Metastatic Potential Of Colorectal Cancer Cells Research Articles

LKB1 loss promotes colorectal cancer cell metastasis through regulating TNIK expression and actin cytoskeleton remodeling.

Colorectal cancer (CRC) is one of the most common malignant tumors. Approximately 5%-6% of CRC cases are associated with hereditary CRC syndromes, including the Peutz-Jeghers syndrome (PJS). Liver kinase B1 (LKB1), also known as STK11, is the major gene responsible for PJS. LKB1 heterozygotic deficiency is involved in intestinal polyps in mice, while the mechanism of LKB1 in CRC remains elusive. In this study, we generated LKB1 knockout (KO) CRC cell lines by using CRISPR-Cas9. LKB1 KO promoted CRC cell motility in vitro and tumor metastases in vivo. LKB1 attenuated expression of TRAF2 and NCK-interacting protein kinase (TNIK) as accessed by RNA-seq and western blots, and similar suppression was also detected in the tumor tissues of azoxymethane/dextran sodium sulfate-induced intestinal-specific LKB1-KO mice. LKB1 repressed TNIK expression through its kinase activity. Moreover, attenuating TNIK by shRNA inhibited cell migration and invasion of CRC cells. LKB1 loss-induced high metastatic potential of CRC cells was depended on TNIK upregulation. Furthermore, TNIK interacted with ARHGAP29 and further affected actin cytoskeleton remodeling. Taken together, LKB1 deficiency promoted CRC cell metastasis via TNIK upregulation and subsequently mediated cytoskeleton remodeling. These results suggest that LKB1-TNIK axis may play a crucial role in CRC progression.

DDX17 induces epithelial-mesenchymal transition and metastasis through the miR-149-3p/CYBRD1 pathway in colorectal cancer

DEAD box helicase 17 (DDX17) has been reported to be involved in the initiation and development of several cancers. However, the functional role and mechanisms of DDX17 in colorectal cancer (CRC) malignant progression and metastasis remain unclear. Here, we reported that DDX17 expression was increased in CRC tissues compared with noncancerous mucosa tissues and further upregulated in CRC liver metastasis compared with patient-paired primary tumors. High levels of DDX17 were significantly correlated with aggressive phenotypes and worse clinical outcomes in CRC patients. Ectopic expression of DDX17 promoted cell migration and invasion in vitro and in vivo, while the opposite results were obtained in DDX17-deficient CRC cells. We identified miR-149-3p as a potential downstream miRNA of DDX17 through RNA sequencing analysis, and miR-149-3p displayed a suppressive effect on the metastatic potential of CRC cells. We demonstrated that CYBRD1 (a ferric reductase that contributes to dietary iron absorption) was a direct target of miR-149-3p and that miR-149-3p was required for DDX17-mediated regulation of CYBRD1 expression. Moreover, DDX17 contributed to the metastasis and epithelial to mesenchymal transition (EMT) of CRC cells via downregulation of miR-149-3p, which resulted in increased CYBRD1 expression. In conclusion, our findings not only highlight the significance of DDX17 in the aggressive development and prognosis of CRC patients, but also reveal a novel mechanism underlying DDX17-mediated CRC cell metastasis and EMT progression through manipulation of the miR-149-3p/CYBRD1 pathway.

Long Non-Coding RNA CASC2 Functions as A Tumor Suppressor in Colorectal Cancer via Modulating The miR-18a-5p/BTG3 Pathway.

