Up-regulation of NKX3.1 expression and inhibition of LNCaP cell proliferation induced by an inhibitory element decoy

NKX3.1 is an androgen-regulated prostate-specific homeobox gene that is thought to play an important role in prostate development and cancerogenesis. NKX3.1 acts as a tumor suppressor gene specifically in the prostate. Up-regulation of NKX3.1 gene offers a promising gene therapy for prostate cancer. The decoy strategy has been developed and is considered a useful tool for regulating gene expression and gene therapy. In our previous studies, we identified a 20 bp inhibitory element upstream of the NKX3.1 promoter.In this study, we focused on using the 20 bp inhibitory element decoy to block negative regulation of the NKX3.1 gene and to up-regulate NKX3.1 expression using synthetic double-stranded oligodeoxynucleotides of the 20 bp inhibitory element. We found in an electrophoretic mobility shift assay experiment that the 20 bp inhibitory decoy presented competitive binding to a specific binding protein of the 20 bp inhibitory element in prostate cancer cell line LNCaP. In luciferase reporter gene assays, we found that the 20 bp inhibitory decoy could enhance NKX3.1 promoter activity, and RT-PCR and Western blot analysis revealed that NKX3.1 expression was up-regulated effectively by the transfection with the 20 bp inhibitory decoy. Furthermore,cell proliferation was inhibited by up-regulated NKX3.1 expression induced by the 20 bp inhibitory decoy.     

Lack of an Additive Effect between the Deletions of Klf5 and Nkx3-1 in Mouse Prostatic Tumorigenesis

Prostate cancer is one of the most common malignancies.The development and progression of prostate cancer are driven by a series of genetic and epigenetic events including gene amplification that activates oncogenes and chromosomal deletion that inactivates tumor suppressor genes.Whereas gene amplification occurs in human prostate cancer,gene deletion is more common,and a large number of chromosomal regions have been identified to have frequent deletion in prostate cancer,suggesting that tumor suppressor inactivation is more common than oncogene activation in prostatic carcinogenesis (Knuutila et al.,1998,1999;Dong,2001).Among the most frequently deleted chromosomal regions in prostate cancer,target genes such as NKX3-1 from 8p21,PTENfrom 10q23 andATBF1 from 16q22 have been identified by different approaches (He et al.,1997;Li et al.,1997;Sun et al.,2005),and deletion of these genes in mouse prostates has been demonstrated to induce and/or promote prostatic carcinogenesis.For example,knockout of Nkx3-1 in mice induces hyperplasia and dysplasia (Bhatia-Gaur et al.,1999;Abdulkadir et al.,2002) and promotes prostatic tumorigenesis (Abate-Shen et al.,2003),while knockout of Pten alone causes prostatic neoplasia (Wang et al.,2003).Therefore,gene deletion plays a causal role in prostatic carcinogenesis (Dong,2001).     

NKX3.1 potentiates TNF-α/CHX-induced apoptosis of prostate cancer cells through increasing caspase-3 expression and its activity

NKX3.1, a prostate-specific homeobox gene, plays an important role in prostate cancer and usually functions as tumor suppressor gene. Previously we have demonstrated that forced expression of NKX3.1 reduced cell growth and invasion in prostate cancer cell line PC-3. Presently, we investigated the effect of NKX3.1 on the sensitivity of the prostate cancer cells to apoptosis inducer tumor necrosis factor-α (TNF-α) and cycloheximide (CHX). PC-3 cells were transfected with NKX3.1 expression plasmid (pcDNA3.1-NKX3.1) and LNCaP cells were transfected with siRNA expression plasmid (pRNAT-RNAi1) targeting NKX3.1. The cell morphology and apoptotic rate were analyzed by Hoechst 33342 staining and Flow Cytometry in absence or presence of TNF-α and CHX. The activity of caspase-3 was determined using DEVD-pNA as substrate. Simultaneously, the effect of NKX3.1 on caspase-3 expression was detected using RT-PCR and Western blot. The results showed that ectopic expression of NKX3.1 promoted TNF-α/CHX-induced apoptosis in PC-3 cells, whereas knockdown of NKX3.1 protected LNCaP cells from apoptosis induced by TNF-α/CHX. The pro-apoptosis activity of NKX3.1 might partially contribute to its elevation of caspase-3 expression and activity. Manipulating NKX3.1 expression should be a promising therapeutic strategy for treating both androgen-dependent and androgen-independent prostate cancer.     

