HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via geranylgeranylation and RhoA activation.

BACKGROUND: Simvastatin is widely used to lower cholesterol levels in patients with cardiovascular diseases, although accumulating evidence suggests that statins, such as simvastatin, also exert numerous anti-tumoral effects. AIM: The aim of this study was to examine the effect of simvastatin on colon cancer cell migration. METHODS: Migration assays were performed to evaluate CCL17-induced colon cancer cell (HT-29) chemotaxis. In vitro tumor growth and apoptosis were assessed using a proliferation assay and annexin V assay, respectively. Active RhoA protein levels in CCL17-stimulated colon cancer cells were quantified using a G-LISA assay. RESULTS: We found that simvastatin dose-dependently decreased CCL17-induced colon cancer cell migration. Simvastatin had no effect on colon cancer cell proliferation or apoptosis. Inhibition of beta chemokine receptor 4, CCR4, reduced CCL17-evoked activation of RhoA in colon cancer cells. Moreover, administration of mevalonate reversed the inhibitory effect of simvastatin on CCL17-induced colon cancer cell migration. Interestingly, co-incubation with geranylgeranyl pyrophosphate (GGPP) antagonized the inhibitory impact of simvastatin on colon cancer cell migration triggered by CCL17. Moreover, we observed that simvastatin decreased CCL17-induced activation of RhoA in colon cancer cells. Administration of mevalonate and GGPP reversed the inhibitory effect of simvastatin on CCL17-provoked RhoA activation in colon cancer cells. CONCLUSIONS: Taken together, our findings show for the first time that HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via inhibition of geranylgeranylation and RhoA activation. Thus, statins, such as simvastatin, might be effective tools to antagonize CCL17-dependent migration and metastasis of colon cancer cells.

CCR4 mediates CCL17 (TARC)-induced migration of human colon cancer cells via RhoA/Rho-kinase signaling.

BACKGROUND: Accumulating data suggest a role of chemokines in tumor cell metastasis. CCR4 has been implicated in hematologic malignancies and recently also in solid tumors. Herein, we hypothesized that CCR4 might be expressed and support migration of colon cancer cells. METHODS: We used quantitative RT-PCR and flow cytometry to determine mRNA and surface expression of CCR4 on colon cancer cell lines (HT-29) and (AZ-97). Total RhoA and active RhoA protein levels in CCL17-stimulated colon cancer cells were quantified using ELISA and G-LISA assays. Migration assays were performed to evaluate colon cancer cells chemotaxis. In vitro tumor growth was assessed using proliferation assay. RESULTS: Our results show clear-cut mRNA levels and surface expression of CCR4 on a colon cancer cell line (HT-29) and on tumor cells (AZ-97). CCR4 ligand CCL17 (TARC) was a potent stimulator of colon cancer cell migration. This CCL17-induced colon cancer cell migration was inhibited by pre-incubation of the colon cancer cells with an antibody directed against CCR4 or an antagonist against CCR4. CCL17-induced signaling in colon cancer cells revealed that CCL17 increased mRNA formation of RhoA-C in colon cancer cells. Our results also found that CCL17 increased total RhoA and active RhoA protein levels in colon cancer cells. The Rho-kinase inhibitor Y-27632 abolished CCL17-induced colon cancer cell chemotaxis. In addition, inhibition of isoprenylation by GGTI-2133 markedly reduced colon cancer cell migration triggered by CCL17. CONCLUSIONS: Our novel data indicate for the first time that the CCL17-CCR4 axis might be involved in the spread of colon cancer cells.

CXCL2-CXCR2 axis mediates αV integrin-dependent peritoneal metastasis of colon cancer cells.

