TWIST1, A novel androgen-regulated gene, is a target for NKX3-1 in prostate cancer cells
© Eide et al.; licensee BioMed Central Ltd. 2013
Received: 16 August 2012
Accepted: 29 January 2013
Published: 31 January 2013
TWIST1 plays a key role in EMT-mediated tumor invasion and metastasis. Since bone metastasis is a hallmark of advanced prostate cancer and is detected in at least 85% of patients who die of this disease, it is of great importance to understand the regulation of the cellular signaling pathways involved in the metastatic process.
Prostatic cell lines were analyzed using real time RT-PCR, chromatin immunoprecipitations (ChIP) and transfection of siRNA’s and reporter constructs.
We report in this paper that TWIST1 is an androgen-regulated gene under tight regulation of NKX3-1. Androgens repress the expression of TWIST1 via NKX3-1, which is a prostate–specific tumor suppressor that is down-regulated in the majority of metastatic prostate tumors. We show that NKX3-1 binds to the TWIST1 promoter and that NKX3-1 over-expression reduces the activity of a TWIST1 promoter reporter construct, whereas NKX3-1 siRNA up-regulates endogenous TWIST1 mRNA in prostate cancer cells.
Our finding that NKX3-1 represses TWIST1 expression emphasizes the functional importance of NKX3-1 in regulating TWIST1 expression during prostate cancer progression to metastatic disease.
KeywordsProstate cancer LNCaP cells TWIST1 NKX3-1
Metastasis is the leading cause of morbidity and mortality among men with prostate cancer (PCa). Metastasis is a complex multistep process controlled by distinct genes and signaling pathways in each step. Epithelial-meschencymal transition (EMT), a critical event for morphogenetic movements during formation of parietal endoderm during gastrulation, may represent the initial phase of metastasis. Furthermore, EMT seems to induce stem-like properties of epithelial cells.
A pivotal step of EMT is the loss of E-cadherin. TWIST1, a master regulator of mesodermal development and a key mediator in the metastatic process, represses the expression of E-cadherin. Furthermore, TWIST1 depletion reduces the expression of N-cadherin in PC3 cells, a metastatic prostate cancer cell line, suggesting that TWIST1 supports EMT in prostate cancer. In support of this, a positive correlation between the level of TWIST1 and prostate cancer metastasis has been reported.
NKX3-1 is a critical gene associated with early stage of prostate tumorigenesis as down-regulation of NKX3-1 is observed in both prostatic intraepithelial neoplasia (PIN) and adenocarsinomas. Whereas high levels of TWIST1 is expressed in prostate cancer metastasis, low levels of NKX3-1 expression is observed in most prostate cancer metastasis examined. NKX3-1 encodes a homeobox gene that is switched on during embryonic development of prostate tissue and is one of the earliest markers of luminal prostate epithelium. NKX3-1 expression is strictly regulated by androgens and also appears to mark a sub-population of prostate stem cells[9, 10].
We report in this paper that TWIST1 is a novel androgen-regulated gene whose expression is tightly controlled by NKX3-1.
TWIST1 is regulated by androgens
TWIST1 is an NKX3-1 target gene
Next, a reporter construct containing 5kb of the mouse TWIST1 promoter (pGL3-TWIST-Luc) was co-transfected with an NKX3-1 expression vector. Over-expression of NKX3-1 reduced the promoter activity of TWIST1 by 50%, indicating that NKX3-1 represses the TWIST1 promoter activity (Figure3C). No effect of NKX3-1 over-expression was observed on cells transfected with pGL3 lacking the TWIST1 promoter (pGL3).
Finally, the effect of NKX3-1 on endogenous TWIST1 mRNA level was studied by transfecting LNCaP cells with siRNA targeting NKX3-1. A 5-fold increase in TWIST1 mRNA level was observed when NKX3-1 expression was down-regulated (Figure3D). The impact of R1881 stimulation, NKX3-1 overexpression and siRNA on NKX3-1 expression in LNCaP cells are summarized in Figure3E. Both R1881 stimulation as well as overexpression of NKX3-1 led to a significant increase in NKX3-1 expression, while siRNA against NKX3-1 reduced the expression markedly.
