Par-4 Transcriptionally Regulates Bcl-2 through a WT1-binding Site on the bcl-2 Promoter

Abstract

Elevated expression levels of the bcl-2 proto-oncogene have been extensively correlated with the appearance of androgen independence in prostate cancer. Although bcl-2 was first cloned as the t(14:18) translocation breakpoint from human follicular B cell lymphoma, the mechanism of overexpression of bcl-2 is largely undefined for advanced prostate cancer because there are no gross alterations in the gene structure. We investigated the role of the product of the prostate apoptosis response gene-4 (Par-4) and the product of the Wilms' tumor 1 gene (WT1) in the regulation of Bcl-2 expression in prostate cancer cell lines. We observed growth arrest and apoptosis, upon decreasing Bcl-2 protein and transcript in the high Bcl-2-expressing, androgen-independent prostate cancer cell line, by all-trans-retinoic acid treatment (ATRA), but this did not occur in the androgen-dependent cell line expressing low levels of Bcl-2. The decrease in the Bcl-2 protein and transcript following all-trans-retinoic acid treatment was accompanied by changes in localization of Par-4 and an induction in the expression of WT1 protein. In stable clones expressing ectopic Par-4 and in ATRA-treated cells, we observed decreased Bcl-2 protein and transcript. This was accompanied by an induction in WT1 expression. The involvement of WT1 in the Par-4-mediated down-modulation of Bcl-2 was further defined by blocking endogenous WT1 expression, which resulted in an increase in Bcl-2 expression. Finally, we detected Par-4 and WT1 proteins binding to a previously identified WT1-binding site on the bcl-2 promoter both in vitro and in vivo leading to a decrease in transcription from the bcl-2 promoter. We conclude that Par-4 regulates Bcl-2 through a WT1-binding site on the bcl-2 promoter. These data also identify Par-4 nuclear localization as a novel mechanism for ATRA-mediated bcl-2 regulation.

HER2/neu increases the expression of Wilms' Tumor 1 (WT1) protein to stimulate S-phase proliferation and inhibit apoptosis in breast cancer cells

“…However, the bcl-2 promoter has also been shown to be negatively regulated by WT1 (Hewitt et al, 1995;Cheema et al, 2003). Differences in cell types, status of WT1 isoforms, and the interaction of WT1 protein with other proteins may explain the differences between our results and those of Hewitt et al (1995) and Cheema et al (2003).…”

PAWR-mediated suppression of BCL2 promotes switching of 3-azido withaferin A (3-AWA)-induced autophagy to apoptosis in prostate cancer cells

“…13 As a binding partner of Wilms Tumor protein, PAWR indirectly acts as a transcriptional repressor and downregulates BCL2 expression through a WT1-binding site in the BCL2 promoter region (-1640 bp). 14 Surprisingly, in this study, we demonstrated that 3-AWA-stimulated or overexpressed PAWR not only inhibited BCL2 protein, but also suppressed another BH3 domain protein BECN1, an essential component in autophagy.…”

“…38 BCL2 activity is tightly regulated in cells undergoing apoptosis and the proapoptotic protein PAWR modulates cellular BCL2 by binding to the BCL2 promoter through the WT1 protein. 14 Therefore, to investigate the modus operandi of interaction between these 2 proteins in a dual perspective encompassing autophagy and apoptosis, a coimmunoprecipitation affinity isolation assay was performed. Immunoprecipitation of BECN1 with BECN1 antibody pulled down BCL2 from whole cell lysate and the BCL2 antibody coimmunoprecipitated BECN1, respectively, when PC-3 cells were treated with 0.75 mM 3-AWA for 12 h. Moreover, compared to DMSO vehicle-exposed cells, wherein the BECN1 antibody immunoprecipitated BECN1 but not BCL2, our results indicated that a direct interaction between BCL2 and BECN1 existed at an optimum autophagy-inducing concentration of 3-AWA (0.75 mM) (Fig.…”

“…13 As a binding partner of the WT1 protein, PAWR indirectly functions as a transcriptional corepressor and is involved in the downregulation of BCL2 expression through binding of the PAWR-WT1 complex in the BCL2 promoter region. 14 Although vast knowledge has emerged in the recent past about the PAWR-BCL2 interaction, a persistent gap still prevails regarding how PAWR controls other death pathways through modulation of BCL2 function.…”

Functional Role of WT1 in Prostate Cancer

“…In vascular cells, WT1 expression was associated with an increase in proangiogenic molecules such as VEGF (95). Similarly, both VEGF and WT1 are elevated in some PC cells (96), consistent with its ability to regulate growth control pathways important in PC (46,50,(55)(56)(57)(58)(59)(60)(61). Additionally, WT1…”