Peritoneal metastasis is an insidious aspect of colorectal cancer. The aim of the present study was to define mechanisms regulating colon cancer cell adhesion and spread to peritoneal wounds after abdominal surgery. Mice was laparotomized and injected intraperitoneally with CT-26 colon carcinoma cells and metastatic noduli in the peritoneal cavity was quantified after treatment with a CXCR2 antagonist or integrin-αV-antibody. CT-26 cells expressed cell surface chemokine receptors CXCR2, CXCR3, CXCR4 and CXCR5. Stimulation with the CXCR2 ligand, CXCL2, dose-dependently increased proliferation and migration of CT-26 cells in vitro. The CXCR2 antagonist, SB225002, dose-dependently decreased CXCL2-induced proliferation and migration of colon cancer cells in vitro. Intraperitoneal administration of CT-26 colon cancer cells resulted in wide-spread growth of metastatic nodules at the peritoneal surface of laparotomized animals. Laparotomy increased gene expression of CXCL2 at the incisional line. Pretreatment with CXCR2 antagonist reduced metastatic nodules by 70%. Moreover, stimulation with CXCL2 increased CT-26 cell adhesion to extracellular matrix (ECM) proteins in a CXCR2-dependent manner. CT-26 cells expressed the αV, ß1 and ß3 integrin subunits and immunoneutralization of αV abolished CXCL2-triggered adhesion of CT-26 to vitronectin, fibronectin and fibrinogen. Finally, inhibition of the αV integrin significantly attenuated the number of carcinomatosis nodules by 69% in laparotomized mice. These results were validated by use of the human colon cancer cell line HT-29 in vitro. Our data show that colon cancer cell adhesion and growth on peritoneal wound sites is mediated by a CXCL2-CXCR2 signaling axis and αV integrin-dependent adhesion to ECM proteins.

Retraction: Neutrophil extracellular traps promote peritoneal metastasis of colon cancer cells.

[This retracts the article DOI: 10.18632/oncotarget.26664.].

Neutrophil extracellular traps promote peritoneal metastasis of colon cancer cells.

Cytoreductive surgery is the only curative option for patients with peritoneal carcinomatosis, however, intraperitoneal recurrence rate is high making new ways to prevent cancer recurrence an urgent need. Recent evidence suggests that neutrophils are involved in cancer progression. The purpose of our study was to examine the role of neutrophils in the spread of colon cancer cells in the peritoneal cavity. The number of metastatic noduli in the peritoneal cavity was quantified in mice injected with murine colon cancer cells (CT-26) intraperitoneally after surgical laparotomy and treated with a neutrophil depleting antibody or DNase I. In addition, peritoneal metastases were harvested from patients with peritoneal carcinomatosis. Scanning and transmission electron microscopy showed extensive neutrophil extracellular trap (NET) formation in peritoneal colon cancer metastases in mice and patients. Neutrophil depletion markedly reduced the number of metastases in laparotomised animals. Administration of DNase I decreased the number of metastatic nodules by 88% in laparotomised animals as well as NET-induced chemokine-dependent colon cancer cell migration and adhesion in vitro. Finally, CT-26 cancer cells were found to express the αvß3 integrin and inhibition of αv integrin abolished NET-induced adhesion of colon cancer cells to vitronectin. Taken together, our data show that NETs play an important role in colon cancer cell metastasis in the peritoneal cavity and regulate colon cancer cell migration and adhesion to extracellular matrix proteins. These novel findings suggest that targeting NETs might be an effective strategy to antagonize intrabdominal recurrences of colon cancer after cytoreductive surgery in patients with peritoneal carcinomatosis.

MiR-155-5p positively regulates CCL17-induced colon cancer cell migration by targeting RhoA.

Colorectal cancer is the second most common cause of cancer-related death, which is due to migration of tumor cells to distant sites of metastasis. Accumulating data indicate that mciroRNAs play an important role in several aspects of colon cancer cell biology. Herein, we examined the role of miR-155-5p in colon cancer cell migration induced by the CCL17-CCR4 axis in HT-29 colon cancer cells. We found that miR-155-5p knockdown in serum starved colon cancer cells decreased CCL17-induced cell chemotaxis. Moreover, knocking down miR-155-5p markedly decreased CCL17-provoked activation of RhoA in colon cancer cells. Bioinformatics analysis predicted two putative binding sites in the AU-rich element at the 3'-UTR of RhoA mRNA. MiR-155-5p binding to RhoA mRNA was verified using a target site blocker and functionally validated by RNA immunoprecipitation assays, showing that miR-155-5p-dependent regulation of RhoA mRNA is mediated by AU-rich elements present in the 3'-UTR region. Taken together, these results show that miR-155-5p positively regulates RhoA mRNA levels and translation as well as cell migration in serum starved colon cancer cells and indicate that targeting miR-155-5p might be a useful strategy to antagonize colon cancer metastasis.