Abstract
TBLR1/TBL1XR1, a core component of the nuclear receptor corepressor (NCoR) complex critical for the regulation of multiple nuclear receptors, is a transcriptional coactivator of androgen receptor (AR) and functions as a tumor suppressor when expressed in the nucleus in prostate. Subcellular localization of a protein is critical for its function, and although TBLR1, as a transcriptional cofactor, has been primarily viewed as a nuclear protein, many cells also express variable levels of cytoplasmic TBLR1 and its cytoplasmic specific functions have not been studied. Prostate cancer (PCa) cells express moderately higher level of cytoplasmic TBLR1 compared to benign prostate cells. When comparing androgen-dependent (AD) to androgen-independent (AI) PCa, AI cells contain very high levels of TBLR1 cytoplasmic expression and low levels of nuclear expression. Overexpression of cytoplasmic TBLR1 in AD cells inhibits apoptosis induced by androgen deprivation therapy, either in an androgen free condition or in the presence of bicalutamide. Additionally, we identified a cytoplasmic specific isoform of TBLR1 (cvTBLR1) approximately 5 kDa lower in molecular weight, that is expressed at higher levels in AI PCa cells. By immunoprecipitation, we purified cvTBLR1 and using mass spectrometry analysis combined with N-terminal TMPP labeling and Edman degradation, we identified the cleavage site of cvTBLR1 at amino acid 89, truncating the first 88 amino acids of the N-terminus of the full length protein. Functionally, cvTBLR1 expressed in the cytoplasm reduced apoptosis in PCa cells and promoted growth, migration, and invasion. Finally, we identified a nuclear export signal sequence for TBLR1 cellular localization by deletion and site-directed mutagenesis. The roles of TBLR1 and cvTBLR1 provide novel insights into the mechanism of castration resistance and new strategies for PCa therapy.
Highlights
Prostate cancer (PCa) is an exceedingly prevalent cancer and is the second leading cause of cancer related death of men in the US [1]
We found that the increased level of Transducin beta like related 1 (TBLR1) in AI cancer cell lines was due to increased cytoplasmic TBLR1 (Figure 1C, 1D)
Our previous study has shown that TBLR1 is a transcriptional activator of androgen receptor (AR) and its nuclear expression is significantly reduced in PCa compared with benign prostate glands
Summary
Prostate cancer (PCa) is an exceedingly prevalent cancer and is the second leading cause of cancer related death of men in the US [1]. TBLR1 and the closely related TBL1X are necessary for activation of Wnt target genes [11]. Overexpression of TBLR1 inhibits growth in 293T and Jurkat cells and is involved in degradation of the anti-apoptotic protein BCL-3 [8, 13]. Immunohistochemical data shows TBLR1 as primarily a nuclear protein in normal prostate but shows reduced nuclear expression in PCa [15]. TBLR1 is a strong coactivator of AR in prostate cancer, leading to selective activation of growth suppressive AR target genes and tumor suppression. Overexpression of TBLR1in NPC cells causes reduced sensitivity to cisplatin-induced apoptosis [18]
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