Reportedly, long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) is involved in regulating colorectal cancer (CRC) progression. However, the function and detailed downstream mechanism of CASC2 in CRC progression are not fully elucidated. The aim of the study was to investigate the potential function and molecular mechanism of CASC2 in CRC progression. In this experimental study, quantitative real-time polymerase chain reaction (qRT-PCR) was adopted to probe CASC2, microRNA-18a-5p (miR-18a-5p) and B cell translocation gene 3 (BTG3) mRNA expression in CRC tissues and cell lines. After CASC2 was overexpressed in Colo-678 and HCT116 cell lines, methylthiazol tetrazolium (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assays were employed to examine the proliferation of CRC cells. Transwell migration and invasion assays were executed to evaluate the metastatic potential of CRC cells. The targeting relationships among CASC2, miR-18a-5p and BTG3 were validated by dual luciferase reporter gene assay. Western blot assay was applied to examine the regulatory effects of CASC2 and miR-18a-5p on BTG3 protein expression. CASC2 was decreased in CRC tissues and cell lines, and its low expression in CRC tissues was associated with larger tumor size and lymph node metastasis. CASC2 overexpression restrained proliferative, migrative and invasive capabilities of CRC cells. CASC2 could function as a molecular sponge for miR-18a-5p and repress the expression of miR-18a-5p. Furthermore, the inhibitory effects of CASC2 on the malignant phenotypes of CRC cells was counteracted by miR-18a-5p mimics. Additionally, CASC2 could positively regulate BTG3 expression via suppressing miR-18a-5p. CASC2 inhibits CRC development by suppressing miR-18a-5p and raising BTG3 expression.

Transposon-activated POU5F1B promotes colorectal cancer growth and metastasis

The treatment of colorectal cancer (CRC) is an unmet medical need in absence of early diagnosis. Here, upon characterizing cancer-specific transposable element-driven transpochimeric gene transcripts (TcGTs) produced by this tumor in the SYSCOL cohort, we find that expression of the hominid-restricted retrogene POU5F1B through aberrant activation of a primate-specific endogenous retroviral promoter is a strong negative prognostic biomarker. Correlating this observation, we demonstrate that POU5F1B fosters the proliferation and metastatic potential of CRC cells. We further determine that POU5F1B, in spite of its phylogenetic relationship with the POU5F1/OCT4 transcription factor, is a membrane-enriched protein that associates with protein kinases and known targets or interactors as well as with cytoskeleton-related molecules, and induces intracellular signaling events and the release of trans-acting factors involved in cell growth and cell adhesion. As POU5F1B is an apparently non-essential gene only lowly expressed in normal tissues, and as POU5F1B-containing TcGTs are detected in other tumors besides CRC, our data provide interesting leads for the development of cancer therapies.

MiR-215-5p Reduces Liver Metastasis in an Experimental Model of Colorectal Cancer through Regulation of ECM-Receptor Interactions and Focal Adhesion.

Background: Growing evidence suggests that miR-215-5p is a tumor suppressor in colorectal cancer (CRC); however, its role in metastasis remains unclear. This study evaluates the effects of miR-215 overexpression on the metastatic potential of CRC. Methods: CRC cell lines were stably transfected with miR-215-5p and used for in vitro and in vivo functional analyses. Next-generation sequencing and RT-qPCR were performed to study changes on the mRNA level. Results: Overexpression of miR-215-5p significantly reduced the clonogenic potential, migration, and invasiveness of CRC cells in vitro and tumor weight and volume, and liver metastasis in vivo. Transcriptome analysis revealed mRNAs regulated by miR-215-5p and RT-qPCR confirmed results for seven selected genes. Significantly elevated levels of CTNNBIP1 were also observed in patients' primary tumors and liver metastases compared to adjacent tissues, indicating its direct regulation by miR-215-5p. Gene Ontology and KEGG pathway analysis identified cellular processes and pathways associated with miR-215-5p deregulation. Conclusions: MiR-215-5p suppresses the metastatic potential of CRC cells through the regulation of divergent molecular pathways, including extracellular-matrix-receptor interaction and focal adhesion. Although the specific targets of miR-215-5p contributing to the formation of distant metastases must be further elucidated, this miRNA could serve as a promising target for CRC patients' future therapeutic strategies.

