Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Feb 3;3(2):e86.
doi: 10.1038/oncsis.2013.48.

Tumor suppressor NDRG2 tips the balance of oncogenic TGF-β via EMT inhibition in colorectal cancer

L Shen  1 , X Qu  2 , Y Ma  1 , J Zheng  3 , D Chu  1 , B Liu  4 , X Li  1 , M Wang  5 , C Xu  3 , N Liu  6 , L Yao  1 , J Zhang  1
Affiliations

Tumor suppressor NDRG2 tips the balance of oncogenic TGF-β via EMT inhibition in colorectal cancer

L Shen et al. Oncogenesis. .

Abstract

Transforming growth factor-beta (TGF-β), a pluripotent cytokine expressed in the colon, has a crucial but paradoxical role in colorectal cancer (CRC). TGF-β is a potent proliferation inhibitor of normal colon epithelial cells and acts as a tumor suppressor. However, TGF-β also promotes invasion and metastasis during late-stage CRC, thereby acting as an oncogene. Thus, understanding the factors behind the paradoxical roles of TGF-β and elucidating the mechanisms by which TGF-β-induced proliferation inhibition is impaired in CRC are necessary. Here, we found that the N-Myc tumor suppressor gene downstream-regulated gene NDRG2 (N-Myc downstream-regulated gene 2), which is a TGF-β-responsive gene, abrogated TGF-β-induced epithelial-mesenchymal transition (EMT) and further inhibited the invasion and migration of CRC cells. TGF-β positively induced NDRG2 expression through direct transactivation mediated by Sp1 and by abrogation of the repressive c-Myc/Miz-1 complex on NDRG2 promoter in normal epithelial cells. Aberrant hypermethylation of NDRG2, which could respond to TGF-β growth inhibition signaling, abrogated the inhibitory effect of NDRG2 in TGF-β-induced EMT in CRCs. Reduced NDRG2 expression was highly correlated with the invasion stage and metastasis of CRC. Our study establishes that NDRG2 is a new tumor suppressor gene that responds to TGF-β anti-proliferative signaling and tips the balance of oncogenic TGF-β during late-stage CRC.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Positive correlation between NDRG2 and p-Smad2/3 in colorectal cancer tissues. (a) IHC staining of NDRG2 and p-Smad2/3 in human adjacent normal and cancerous colon samples from patients at different stages. Case 1 is from a stage III patient, and Case 2 is from a stage II patient. Original magnification: × 5, × 40 or × 100. (b, c) Relative expression level of p-Smad2/3 and NDRG2 in human tissue samples. Student's t-test was applied for statistical analyses. *P<0.05, which was considered significantly different. (d) Correlation between NDRG2 and p-Smad2/3 expression with linear regression and Pearson's correlation significance (P<0.001, ANOVA test).
Figure 2
Figure 2
TGF-β directly transactivates NDRG2 at the transcriptional level. (a, b) HaCaT cells were treated with TGF-β for 24 h, and the protein and mRNA expression levels of NDRG2 and p21Cip1 were detected. β-Actin served as a control to ensure equal loading. (c) HaCaT cells were treated with TGF-β for the indicated time, and the c-Myc and NDRG2 protein levels were detected. β-Actin served as a control to ensure equal loading. (d) HaCaT cells were treated with 10 ng/ml TGF-β for 48 h. Relative cell viability was detected with an MTT assay (n=3). (e) A panel of NDRG2 promoter constructs were transfected into HaCaT cells. At 24 h post transfection, the cells were treated with 10 ng/ml TGF-β for 20 h. The data shown are the means±s.d. from triplicate analysis. (f) Top: Representation of the human NDRG2 promoter regions depicting the sequence of the Smad-binding region (SBR). Bottom: HaCaT cells after TGF-β treatment were extracted and subjected to a chromatin immunoprecipitation assay. The presence of the NDRG2 promoter was detected with RT–PCR and visualized with ethidium bromide staining.
Figure 3
Figure 3
TGF-β-induced NDRG2 induction requires the Sp1 transcription factor. (a) Cells were transfected with pcDNA3 or pcDNA3-HA-Sp1. At 12 or 24 h after transfection, protein was extracted for western blotting (top), quantitative real-time PCR (middle) and a reporter assay using NDRG2 (−1455/+274) (bottom). (b) HaCaT cells were transfected with the indicated NDRG2 reporter constructs with or without a Sp1 expression vector. Luciferase activities were detected 24 h after transfection. (c) Human intestinal epithelial cells (HIEC) transfected with pcDNA3 or pcDNA3-HA-Sp1 were immunoprecipitated with the indicated antibodies. The presence of the NDRG2 promoter containing Sp1 binding sites was detected by RT–PCR and visualized with ethidium bromide staining. (d, e) HaCaT cells were transfected with the indicated NDRG2 reporter constructs with or without Sp1 expression or shRNA vector. The cells were incubated in the presence or absence of 10 ng/ml TGF-β for 20 h before lysis and then analyzed for luciferase activity. (f) Quantitative real-time PCR (left) and luciferase reporter assay (right) analysis for the involvement of mithramycin in TGF-β-mediated NDRG2 expression. HaCaT cells were pretreated with or without mithramycin for 1 h at the indicated concentrations and then stimulated with 10 ng/ml TGF-β for 20 h. (a–f) The data are presented as the means±s.d. from triplicate analyses.
Figure 4
Figure 4