In this study we show both that TWIST1 mRNA is up-regulated by androgen via AR and that NKX3-1, a well-known androgen-regulated gene, binds the upstream regulatory region of the TWIST1 gene and represses the expression of TWIST1.
In a recent study, Takayama et al. identified putative direct target genes of AR using ChIP-on ChIP and Cap Analysis Gene Expression (CAGE). One of the genes identified by in silico analysis was TWIST1. Our results support this report as increased levels of TWIST1 mRNA was observed in R1881 stimulated LNCaP cells, while reduced expression of TWISTl was observed in cells transfected with siRNA targeting AR.
Furthermore, our data shows binding of NKX3-1 to the promoter region of TWIST1. We therefore suggest that NKX3-1 mediates an indirect and late effect of androgen stimulation on TWIST1 expression. Interestingly, we show that siRNA targeting AR reduces the level of TWIST1, whereas siRNA targeting NKX3-1 increases TWIST1 expression suggesting that TWIST1 expression is tightly controlled by androgen. NKX3-1 has been shown to function both as an activator and a repressor of transcription, but few target genes have been identified[13–15].
The physical binding of NKX3-1 to the TWIST1 promoter might block the mesenchymal drive of TWIST1, until NKX3-1 expression is down-regulated or lost in PIN or adenocarcinoma lesions. Loss of NKX3-1 expression has been observed in ~20% of PIN lesions, ~40% of advanced prostate tumors and up to 80% of metastatic prostate cancer. Androgen deprivation therapy as the most widely used treatment for advanced prostate cancer is likely to abolish androgen-stimulation of NKX3-1, leading eventually to down-regulation of repressor protein and de-repression of TWIST1’s metastatic potential.
In an attempt to identify genes whose regulation are altered by NKX3-1, Song et al. performed gene expression profiling analyses on micro dissected glands from NKX3-1-deficient prostate tissues during prostate cancer progression. They observed similarities between the expression profile of the micro dissected glands and constitutive activated AKT-transgenic mice as well as PTEN-deficient mice, suggesting that the PTEN-AKT-NKX3-1 axis serve as a major molecular path of prostate tumorigenesis. Li and Zhou showed that activation of the AKT pathway by TWIST1 is critical for the sustention of cancer stem cell-like traits generated by EMT, again suggesting a link between loss of NKX3-1 expression, relive of TWIST1 expression and eventually activation of AKT pathway.
We report in this paper that TWIST1 is an androgen-regulated gene, tightly regulated by NKX3-1. We show that NKX3-1 binds to the TWIST1 promoter and that NKX3-1 over-expression reduces the activity of a TWIST1 promoter reporter construct, whereas NKX3-1 siRNA up-regulated endogenous TWIST1 mRNA in prostate cancer cells. Our finding that NKX3-1 represses TWIST1 expression emphasizes the functional importance of NKX3-1 in regulating TWIST1 expression during prostate cancer progression to metastatic disease.
LNCaP and RWPE-1 cells were purchased from ATCC (Rockville, MD) and cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS) or Keratinocyte-SFM medium from Invitrogen (Carlsbad, CA, USA) supplemented with 2.5 μg Epidermal Growth Factor (EGF) and 25 mg Bovine Pituitary Extract (BPE), respectively, and stimulation with synthetic androgen R1881 was performed as previously described.
Semi-quantitative real time RT-PCR (sqRT-PCR)
Total RNA was isolated using Trizol™ from Invitrogen (Carlsbad, CA), and 100 ng of total RNA was used in a one-step RT-PCR reaction (QIAGEN Quantitect SYBR Green RT-PCR kit) that was performed using an MJ Research DNA Engine Opticon Continuous Fluorescence Detection System (MJ Research Inc., Waltham, MA). RT-PCR cycles were performed as previously described by Ramberg et al.. G6PD was used for normalization. The ΔΔCt formula was used as described in the protocol from Applied Biosystems (Foster City, CA). All the PCR-products were verified by sequencing.
