Stimulation of Prostate Cancer Cellular Proliferation and Invasion by the Androgen Receptor Co-Activator ARA70β

Abstract

ARA70 was first identified as a gene fused to the ret oncogene in thyroid carcinoma and subsequently as a co-activator for androgen receptor (AR). Two isoforms of ARA70 have been identified: a 70-kDa version called ARA70␣ and an internally spliced 35-kDa variant termed ARA70␤. We have previously reported that ARA70␣ expression is reduced in prostate cancer, and its overexpression inhibits proliferation of LNCaP prostate cancer cells. However, the function of the ARA70␤ isoform in prostate cancer is not understood. In this report we examined the effects of ARA70␤ on AR transcriptional regulation as well as prostate cancer cellular proliferation and invasion. Although both ARA70␣ and ARA70␤ functioned as transcriptional co-activators of AR in cell-based reporter assays, ARA70␤ overexpression, in contrast to ARA70␣, promoted prostate cancer cellular proliferation and invasion through Matrigel. Interestingly, genome-wide expression profiling of cells expressing ARA70␤ revealed an increase in the expression of genes involved in the control of cell division and adhesion, compatible with a role for ARA70␤ in proliferation and invasion. Consistent with its function in promoting cell growth and invasion, ARA70␤ expression was increased in prostate cancer. Our findings implicate ARA70␤ as a regulator of tumor cell growth and metastasis by affecting gene expression. The androgen receptor (AR) is a transcription factor that regulates prostate cell growth and differentiation.1 AR mediates transcription through a series of events including ligand binding, DNA binding to cognate androgen response elements (AREs), and interaction with various co-activators. This results in activation of the general transcriptional machinery. 2 Co-activators are components that are required for activator-dependent transcription but appear to be dispensable for basal transcription. AR co-activators are thought to facilitate AR-dependent transcription by linking the receptor to the basal transcription machinery or by modulating chromatin through methylation and acetylation.3,4 An increasing number of AR-interacting proteins have been identified, and several have been shown to function as AR co-activators in cellbased reporter assay. [5][6][7] Interestingly, a number of AR co-activators display diverse patterns of expression in prostate cancer. For example, the AR cofactors ARA24, PIAS1, 8 cyclinD1, 9 SRC1, 10 and FHL2 11 appear overexpressed, whereas ARA70 8 and ART-27 12 are reduced in human prostate cancers compared to adjacent benign tissue. In addition, although most co-factors are expressed in prostatic epithelium, ARA55 is expressed exclusively in prostatic stroma, with reduced expression in prostate cancer. These findings suggest that AR coactivators perform distinct functions in prostate growth.One of the earliest described AR co-activators is ARA70. It was initially identified as a gene fused to the ret oncogene 13,14 and subsequently characterized as an AR co-activator.15 ARA70 interacts with the AR ligand-binding domain (LBD) via an FXX...

Protein arginine methyltransferase 6-dependent gene expression and splicing: association with breast cancer outcomes

“…NCOA4, YWHAE, ARL6IP1 and MINA are overexpressed in a number of different cancers (Hu et al 2004, Teye et al 2004, Cimino et al 2008, Peng et al 2008, Guo et al 2010. The up-regulation of YWHAE in breast cancer is associated with diseasefree and overall survival, and therefore, the downregulation of YWHAE from reduced PRMT6 levels may be unfavourable in relation to breast cancer prognosis (Cimino et al 2008).…”

“…These genes, taken from the list of 159 differentially expressed genes, comprised six genes with decreased expression and two with increased expression following PRMT6 knockdown. The genes were chosen for their biological significance and had relevance to either steroid hormone-dependent gene expression (FK506 binding protein 4, 59 kDa: FKBP4; homeodomain interacting protein kinase 3: HIPK3 and nuclear receptor co-activator 4: NCOA4) (Yeh & Chang 1996, Moilanen et al 1998, Riggs et al 2003 and/or cancer (NCOA4, ADP-ribosylation factor-like 6 interacting protein 1: ARL6IP1; MYC-induced nuclear antigen: MINA; tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide: YWHAE; phosphatase and tensin homolog: PTEN and insulin-like growth factor binding protein 3: IGFBP3) (Eng 2003, Hu et al 2004, Teye et al 2004, Cimino et al 2008, Peng et al 2008, Guo et al 2010, Ingermann et al 2010. PRMT6 levels were decreased in MCF-7 or T-47D breast cancer cells by siRNA for 48 h. We conducted further validation, utilising two (different) non-overlapping siRNAs targeting PRMT6 (individually) to decrease the likelihood of observing non-specific off-target effects.…”

Tumor Suppressor Function of Androgen Receptor Coactivator ARA70α in Prostate Cancer

“…Indeed, we showed that ARA70␤ functions as an oncogene in promoting prostate cancer cell growth and invasion. 20 In this study, we demonstrated that ARA70␣ functions as a tumor suppressor gene in prostate cancer in in vitro and in vivo experiments.…”

“…ARA70␤ promotes androgen-dependent growth and invasion of prostate cell lines. 20 It appears that the changes in splicing of ARA70 into its two distinct variants may be a part of such an AR-dependent mechanism, which switches between either proliferation or growth arrest and/or differentiation.…”

“…Immunoblots were blocked for 30 minutes in 3% nonfat dry milk in Trisbuffered saline Tween 20 (20 mmol/L of Tris-HCl, pH 7.6, 150 mmol/L of NaCl, and 0.1% Tween 20). Blots were incubated with antibodies raised against ARA70␣, 20 androgen receptor and ␤-actin (Sigma-Aldrich, St. Louis, MO), apoptosis-inducing factor, Bax, Bcl-XL, cleaved caspase 3, caspase 8, and p53 upregulated modulator of apoptosis (PUMA; Cell Signaling Technology, Danvers, MA) for 2 hours at room temperature, washed with Trisbuffered saline Tween 20 three times, and incubated for 1.5 hours with the secondary antibody (1:5000; Amersham Biosciences). Antibodies were diluted with 2% bovine serum albumin in Tris-buffered saline Tween 20.…”