881 could trigger ARD1 production in AR-null PC-3 cell lines in which AR was ectopically expressed. In these cells the amount of ARD1 produced was dependent on the dose of R1881 and level of AR expression.
Tests in the androgen-sensitive human prostate adenocarcinoma cell line LNCaP demonstrated that knockdown of ARD1 using shRNA was associated with significantly reduced cell growth rates, markedly reduced or even absent colony formation, and an inability to generate foci in anchorage-independent colony formation assays on soft agar. Stable ARD1 shRNA transfected cells were also incapable of forming xenograft tumor growth in experimental mice.
In contrast, LNCaP cells transfected using control shRNAs generated xenograft tumors that increased in volume daily. “Together, these data strongly suggest that ARD1 is an oncoprotein in prostate cancer and that silencing of ARD1 reduces prostate cancer cell proliferation and oncogenicity,” Dr Liu et al. state.
Because AR-mediated transcription is believed to regulate prostate cancer cell growth, the team moved on to examined the role of ARD1 in transcriptional regulation of AR target genes. Tests using prostate-specific antigen (PSA) and mouse mammary tumor virus (MMTV) luciferase reporter assays confirmed that up-regulation of ARD1 in prostate cancer is induced by androgen in an AR-dependent manner. In these assays the luciferase activities of both reporters was significantly induced in LNCaP cells stimulated with R1881 and in PC-3 cells following ectopic expression of AR.
In contrast, activity of the two reporters was markedly reduced in LNCaP cells after ARD1 silencing but was increased in PC-3 cells after co-expression of AR and ARD1. qRT-PCR analyses added more detail into the picture. “qRT-PCR analysis of 12 known AR target genes revealed that following R1881 treatment and ARD1 silencing in LNCaP cells, the mRNA levels of the 4 AR down-regulated genes (FN1, ACPP, UGT2B13, and UGT2B17) were activate,” the authors write.
“In contrast, the eight AR upregulated genes including PSA, TMPRSS2, SLC45A3, FASN, ABHD2, ALDH1A3, FKBP5, and NDRG1 were down-regulated, respectively, in comparison with the mRNA levels in the control cells without silencing of ARD.”
To confirm that ARD1 acts as an AR co-activator the researchers then carried out PSA luciferase reporter assays in LNCaP cells that express AR and in PC-3 cells that lack endogenous AR expression. In these experiments overexpression of ARD1 in the LNCaP cells resulted in a roughly threefold activation of the PSA reporter, whereas silencing AR using an AR-specific siRNA abolished the effect of ARD1 on the reporter activity. In the AR-deficient PC-3 cells, expression of ARD1 alone had no significant effect on the reporter, but co-expression of ARD1 and AR led to a more than fivefold higher reporter activation.
In support of the reporter assay data, qRT-PCR analysis showed that ARD1 expression was associated with a 3-4-fold increase in transcription levels of two AR target genes, PSA and TMPRSS2. This increase in target gene transcription was prevented when AR was silenced. ChiP analysis further demonstrated that overexpression of ARD1 in LNCaP cells facilitated the interaction of AR with the PSA or TMPRSS2 promoter.
Interestingly, silencing ARD1 attenuated PSA levels but appeared to have no effect on AR levels. Given that ARD1 is an acetyltransferase, the team hypothesized that ARD1’s regulatory effect might relate to its acetylation of AR. Using co-immunoprecipitation techniques the researchers confirmed that ARD1 physically interacts with AR in vivo and in vitro. Studies with an acetylation-specific antibody demonstrated that silencing ARD1 in LNCaP cells markedly blocked AR acetylation. Further tests in cells expressing an acetylation-incompetent ARD1 mutant confirmed that the protein is directly responsible for AR acetylation and the activation of AR target gene transcription.
Dr. Liu et al. claim their data has uncovered a unique role for ARD1 as an AR regulator in prostate tumorigenesis. “We show that AR-ARD1 interaction and ARD1-dependent AR acetylation are required for transcriptional activation of AR target genes in vivo and in vitro,” they write. “By linking the overexpression of ARD1 in prostate cancer and ARD1-dependent acetylation of AR to AR-mediated transcription, our study provides a unique avenue for controlling AR-mediated prostate tumorigenesis by direct inhibition of ARD1 expression or AR-ARD1 interaction. Therefore, developing ARD1-specific inhibitor or AR-ARD1 interaction-disrupting peptide may be of therapeutic benefit in the treatment of prostate cancer.”