Primer sets used in sqRT-PCR
G6PD (NM_000402); Left: tgcatgagccagataggc and right: acagggaggagatgtggttg, NKX3-1 (NM_006167); Left: gagacgctggcagagacc and right: ttctgcggctgcttaggg, AR (NM_000044); Left: gcgatccttcaccaatgtca and right: cattcggacacactggctgt, TWIST1 (NM_000474); Left: cttctcggtctggaggatgg and right: ctccttctctggaaacaatgaca.
Protein extraction followed by Western blot analysis was performed as previously described by Kvissel et al.. Primary antibodies used were the following: anti-TWIST1 (H-81, sc-15393), anti-AR (N-20, sc-816), both from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-NKX3-1 antibody was kindly provided by Professor Fahri Saatcioglu at Department of Molecular Biosciences, University of Oslo, Norway. For loading control we used anti-PRKAR1A (610609, BD Transduction Laboratories) or anti-α-tubulin antibody from Sigma (St. Louis, MO).
Transfection and luciferase assay
LNCaP cells were cultured at 300.000 cells per 6-well dish and transfected with a luciferase reporter plasmid including 5 kb of the mouse TWIST1 promoter- pGL3-TWIST (kindly provided by Steven Kendall and Carlotta Glackin, Beckman Research Institute, City of Hope, USA) or pGL3 as negative control using Lipofectamine 2000 (INVT11668019, Invitrogen) according to the manufactures protocol. For overexpression of NKX3-1, a commercial transfection-ready TRUE clone (sc116287, ORIGENE, Rockville, MD, USA) was purchased. Cells were co-transfected with pCMV β-gal (Clontech) to monitor the transfection efficiency. After 72 hours, luciferase and β-galactosidase activity were measured as previously described.
Preparation and transfection of synthetic small interfering RNA (siRNA)
siRNA targeting the following sequence within the androgen receptor mRNA: 5’-AAAAGCCCATCGTAGAGGCCCCA-3’ was purchased from Dharmacon (Dharmacon Inc. (Lafayette, CO). A non-silencing siRNA (cat.no D-001810-10-20, Dharmacon) was included in all siRNA experiments. siRNA against NKX3-1 was purchased from QIAGEN (2-for-silencing) and included the following sequences: NKX3-1-1: 5’-CAGGCTATCATATATACTGTA-3’, NKX3-1-2: 5’-ACGCTATAAGACTAAGCGAAA-3’. All siRNAs were transfected into cells using DharmaFECT ™ 3 transfection reagent according to the manufacture’s protocol (cat.no T-2003, Dharmacon).
ChIP assays were carried out according to the QuickChIP protocol (Imgenex, San Diego, CA) with a crosslinking time of 10 minutes using 1% formaldehyde for the LNCaP cells and using an anti human NKX3-1 monoclonal antibody (cat.no 35–9700, Zymed Laboratories Inc., San Fransisco, CA). Following DNA purification (QuickChIP DNA Purification kit), sqPCR was performed using QIAGEN Quantitect SYBR Green RT-PCR kit with the following primers:
NKX3-1 BS3; Left: cccagttacacttggatgcagta and right: tcccctggtgagatcatacatac. Negative control primers from human chromosome 12 genomic contig; Left: atggttgccactggggatct and right: tgccaaagcctaggggaaga. Chromatin immunoprecipitated with IgG was used as a negative control. The ΔΔCt formula was used as described in the protocol from Applied Biosystems (Foster City, CA).
Prostatic Intraepithelial Neoplasia
Chromatin Immunopreciptation Analyses.
We thank the Department of Urology at the Mayo Clinic and Foundation in Rochester, Minnesota, USA and Department of Urology at the Oslo University Hospital, Aker. Furthermore, we thank Lucy Smith (Mayo Clinic, Rochester, USA) for skilled technical assistance and Fahri Saatcioglu (University of Oslo, Norway)for providing us with anti human NKX3-1 antibody. This work was supported by The Norwegian Research Council, Health Region South East, The Norwegian Cancer Society, University of Oslo and Oslo University Hospital, Aker. DJT was supported by NIH grants CA121277, CA91956, CA125747 and the T.J. Martell Foundation.
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