ARHGAP25: A negative regulator of colorectal cancer (CRC) metastasis via the Wnt/β-catenin pathway

Herein, we found ARHGAP25 was down-regulated in colon biopsies of patients with colorectal cancer (CRC). Gene set enrichment analysis (GSEA) also showed that ARHGAP25 was negatively correlated with Wnt/β-catenin activation. To study the role of ARHGAP25 in CRC and its possible mechanism, we established lentiviral-mediated ARHGAP25 overexpression in HCT116 and RKO cells along with siRNA-mediated ARHGAP25 knockdown in SW620 cells. The metastatic capacity of the CRC cell lines in vitro was assessed by measuring cell proliferation, migration and invasion. Additionally, expression of matrix metalloproteinases (MMP2, MMP7 and MMP9), EMT-associated factors (E-cadherin, ZEB1, Snail and Twist1) and β-catenin (a core part of Wnt/β-catenin signaling) was determined. XAV939, a Wnt/β-catenin inhibitor, was used for treatment. Our data suggests that ARHGAP25 overexpression significantly inhibits CRC cell growth, suppresses cell migration and invasion, and reduces expression of MMPs, EMT-associated factors and β-catenin. Our results suggest not only that ARHGAP25 has an anti-metastatic role in CRC cells, but also that the inactivation of the Wnt/β-catenin pathway is important in this process. Accordingly, siRNA-ARHGAP25 resulted in the opposite effect, favoring CRC metastasis in vitro, and activating the Wnt/β-catenin pathway in CRC cells. In addition, the siRNA-ARHGAP25 induced changes on cell proliferation, migration, and invasion were significantly reversed with XAV939 treatment. Importantly, the anti-metastatic effects of ARHGAP25 were further substantiated in a CRC lung metastasis xenograft model. We conclude that ARHGAP25 negatively regulates the metastatic potential of CRC cells via the Wnt/β-catenin pathway. Thus, our findings implicate ARHGAP25 as a potential therapeutic target in CRC metastasis.

Alcohol, carcinoembryonic antigen processing and colorectal liver metastases.

It is well established that alcohol consumption is related to the development of alcoholic liver disease. Additionally, it is appreciated that other major health issues are associated with alcohol abuse, including colorectal cancer (CRC) and its metastatic growth to the liver. Although a correlation exists between alcohol use and the development of diseases, the search continues for a better understanding of specific mechanisms. Concerning the role of alcohol in CRC liver metastases, recent research is aimed at characterizing the processing of carcinoembryonic antigen (CEA), a glycoprotein that is associated with and secreted by CRC cells. A positive correlation exists between serum CEA levels, liver metastasis, and alcohol consumption in CRC patients, although the mechanism is not understood. It is known that circulating CEA is processed primarily by the liver, first by nonparenchymal Kupffer cells (KCs) and secondarily, by hepatocytes via the asialoglycoprotein receptor (ASGPR). Since both KCs and hepatocytes are known to be significantly impacted by alcohol, it is hypothesized that alcohol-related effects to these liver cells will lead to altered CEA processing, including impaired asialo-CEA degradation, resulting in changes to the liver microenvironment and the metastatic potential of CRC cells. Also, it is predicted that CEA processing will affect cytokine production in the alcohol-injured liver, resulting in pro-metastatic changes such as enhanced adhesion molecule expression on the hepatic sinusoidal endothelium. This chapter examines the potential role that alcohol-induced liver cell impairments can have in the processing of CEA and associated mechanisms involved in CEA-related colorectal cancer liver metastasis.

CXC receptor-4 mRNA silencing abrogates CXCL12-induced migration of colorectal cancer cells

