Limits...
NDRG1 overexpression promotes the progression of esophageal squamous cell carcinoma through modulating Wnt signaling pathway

View Article: PubMed Central - PubMed

ABSTRACT

N-myc down-regulated gene 1 (NDRG1) has been shown to regulate tumor growth and metastasis in various malignant tumors and also to be dysregulated in esophageal squamous cell carcinoma (ESCC). Here, we show that NDRG1 overexpression (91.9%, 79/86) in ESCC tumor tissues is associated with poor overall survival of esophageal cancer patients. When placed in stable transfectants of the KYSE 30 ESCC cell line generated by lentiviral transduction with the ectopic overexpression of NDRG1, the expression of transducin-like enhancer of Split 2 (TLE2) was decreased sharply, however β−catenin was increased. Mechanistically, NDRG1 physically associates with TLE2 and β−catenin to affect the Wnt pathway. RNA interference and TLE2 overexpression studies demonstrate that NDRG1 fails to active Wnt pathway compared with isogenic wild-type controls. Strikingly, NDRG1 overexpression induces the epithelial mesenchymal transition (EMT) through activating the Wnt signaling pathway in ESCC cells, decreased the expression of E-cadherin and enhanced the expression of Snail. Our study elucidates a mechanism of NDRG1-regulated Wnt pathway activation and EMT via affecting TLE2 and  β-catenin expression in esophageal cancer cells. This indicates a pro-oncogenic role for NDRG1 in esophageal cancer cells whereby it modulates tumor progression.

No MeSH data available.


Related in: MedlinePlus

NDRG1 impacts the Wnt pathway via TLE2 and β-catenin in esophageal cancer cells. (A) Relative gene expression levels of FZD8, TLE2, LEF, and WNT3A were analyzed in KYSE 30 cells overexpressed NDRG1 (KYSE 30-NDRG1) compared with those in mock cells (KYSE 30-Ctrl) using real-time PCR arrays. (B) mRNA levels of Wnt pathway-associated genes in NDRG1 knock-down cells (KYSE 30-shNDRG1, shNDRG1) were assessed by quantitative RT-PCR and compared with mock cells (KYSE 30-vec, vec). (C) Protein levels of NDRG1, TLE2 and β-catenin from KYSE 30-NDRG1 and KYSE 30-Ctrl cells were measured by Western blot. (D) Quantitative RT-PCR results validated the mRNA levels of the TLE2 in KYSE 30-NDRG1 and NDRG1 knock-down (KYSE 30-shNDRG1) cells. (E) The colocalization of NDRG1 and TLE2 was analyzed by confocal microscopy in KYSE 30-NDRG1 and KYSE 30-Ctrl cells. TLE2 was mainly localized in the nuclei, while NDRG1 was expressed in both the nuclei and cytoplasma. NDRG1 and TLE2 colocalized in the nuclei.Green fluorescence, TLE2; red fluorescence, NDRG1; blue fluorescence, nuclei stained with DAPI. Scale bar, 10 μm. (F) Cytosolic and nuclear proteins were isolated from KYSE 30-NDRG1 and KYSE 30-Ctrl cells, and NDRG1, β-catenin and TLE2 expression was analyzed by western blot. Lamin B, nuclear marker; β-actin, cytoplasmic marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036407&req=5

f0003: NDRG1 impacts the Wnt pathway via TLE2 and β-catenin in esophageal cancer cells. (A) Relative gene expression levels of FZD8, TLE2, LEF, and WNT3A were analyzed in KYSE 30 cells overexpressed NDRG1 (KYSE 30-NDRG1) compared with those in mock cells (KYSE 30-Ctrl) using real-time PCR arrays. (B) mRNA levels of Wnt pathway-associated genes in NDRG1 knock-down cells (KYSE 30-shNDRG1, shNDRG1) were assessed by quantitative RT-PCR and compared with mock cells (KYSE 30-vec, vec). (C) Protein levels of NDRG1, TLE2 and β-catenin from KYSE 30-NDRG1 and KYSE 30-Ctrl cells were measured by Western blot. (D) Quantitative RT-PCR results validated the mRNA levels of the TLE2 in KYSE 30-NDRG1 and NDRG1 knock-down (KYSE 30-shNDRG1) cells. (E) The colocalization of NDRG1 and TLE2 was analyzed by confocal microscopy in KYSE 30-NDRG1 and KYSE 30-Ctrl cells. TLE2 was mainly localized in the nuclei, while NDRG1 was expressed in both the nuclei and cytoplasma. NDRG1 and TLE2 colocalized in the nuclei.Green fluorescence, TLE2; red fluorescence, NDRG1; blue fluorescence, nuclei stained with DAPI. Scale bar, 10 μm. (F) Cytosolic and nuclear proteins were isolated from KYSE 30-NDRG1 and KYSE 30-Ctrl cells, and NDRG1, β-catenin and TLE2 expression was analyzed by western blot. Lamin B, nuclear marker; β-actin, cytoplasmic marker.