TGF-β abrogates the c-Myc/Miz-1 complex and upregulates NDRG2 promoter activity. (a–d) HaCaT cells were incubated for 20 h in the presence or absence of 10 ng/ml TGF-β, and the mRNA levels and luciferase activity were determined. The data are presented as the means±s.d. from triplicate analyses. (a) Cells were transfected with either a control or a c-Myc expression vector. Quantitative real-time PCR was performed. (b) Cells were transfected with the indicated NDRG2 reporter constructs with or without the c-Myc expression vector. (c) Cells were transfected with the NDRG2 reporter construct together with wild-type Miz-1 and its mutant construct, as indicated. (d) Cells were transfected with the NDRG2 reporter construct with or without a c-Myc expression plasmid. Increasing amounts (0.01, 0.05 and 0.1 μg) of Miz-1 construct were co-transfected as indicated. (e, f) HaCaT cells treated with TGF-β were extracted and subjected to chromatin immunoprecipitation. The presence of the NDRG2 promoter was detected by RT–PCR and visualization by ethidium bromide staining.
Figure 5
Figure 5
NDRG2 overexpression attenuates TGF-β-induced EMT. (a) Phase-contrast photomicrographs of control cells and cells treated with 10 ng/ml TGF-β for 48 h. (b) Circularity index of untreated and TGF-β-treated cells. (c) Western blot analysis of E-cadherin and vimentin expression from total lysates of untreated cells and cells treated with 10 ng/ml TGF-β for 48 h. (d) Relative mRNA levels of E-cadherin and vimentin in control cells and cells treated with 10 ng/ml TGF-β for 48 h. The control values were normalized to 1, and the data are expressed as fold-change in treated cells. (e, f) The migratory and invasive behavior of untreated cells and cells treated with 10 ng/ml TGF-β for 48 h. The control values were normalized to 1, and the data are expressed as fold-change in treated cells. (d–f) The data are presented as the means±s.d. from triplicate analyses.
Figure 6
Figure 6
NDRG2 knockdown promotes TGF-β-induced EMT. (a) Phase-contrast photomicrographs of control cells and cells treated with TGF-β demonstrated that NDRG2 and p21Cip1 knockdown mimics the EMT phenotype in HT29 cells. (b) Circularity index of untreated and TGF-β1-treated cells. (c) Western blot analysis of E-cadherin and vimentin expression in total lysates from untreated cells and cells treated with 10 ng/ml TGF-β for 48 h. (d, e) Migratory and invasive behavior of untreated cells and cells treated with 10 ng/ml TGF-β for 48 h. The control values were normalized to 1, and the data are expressed as the fold-change in treated cells. (f) The expression level of mRNAs encoding E-cadherin, N-cadherin, fibronectin, Twist, Snail and vimentin in control cells and cells treated with 10 ng/ml TGF-β for 48 h. (d–f) The data are presented as the means±s.d. from triplicate analysis.
Figure 7
Figure 7
Promoter hypermethylation reduces NDRG2 expression and hinders the progression and metastasis of colorectal cancer. (a) The RNA and protein levels of NDRG2 in multiple colorectal cancer cell lines and the normal human intestinal epithelial cells (HIEC) were detected by comparative RT–PCR and western blotting. (b) Bisulfite sequencing analysis of the NDRG2 promoter in a panel of cell lines. (Top) Schematic of the NDRG2 promoter. The positions of the CpG dinucleotides are shown to scale by vertical lines. (Bottom) Each circle represents a CpG dinucleotide: open (white) circles denote unmethylated CpG sites and filled (black) circles indicate methylated CpG sites. Each row represents a single clone. (c) The methylation status of the NDRG2 promoter was analyzed by methylation-specific PCR in each of the 24 normal colon and colorectal cancer tissues. The white and black colors represent hypomethylation and hypermethylation, respectively. Each experiment was independently repeated three times. (d) The binding activity of Spl to methylated and unmethylated recognition sites in vitro. (Top) Double-stranded oligonucleotides were derived from the Spl recognition sites in the NDRG2 promoter. The methylated C (Cm) is shown in red. A, no methylation; A1, single methylation on recognition site; A2, double methylation on recognition site; A3, methylation on adjacent region; A4, methylation of both CpG dinucleotides within its binding site and adjacent CpG sites. (Bottom) Electrophoretic mobility shift analysis of HT29 cell nuclear extracts. The Sp1 complex and free probe are indicated by the arrow. (e) HCT116 cells were transfected with or without a Sp1 expression vector. Twenty-four hours post transfection, the cells were treated with 5-aza-dC at the indicated concentrations for 48 h before harvesting for western blotting and quantitative real-time PCR analysis. The data are the means±s.d. from triplicate analyses. (f) The immunoreactivity score (IRS) distribution of NDRG2 IHC staining among tumors of different invasion statuses. (g) The IRS distribution of NDRG2 IHC staining between tumors of different lymph-node metastasis statuses. (h) The IRS distribution of NDRG2 IHC staining between tumors of different distant metastasis statuses. (i) Model of NDRG2 tipping the balance of oncogenic TGF-β via EMT inhibition in colorectal cancer.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62:10–29. - PubMed
    1. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61:759–767. - PubMed
    1. Derynck R, Akhurst RJ, Balmain A. TGF-beta signaling in tumor suppression and cancer progression. Nat Genet. 2001;29:117–129. - PubMed
    1. Engel ME, Datta PK, Moses HL. Signal transduction by transforming growth factor-beta: a cooperative paradigm with extensive negative regulation. J Cell Biochem Suppl. 1998;30-31:111–122. - PubMed
    1. Shin I, Bakin AV, Rodeck U, Brunet A, Arteaga CL. Transforming growth factor beta enhances epithelial cell survival via Akt-dependent regulation of FKHRL1. Mol Biol Cell. 2001;12:3328–3339. - PMC - PubMed

LinkOut - more resources

-