Transcriptional repression of tumor suppressor CDC73, encoding an RNA polymerase II interactor, by Wilms tumor 1 protein (WT1) promotes cell proliferation: implication for cancer therapeutics

Abstract

The Wilms tumor 1 gene (WT1) can either repress or induce the expression of genes. Inconsistent with its tumor suppressor role, elevated WT1 levels have been observed in leukemia and solid tumors. WT1 has also been suggested to act as an oncogene by inducing the expression of MYC and BCL-2. However, these are only the correlational studies, and no functional study has been performed to date. Consistent with its tumor suppressor role, CDC73 binds to RNA polymerase II as part of a PAF1 transcriptional regulatory complex and causes transcriptional repression of oncogenes MYC and CCND1. It also represses β-catenin-mediated transcription. Based on the reduced level of CDC73 in oral squamous cell carcinoma (OSCC) samples in the absence of loss-of-heterozygosity, promoter methylation, and mutations, we speculated that an inhibitory transcription factor is regulating its expression. The bioinformatics analysis predicted WT1 as an inhibitory transcription factor to regulate the CDC73 level. Our results showed that overexpression of WT1 decreased CDC73 levels and promoted proliferation of OSCC cells. ChIP and EMSA results demonstrated binding of WT1 to the CDC73 promoter. The 5-azacytidine treatment of OSCC cells led to an up-regulation of WT1 with a concomitant down-regulation of CDC73, further suggesting regulation of CDC73 by WT1. Exogenous CDC73 attenuated the protumorigenic activity of WT1 by apoptosis induction. An inverse correlation between expression levels of CDC73 and WT1 was observed in OSCC samples. These observations indicated that WT1 functions as an oncogene by repressing the expression of CDC73 in OSCC. We suggest that targeting WT1 could be a therapeutic strategy for cancer, including OSCC.

Keywords: Apoptosis; CDC73; Cancer Prevention; HRPT2; OSCC; Oncogene; Parafibromin; Transcription Promoter; Tumor Suppressor Gene; WT1.

FIGURE 1.

Analysis of the CDC73 promoter. A, in silico analysis of the putative CDC73 promoter and binding sites for transcription factors, including that of WT1. Downward arrow represents the start of exon 1 and TSS. TSS is numbered as +1, and the rest of the sequence is numbered relative to it. Putative transcription factor binding are boxed. Primer sequences used for the CDC73 promoter cloning are underlined. B, schematic representation of the promoter construct pGL3-PCDC73 harboring a fragment from −425 to +400 bp of the CDC73 promoter upstream from the luciferase reporter gene (Luc) in the pGL3-Basic vector. C, functional characterization of the putative CDC73 promoter region by the Dual Luciferase Reporter assay. The graph shows relative luciferase units of constructs over the Renilla luciferase construct used for normalization of transfection efficiency. Note, the construct pGL3-PCDC73 shows a significantly higher luciferase activity over the pGL3-Basic vector. Each bar represents average data from three experiments. Error bars, S.D. ***, p < 0.001. D, conserved putative WT1 binding site in the CDC73 promoter in different species. This site is almost identical to the consensus WT1 binding site (shaded).

FIGURE 2.

Effect of WT1 overexpression on CDC73 expression and characterization of its binding to the CDC73 promoter. A, effect of WT1 overexpression on CDC73 expression in SCC131 cells. Note that the WT1 overexpression knocks down CDC73 expression in a dose-dependent manner. Mock represents cells co-transfected with pcDNA3.1(+) and pcDNA3-GFP. B, results of co-transfection of pGL3-PCDC73 or pGL3-PCDC73(mut) with WT1 construct pcDNA3.1(+)-WT1^+/+ in SCC131 cells. Note that the luciferase reporter driven by the CDC73 promoter is knocked down by WT1 overexpression in a dose-dependent manner. C, effect of the 5-aza-dC (AZA) treatment on WT1 and CDC73 expression in SCC131 cells. Note an up-regulation of WT1 with a concomitant down-regulation of CDC73 following the treatment. β-Actin was used as a normalizing control. DMSO, dimethyl sulfoxide. Each bar represents average data from three experiments ± S.E. (error bars). D, analysis of WT1 promoter methylation by COBRA following digestion of a 315-bp amplicon with AciI. Note promoter methylation (digestion of amplicon) in SCC131 cells and the absence of promoter methylation (no digestion of amplicons) in KB and SCC084 cells. −veC represents digestion of a 315-bp WT1 amplicon with AciI using the bisulfite-treated DNA template from the peripheral blood of a normal individual. +veC represents digestion of a 433-bp WRAP73 amplicon with AciI using the untreated DNA template from the peripheral blood sample of a normal individual. E, characterization of WT1 binding to the CDC73 promoter by the ChIP assay. The relative quantity of promoter enriched by ChIP was quantified by qPCR and expressed as the -fold enrichment of the CDC73 and MYC promoters after normalization to rabbit IgG. Data represent the means of two independent experiments. F, characterization of WT1 binding to the CDC73 promoter by EMSA. Lanes 1–5 were loaded with an end-labeled probe from the CDC73 promoter. Lanes 6–10 were loaded with an end-labeled probe from the MYC promoter. Note the shift and supershift in lane 3 showing the binding of WT1 to the CDC73 promoter. As expected, also note the shift and supershift in lane 8, indicating the binding of WT1 to the MYC promoter (positive control). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, nonsignificant.

FIGURE 3.

qRT-PCR analysis in HEK293 cells transfected with siRNAs targeting WT1 and IGFBP2. Note that HEK293 cells transfected with WT1 siRNA at a final concentration of 100 nm show a significant knockdown of WT1 with a concomitant up-regulation of CDC73 compared with mock-transfected cells. siRNA targeting IGFB2 was used as a nonspecific control. Also note that knockdown of WT1 does not affect the expression of an irrelevant target ESRRA. *, p < 0.05; **, p < 0.01; ns, nonsignificant. β-Actin was used as a normalizing control.

FIGURE 4.

Effect of WT1 overexpression on CDC73 expression, proliferation, and apoptosis of SCC131 cells. Left panel, immunoblots (IB) representing the effect of WT1 overexpression on CDC73 expression. Note a reduced expression of CDC73 upon WT1 overexpression (lane 2) compared with cells transfected with the control vector pcDNA3-HA (lane 1). The CDC73 down-regulation by WT1 overexpression is rescued by exogenous CDC73 expression (lane 3). β-Actin was used as a normalizing control. Middle panel, rate of proliferation of SCC131 cells transiently transfected with the empty vector pcDNA-HA, pcDNA3.1(+)-WT1^+/+, or pcDNA3-HA-CDC73 or co-transfected with pcDNA3.1(+)-WT1^+/+ and pcDNA3-HA-CDC73. Right panel, rate of apoptosis of SCC131 cells transiently transfected with the empty vector pcDNA-HA, pcDNA3.1(+)-WT1^+/+, or pcDNA3-HA-CDC73 or co-transfected with pcDNA3.1(+)-WT1^+/+ and pcDNA3-HA-CDC73. Each bar represents average data from three experiments. Error bars, S.E. *, p < 0.05; **, p < 0.01; ns, nonsignificant).

FIGURE 5.

Comparative analysis of WT1 and CDC73 expression in OSCC samples. The graph represents relative expression of WT1 (upper panel) and CDC73 (lower panel) in 24 OSCC samples compared with their normal counterparts, normalized to GAPDH. Numbers along x-axis represent patients. T2, T3, and T4 represent stages of OSCC. Each bar is an average of three experiments. *, p < 0.05; ns, nonsignificant.