The epithelial-mesenchymal transition (EMT) is a key step for cancer cell migration, invasion, and metastasis. Transforming growth factor-β (TGF-β) regulates the EMT and the metastasis suppressor gene, N-myc downstream-regulated gene-1 (NDRG1), could play a role in regulating the TGF-β pathway. NDRG1 expression is markedly increased after chelator-mediated iron depletion via hypoxia-inducible factor 1α-dependent and independent pathways (Le, N. T. and Richardson, D. R. (2004) Blood 104, 2967-2975). Moreover, novel iron chelators show marked and selective anti-tumor activity and are a potential new class of anti-metabolites. Considering this, the current study investigated the relationship between NDRG1 and the EMT to examine if iron chelators can inhibit the EMT via NDRG1 up-regulation. We demonstrated that TGF-β induces the EMT in HT29 and DU145 cells. Further, the chelators, desferrioxamine (DFO) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), inhibited the TGF-β-induced EMT by maintaining E-cadherin and β-catenin, at the cell membrane. We then established stable clones with NDRG1 overexpression and knock-down in HT29 and DU145 cells. These data showed that NDRG1 overexpression maintained membrane E-cadherin and β-catenin and inhibited TGF-β-stimulated cell migration and invasion. Conversely, NDRG1 knock-down caused morphological changes from an epithelial- to fibroblastic-like phenotype and also increased migration and invasion, demonstrating NDRG1 knockdown induced the EMT and enhanced TGF-β effects. We also investigated the mechanisms involved and showed the TGF-β/SMAD and Wnt pathways were implicated in NDRG1 regulation of E-cadherin and β-catenin expression and translocation. This study demonstrates that chelators inhibit the TGF-β-induced EMT via a process consistent with NDRG1 up-regulation and elucidates the mechanism of their activity.

Metastasis is a major cause of death in human colorectal cancer patients. However, the contribution of chemokines in the tumor microenvironment to tumor metastasis is not fully understood. Herein, we examinined several chemokines in colorectal cancer patients using chemokine ELISA array. Immunohistochemistry was used to detect expression of CXCL5 in colorectal cancer patients tissues. Human HCT116 and SW480 cell lines stably transfected with CXCL5, shCXCL5 and shCXCR2 lentivirus plasmids were used in our in vitro study. Immunoblot, immunofluorescence and transwell assay were used to examine the molecular biology and morphological changes in these cells. In addition, we used nude mice to detect the influence of CXCL5 on tumor metastasis in vivo. We found that CXCL5 was overexpressed in tumor tissues and associated with advanced tumor stage as well as poor prognosis in colorectal cancer patients. We also demonstrated that CXCL5 was primarily expressed in the tumor cell cytoplasm and cell membranes, which may indicate that the CXCL5 was predominantly produced by cancer epithelial cells instead of fibroblasts in the tumor mesenchyme. Additionally, overexpression of CXCL5 enhanced the migration and invasion of colorectal cancer cells by inducing the epithelial-mesenchymal transition (EMT) through activation of the ERK/Elk-1/Snail pathway and the AKT/GSK3β/β-catenin pathway in a CXCR2-dependent manner. The silencing of Snail and β-catenin attenuated CXCL5/CXCR2-enhanced cell migration and invasion in vitro. The elevated expression of CXCL5 can also potentiate the metastasis of colorectal cancer cells to the liver in vivo in nude mice intrasplenic injection model. In conclusion, our findings support CXCL5 as a promoter of colorectal cancer metastasis and a predictor of poor clinical outcomes in colorectal cancer patients.

N-myc downstream-regulated gene 1 (NDRG1) is a potent metastasis suppressor that has been demonstrated to inhibit the transforming growth factor-β (TGF-β)-induced epithelial mesenchymal transition (EMT) by maintaining E-cadherin and β-catenin at the cell membrane in prostate and colon cancer cells. However, the precise molecular mechanism remains unclear. In this investigation, we demonstrated that NDRG1 inhibited the phosphorylation of β-catenin at Ser33/37, Thr41 and increased non-phosphorylated β-catenin levels on the plasma membrane in DU145 prostate cancer cells and HT29 colon cancer cells. The mechanism of inhibiting β-catenin phosphorylation involved the NDRG1-mediated up-regulation of the GSK3β-binding protein, FRAT1, which prevents the association of GSK3β with the Axin1/APC/CK1 destruction complex and subsequent phosphorylation of β-catenin. Additionally, NDRG1 was shown to modulate the WNT/β-catenin pathway by inhibiting β-catenin nuclear translocation. This was mediated through its effect of reducing the nuclear localization of p21 activated kinase 4 (PAK4), which is known to act as a transporter for β-catenin nuclear translocation. The current study is the first to elucidate a unique molecular mechanism involved in the regulation of β-catenin phosphorylation and distribution by NDRG1.

N-myc downstream-regulated gene 1 (NDRG1) has been reported to act as a key regulatory molecule in tumor progression-related signaling pathways, especially in tumor metastasis. However, the related mechanism has not been fully discovered yet. Herein we demonstrated that the novel molecule of cell migration and invasion, caveolin-1, has direct interaction with NDRG1 in human colorectal cancer (CRC) cells. Moreover, we discovered that NDRG1 reduces caveolin-1 protein expression through promoting its ubiquitylation and subsequent degradation via the proteasome in CRC cells. In addition, caveolin-1 mediates the suppressive function of NDRG1 in epithelial–mesenchymal transition, migration and invasion in vitro and metastasis in vivo. These results help to fulfill the potential mechanisms of NDRG1 in anti-metastatic treatment for human colorectal cancer.

Laparoscopic versus open right hemicolectomy with curative intent for colon carcinoma

MicroRNAs (miRNAs) have been reported to play crucial roles in regulating a variety of genes pivotal for tumor metastasis. MicroRNA-301a (miR-301a) is overexpressed and displays oncogenic activity in many cancers. However, little is known about the potential roles of miR-301a in colorectal cancer (CRC). Taqman probe stem-loop real-time PCR was used to quantitatively measure the expression level of miR-301a in 48 cases of CRC tissues and the matched adjacent non-tumor mucosa as well as in CRC cell lines. miR-301a mimics and inhibitors were used to up-regulate and down-regulate miR-301a in CRC cells, respectively; lentivirus was used to construct miR-301a stably up- and down-regulated CRC cell lines. Metastasis ability was evaluated by transwell and wound healing assays while invasion was measured by transwell coated with matrix gel in vitro; in vivo metastasis was performed on nude mice model. The target of miR-301a was predicted by TargetScan software and validated by qRT-PCR, immunohistochemistry, western blot and luciferase reporter gene assay. The expression of miR-301a was significantly higher in lymph node metastasis positive CRC samples compared with negative ones. Downregulation of miR-301a significantly inhibited the migration and invasion both in vitro and in vivo while forced up-regulation of miR-301a promoted migration and invasion. TGFBR2 was identified to be the downstream target of miR-301a. Knockdown of TGFBR2 in cells treated by miR-301a inhibitor elevated the previously abrogated migration and invasion. Our data indicated that miR-301a correlated with the metastatic and invasive ability in human colorectal cancers and miR-301a exerted its role as oncogene by targeting TGFBR2.