p53过表达抑制同源盒基因NKX3.1启动子活性

NKX3.1是前列腺特异表达的同源盒基因,在前列腺癌的发生发展中起重要作用,而在前列腺癌进展中常会发生p53的基因突变.为研究两者之间的关系,构建NKX-3.1启动子(1 040bp)-荧光素酶报告基因重组质粒（pGL3-1040）及其缺失突变体,瞬时转染前列腺癌细胞LNCaP.通过荧光素酶表达活性分析,检测p53过表达对NKX3.1启动子活性的影响.结果表明：p53在LNCaP细胞中过表达可明显抑制NKX3.1启动子活性;RT-PCR及Western印迹检测p53过表达对NKX3.1表达的影响.结果表明,p53过表达可以明显抑制同源盒基因NKX3.1的表达.通过TRANSFAC软件分析,在NKX3.1基因上游-526至-507区存在一个p53反应元件的5′核心序列.缺失pGL3-1040中的p53反应元件核心序列并不能消除p53对NKX3.1启动子的抑制作用,表明p53不是通过p53反应元件直接抑制NKX3.1启动子活性.进一步通过5′缺失突变分析,发现NKX3.1启动子-140～+8 bp区仍受p53负调控.此148 bp区域中含有一个Sp1和一个CREB元件,瞬时共转染Sp1表达载体或CREB表达载体的结果表明,p53并不是通过与Sp1或CREB相互作用对NKX3.1启动子发挥抑制作用的.上述结果表明,p53过表达可以抑制同源盒基因NKX3.1启动子活性,下调NKX3.1基因的转录,其调控机制有待进一步研究.     

Cloning, expression and chromosomal location of NKX6B to 10q26, a region frequently deleted in brain tumors

Nkx6-2 (former Gtx) is a murine-homeobox-containing gene localized distally on Chromosome (Chr) 7. Analysis of the expression pattern, together with DNA binding assays, suggests that this gene product might be important for differentiated oligodendrocyte function and in the regulation of myelin gene expression. We now report on the cloning and characterization of the human homolog (NKX6B). DNA sequence analysis of an 11-kb genomic fragment revealed that the complete human gene spans 1.2 kb and is composed of three exons. NKX6B is predicted to encode a polypeptide of 277 amino acids with 97% identity to mouse Nkx6-2. Northern blot experiments showed that NKX6B expression is tightly controlled in a tissue-specific fashion with the highest site of expression being the brain. Finally, using STS content mapping and RH analyis, we demonstrated that NKX6B maps to the 10q26, a region where frequent loss of heterozygosity has been observed in various malignant brain tumors. These results may implicate NKX6B as a candidate tumor suppressor gene for brain tumors, particularly for oligodendrogliomas. Received: 14 June 2000 / Accepted: 21 September 2000     

NKX3.1 is regulated by protein kinase CK2 in prostate tumor cells

Diminished expression of NKX3.1 is associated with prostate cancer progression in humans, and in mice, loss of nkx3.1 leads to epithelial cell proliferation and altered gene expression patterns. The NKX3.1 amino acid sequence includes multiple potential phosphoacceptor sites for protein kinase CK2. To investigate posttranslational regulation of NKX3.1, phosphorylation of NKX3.1 by CK2 was studied. In vitro kinase assays followed by mass spectrometric analyses demonstrated that CK2 phosphorylated recombinant NKX3.1 on Thr89 and Thr93. Blocking CK2 activity in LNCaP cells with apigenin or 5,6-dichlorobenzimidazole riboside led to a rapid decrease in NKX3.1 accumulation that was rescued by proteasome inhibition. Replacing Thr89 and Thr93 with alanines decreased NKX3.1 stability in vivo. Small interfering RNA knockdown of CK2alpha' but not CK2alpha also led to a decrease in NKX3.1 steady-state level. In-gel kinase assays and Western blot analyses using fractionated extracts of LNCaP cells demonstrated that free CK2alpha' could phosphorylate recombinant human and mouse NKX3.1, whereas CK2alpha' liberated from the holoenzyme could not. These data establish CK2 as a regulator of NKX3.1 in prostate tumor cells and provide evidence for functionally distinct pools of CK2alpha' in LNCaP cells.     

Identification of a positive Cis-element upstream of human NKX3.1 gene

NKX3.1 is a prostate-specific homeobox gene related to prostate development and prostate cancer. In this work, we aimed to identify precisely the functional cis-element in the 197 bp region （from -1032 to -836 bp） of the NKX3.1 promoter （from -1032 to ＋8 bp）, which was previously identified to present positive regulatory activity on NKX3.1 expression, by deletion mutagenesis analysis and electrophoretic mobility shift assay （EMSA）. A 16 bp positive cis-element located between -920 and -905 bp upstream of the NKX3.1 gene was identified by deletion mutation analysis and proved to be a functional positive cis-element by EMSA. It will be important to further study the functions and regulatory mechanisms of this positive cis-element in NKX3.1 gene expression.     

Identification of Sp1-elements in the promoter region of human homeobox gene <Emphasis Type="Italic">NKX3.1</Emphasis>

NKX3.1 is a prostate-specific homeobox gene related strongly to prostate development and prostate cancer. However, little is known about the mechanism for regulation of NKX3.1 in prostate cancer. With RT-PCR and western blot, we found that NKX3.1 expression was enhanced by over-expression of Sp1 at both the mRNA and protein levels in prostate cancer LNCaP cells. To identify the Sp1-elements in the promoter region of NKX3.1, a 521 bp-promoter of human NKX3.1 gene containing three possible Sp1-elements was cloned into the upstream of the luciferase reporter gene in pGL₃-basic plasmid. With deletion mutation analysis, plasmid construction, EMSA and oligonucleotide decoy technique, two Sp1-elements which located between +29 to +43 and −60 to −46 of NKX3.1 gene were identified and proven to be functional elements. It will be important to further study on the functions and the regulatory mechanisms of Sp1 element in NKX3.1 gene expression.     