Mentions: A key event following Wnt pathway activation is the molecular switch from transcriptional repression of TLE to the activation of TCF/Lef.34 Through quantitative gene expression analysis of key genes involved in metastasis, we found that Wnt pathway-associated genes, including Frizzled Class Receptor 8 (FZD8), Transducin-Like Enhancer of Split 2 (TLE2), Lymphoid Enhancer-Binding Factor 1 (LEF1) and Wingless-Type MMTV Integration Site Family, Member 3A (WNT3A), were changed; TLE2 expression was significantly decreased and other genes were greatly increased in NDRG1 overexpressing cells (KYSE 30-NDRG1) (Fig. 3A). The mRNA levels of Wnt pathway-associated genes in NDRG1 knock-down cells (KYSE 30-shNDRG1, shNDRG1) were assessed by quantitative RT-PCR and compared with mock cells (KYSE 30-vec, vec)(Fig. 3B). To identify functional links between NDRG1 and TLE2, we examined the effects of NDRG1 overexpression and knock-down on TLE expression. As shown in Fig. 3C and 3D, NDRG1 overexpression suppressed TLE2 expression at both the transcript and protein levels. Inversely, NDRG1 knock-down induced TLE2 upregulation at the transcript level in KYSE 30 cells. To further confirm that TLE expression is regulated by NDRG1, we assessed the co-localization of NDRG1 and TLE2 using immunofluorescence and western blot analysis following NDRG1 ectopic expression. As shown in Fig. 3E, we observed that NDRG1 overexpression slightly increased the nuclear localization of NDRG1 and TLE2 compared with that of the control group. Total cytosolic and nuclear fractions were isolated and tested for the expression of NDRG1, TLE2 and β-catenin by protein gel blot analysis. Significant accumulation of β-catenin and NDRG1 and a decrease in TLE protein levels were observed in the nuclear fractions of KYSE 30 cells (KYSE 30-NDRG1), indicating that NDRG1 regulates TLE2 transcript and protein expression (Fig. 3F).Figure 3.


NDRG1 overexpression promotes the progression of esophageal squamous cell carcinoma through modulating Wnt signaling pathway
NDRG1 impacts the Wnt pathway via TLE2 and β-catenin in esophageal cancer cells. (A) Relative gene expression levels of FZD8, TLE2, LEF, and WNT3A were analyzed in KYSE 30 cells overexpressed NDRG1 (KYSE 30-NDRG1) compared with those in mock cells (KYSE 30-Ctrl) using real-time PCR arrays. (B) mRNA levels of Wnt pathway-associated genes in NDRG1 knock-down cells (KYSE 30-shNDRG1, shNDRG1) were assessed by quantitative RT-PCR and compared with mock cells (KYSE 30-vec, vec). (C) Protein levels of NDRG1, TLE2 and β-catenin from KYSE 30-NDRG1 and KYSE 30-Ctrl cells were measured by Western blot. (D) Quantitative RT-PCR results validated the mRNA levels of the TLE2 in KYSE 30-NDRG1 and NDRG1 knock-down (KYSE 30-shNDRG1) cells. (E) The colocalization of NDRG1 and TLE2 was analyzed by confocal microscopy in KYSE 30-NDRG1 and KYSE 30-Ctrl cells. TLE2 was mainly localized in the nuclei, while NDRG1 was expressed in both the nuclei and cytoplasma. NDRG1 and TLE2 colocalized in the nuclei.Green fluorescence, TLE2; red fluorescence, NDRG1; blue fluorescence, nuclei stained with DAPI. Scale bar, 10 μm. (F) Cytosolic and nuclear proteins were isolated from KYSE 30-NDRG1 and KYSE 30-Ctrl cells, and NDRG1, β-catenin and TLE2 expression was analyzed by western blot. Lamin B, nuclear marker; β-actin, cytoplasmic marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036407&req=5