BackgroundInteractions between CXCR4 and its ligand CXCL12 have been shown to be involved in cancer progression in colorectal cancer (CRC). We performed a comparative CXCL12/CXCR4 expression analysis and assessed the effect of external CXCL12 stimulation on migration of CRC cells without and with CXCR4 inhibition.MethodsExpression of CXCL12/CXCR4 was assessed by quantitative real-time PCR, ELISA and immunohistochemistry in resection specimens of 50 CRC patients as well as in the corresponding normal tissues and in three human CRC cell lines with different metastatic potential (Caco-2, SW480 and HT-29). Migration assays were performed after stimulation with CXCL12 and CXCR4 was inhibited by siRNA and neutralizing antibodies.ResultsIn CRC tissues CXCL12 was significantly down-regulated and CXCR4 was significantly up-regulated compared to the corresponding normal tissues. In cell lines CXCR4 was predominantly expressed in SW480 and less pronounced in HT-29 cells. CXCL12 was only detectable in Caco-2 cells. CXCL12 stimulation had no impact on Caco-2 cells but significantly increased migration of CXCR4 bearing SW480 and HT-29 cells. This effect was significantly abrogated by neutralizing anti-CXCR4 antibody as well as by CXCR4 siRNAs (P < 0.05).ConclusionsCXCR4 expression was up-regulated in CRC and CXCL12 stimulation increased migration in CXCR4 bearing cell lines. Migration was inhibited by both neutralizing CXCR4 antibodies and CXCR4 siRNAs. Thus, the expression and functionality of CXCR4 might be associated with the metastatic potential of CRC cells and CXCL12/CXCR4 interactions might therefore constitute a promising target for specific treatment interventions.

PTCH1, a receptor of Hedgehog signaling pathway, is correlated with metastatic potential of colorectal cancer

Background. Approximately 90% of colorectal cancer (CRC) deaths arise from the metastatic dissemination of primary tumors. It is difficult to predict metastasis of colorectal cancer, especially for patients with the same pathological subtype and differentiation.Aims. To identify biomarkers for predicting CRC metastasis.Patients and methods. We collected 19 primary tumors of CRC with identical pathological subtype, differentiation, and comparable Dukes' stages from patients with matched age and gender but completely different prognosis. Patients were divided into one high-risk and one low-risk group for metastasis. The expression levels of SHH, PTCH1, and sFRP1, which are components of the Hedgehog signaling pathway, were determined by real-time reverse transcription polymerase chain reaction (RT-PCR). To investigate further the correlation between expression level of PTCH1 and metastatic potential of CRC cells, we compared the mRNA and protein levels of the PTCH1 gene in LoVo cells with high metastatic potential and in HT-29, SW480, and SW620 cells with low metastatic potential by RT-PCR and flow cytometry.Results. We found that tumor tissues in the high-risk group for metastasis expressed lower levels of PTCH1 mRNA than did those in the low-risk group. Similarly, mRNA and protein levels of PTCH1 were inversely correlated with the metastatic potential of CRC cell lines. Expression levels of SHH and sFRP1 genes did not differ between the two groups.Conclusion. Our data suggest that PTCH1 might be a potential biomarker that could discriminate CRC with high from that with low metastatic risk.

Comparative analysis of intraperitoneal minimal free cancer cells between colorectal and gastric cancer patients using quantitative RT-PCR: possible reason for rare peritoneal recurrence in colorectal cancer

Peritoneal recurrence has a much lower incidence in colorectal cancer (CRC) patients than gastric cancer (GC) patients. The aim of this study is to clarify the reason for the rare peritoneal recurrence in CRC as compared with GC. The incidence and the abundance of free tumor cells in the peritoneal lavages from 102 CRC and 126 GC patients who underwent curative surgery were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) as genetic markers. Prognostic significance of CEA and CK20 mRNA was also compared between CRC and GC after 2 years of follow-up by Kaplan-Meyer method with overall and peritoneal recurrence-free survival as endpoints. Positivity rate and average values of CEA and CK20 mRNA in peritoneal lavages of CRC patients, which are correlated to the depth of tumor invasion (pT category), were essentially the same as those of GC cases. Overall survival was significantly (marginally) worse in CEA mRNA (CK20 mRNA)-positive CRC patients than negatives like GC. However, peritoneal recurrence-free survival was not different between CEA (CK20) mRNA-positive and -negative CRC patients, in quite contrast to GC cases. Multivariate analysis showed that CEA mRNA was an independent prognostic factor for overall survival in GC patients, but not in CRC patients. These results suggest that the rare peritoneal recurrence in CRC patients is not due to the low incidence or the small number of intraperitoneal free cancer cells, but more likely reflects due to the low-peritoneal metastatic potential of CRC cells.