Gene expression profiles in the PC-3 human prostate cancer cells induced by NKX3.1

NKX3.1, a prostate-specific gene, plays an important role in prostate development and carcinogenesis. However, its precise function has not been established. In present study, we transfected the NKX3.1 eukaryotic expression plasmid (pcDNA3.1-NKX3.1) into human prostate cancer cells PC-3, which lack of NKX3.1 expression, and established stable transfectants. Then, we investigated the influence of NKX3.1 on the cell growth, cell migration and colony formation efficiency. The results showed that restoration of NKX3.1 expression inhibited proliferation and invasion activities of PC-3 cells. Further, a cDNA microarray containing 22,000 human genes was used to identify the gene expression differences. The results showed that there were 1,953 genes showing more than a two-fold difference in expression. Subsequent ontological analysis revealed that a large proportion of the classified genes were related to cell growth, cell signal and cell invasion. Finally, the expression of Caspase-3, Bcl-2, P27, Cdk6 and AMACR, randomly selected genes from microarray data, was validated by RT-PCR and western blot. Collectively, our results first analyzed the gene expression profile in PC-3 cells induced by NKX3.1 and indicated that NKX3.1 might exert its function by regulating the expression of relative genes.     

TNFα-Mediated Loss of β-Catenin/E-Cadherin Association and Subsequent Increase in Cell Migration Is Partially Restored by NKX3.1 Expression in Prostate Cells

Inflammation-induced carcinogenesis is associated with increased proliferation and migration/invasion of various types of tumor cells. In this study, altered β-catenin signaling upon TNFα exposure, and relation to loss of function of the tumor suppressor NKX3.1 was examined in prostate cancer cells. We used an in vitro prostate inflammation model to demonstrate altered sub-cellular localization of β-catenin following increased phosphorylation of Akt^(S473) and GSK3β^(S9). Consistently, we observed that subsequent increase in β-catenin transactivation enhanced c-myc, cyclin D1 and MMP2 expressions. Consequently, it was also observed that the β-catenin-E-cadherin association at the plasma membrane was disrupted during acute cytokine exposure. Additionally, it was demonstrated that disrupting cell-cell interactions led to increased migration of LNCaP cells in real-time migration assay. Nevertheless, ectopic expression of NKX3.1, which is degraded upon proinflammatory cytokine exposure in inflammation, was found to induce the degradation of β-catenin by inhibiting Akt^(S473) phosphorylation, therefore, partially rescued the disrupted β-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure. As, the disrupted localization of β-catenin at the cell membrane as well as increased Akt^(S308) priming phosphorylation was observed in human prostate tissues with prostatic inflammatory atrophy (PIA), high-grade prostatic intraepithelial neoplasia (H-PIN) and carcinoma lesions correlated with loss of NKX3.1 expression. Thus, the data indicate that the β-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.     

The Pace of Prostatic Intraepithelial Neoplasia Development Is Determined by the Timing of Pten Tumor Suppressor Gene Excision

Loss of the PTEN tumor suppressor is a common occurrence in human prostate cancer, particularly in advanced disease. In keeping with its role as a pivotal upstream regulator of the phosphatidylinositol 3-kinase signaling pathway, experimentally-induced deletion of Pten in the murine prostate invariably results in neoplasia. However, and unlike humans where prostate tumorigenesis likely evolves over decades, disease progression in the constitutively Pten deficient mouse prostate is relatively rapid, culminating in invasive cancer within several weeks post-puberty. Given that the prostate undergoes rapid androgen-dependent growth at puberty, and that Pten excisions during this time might be especially tumorigenic, we hypothesized that delaying prostate-specific Pten deletions until immediately after puberty might alter the pace of tumorigenesis. To this end we generated mice with a tamoxifen-inducible Cre recombinase transgene enabling temporal control over prostate-specific gene alterations. This line was then interbred with mice carrying floxed Pten alleles. Despite evidence of increased Akt/mTOR/S6K axis activity at early time points in Pten-deficient epithelial cells, excisions induced in the post-pubertal (6 wk-old) prostate yielded gradual acquisition of a range of lesions. These progressed from pre-malignant changes (nuclear atypia, focal hyperplasia) and low grade prostatic intraepithelial neoplasia (PIN) at 16–20 wks post-tamoxifen exposure, to overtly malignant lesions by ∼1 yr of age, characterized by high-grade PIN and microinvasive carcinoma. In contrast, when Pten excisions were triggered in the pre-pubertal (2 week-old) prostate, neoplasia evolved over a more abbreviated time-frame, with a spectrum of premalignant lesions, as well as overt PIN and microinvasive carcinoma by 10–12 wks post-tamoxifen exposure. These results indicate that the developmental stage at which Pten deletions are induced dictates the pace of PIN development.