f0003: NDRG1 impacts the Wnt pathway via TLE2 and β-catenin in esophageal cancer cells. (A) Relative gene expression levels of FZD8, TLE2, LEF, and WNT3A were analyzed in KYSE 30 cells overexpressed NDRG1 (KYSE 30-NDRG1) compared with those in mock cells (KYSE 30-Ctrl) using real-time PCR arrays. (B) mRNA levels of Wnt pathway-associated genes in NDRG1 knock-down cells (KYSE 30-shNDRG1, shNDRG1) were assessed by quantitative RT-PCR and compared with mock cells (KYSE 30-vec, vec). (C) Protein levels of NDRG1, TLE2 and β-catenin from KYSE 30-NDRG1 and KYSE 30-Ctrl cells were measured by Western blot. (D) Quantitative RT-PCR results validated the mRNA levels of the TLE2 in KYSE 30-NDRG1 and NDRG1 knock-down (KYSE 30-shNDRG1) cells. (E) The colocalization of NDRG1 and TLE2 was analyzed by confocal microscopy in KYSE 30-NDRG1 and KYSE 30-Ctrl cells. TLE2 was mainly localized in the nuclei, while NDRG1 was expressed in both the nuclei and cytoplasma. NDRG1 and TLE2 colocalized in the nuclei.Green fluorescence, TLE2; red fluorescence, NDRG1; blue fluorescence, nuclei stained with DAPI. Scale bar, 10 μm. (F) Cytosolic and nuclear proteins were isolated from KYSE 30-NDRG1 and KYSE 30-Ctrl cells, and NDRG1, β-catenin and TLE2 expression was analyzed by western blot. Lamin B, nuclear marker; β-actin, cytoplasmic marker.
Mentions: A key event following Wnt pathway activation is the molecular switch from transcriptional repression of TLE to the activation of TCF/Lef.34 Through quantitative gene expression analysis of key genes involved in metastasis, we found that Wnt pathway-associated genes, including Frizzled Class Receptor 8 (FZD8), Transducin-Like Enhancer of Split 2 (TLE2), Lymphoid Enhancer-Binding Factor 1 (LEF1) and Wingless-Type MMTV Integration Site Family, Member 3A (WNT3A), were changed; TLE2 expression was significantly decreased and other genes were greatly increased in NDRG1 overexpressing cells (KYSE 30-NDRG1) (Fig. 3A). The mRNA levels of Wnt pathway-associated genes in NDRG1 knock-down cells (KYSE 30-shNDRG1, shNDRG1) were assessed by quantitative RT-PCR and compared with mock cells (KYSE 30-vec, vec)(Fig. 3B). To identify functional links between NDRG1 and TLE2, we examined the effects of NDRG1 overexpression and knock-down on TLE expression. As shown in Fig. 3C and 3D, NDRG1 overexpression suppressed TLE2 expression at both the transcript and protein levels. Inversely, NDRG1 knock-down induced TLE2 upregulation at the transcript level in KYSE 30 cells. To further confirm that TLE expression is regulated by NDRG1, we assessed the co-localization of NDRG1 and TLE2 using immunofluorescence and western blot analysis following NDRG1 ectopic expression. As shown in Fig. 3E, we observed that NDRG1 overexpression slightly increased the nuclear localization of NDRG1 and TLE2 compared with that of the control group. Total cytosolic and nuclear fractions were isolated and tested for the expression of NDRG1, TLE2 and β-catenin by protein gel blot analysis. Significant accumulation of β-catenin and NDRG1 and a decrease in TLE protein levels were observed in the nuclear fractions of KYSE 30 cells (KYSE 30-NDRG1), indicating that NDRG1 regulates TLE2 transcript and protein expression (Fig. 3F).Figure 3.

View Article: PubMed Central - PubMed

ABSTRACT

N-myc down-regulated gene 1 (NDRG1) has been shown to regulate tumor growth and metastasis in various malignant tumors and also to be dysregulated in esophageal squamous cell carcinoma (ESCC). Here, we show that NDRG1 overexpression (91.9%, 79/86) in ESCC tumor tissues is associated with poor overall survival of esophageal cancer patients. When placed in stable transfectants of the KYSE 30 ESCC cell line generated by lentiviral transduction with the ectopic overexpression of NDRG1, the expression of transducin-like enhancer of Split 2 (TLE2) was decreased sharply, however β−catenin was increased. Mechanistically, NDRG1 physically associates with TLE2 and β−catenin to affect the Wnt pathway. RNA interference and TLE2 overexpression studies demonstrate that NDRG1 fails to active Wnt pathway compared with isogenic wild-type controls. Strikingly, NDRG1 overexpression induces the epithelial mesenchymal transition (EMT) through activating the Wnt signaling pathway in ESCC cells, decreased the expression of E-cadherin and enhanced the expression of Snail. Our study elucidates a mechanism of NDRG1-regulated Wnt pathway activation and EMT via affecting TLE2 and  β-catenin expression in esophageal cancer cells. This indicates a pro-oncogenic role for NDRG1 in esophageal cancer cells whereby it modulates tumor progression.

No MeSH data available.


Related in: MedlinePlus