Role In Colorectal Cancer Metastasis Research Articles

LncRNA HOTTIP mediated DKK1 downregulation confers metastasis and invasion in colorectal cancer cells.

Recent studies highlight long non-coding RNAs (lncRNAs) as key regulators of cancer biology that contribute to carcinogenesis. The lncRNA HOXA transcript at the distal tip (HOTTIP) is involved in the development of several cancers. Previous studies demonstrated that HOTTIP could promote colorectal cancer (CRC) cell proliferation via silencing of p21 expression. However, the potential role of HOTTIP in CRC metastasis has not yet been discussed. Here, we found that HOTTIP level was significantly higher in CRC than in corresponding adjacent normal tissues, and patients with a larger tumor size, advanced pathological stage, or distant metastasis had higher HOTTIP expression. Moreover, silencing HOTTIP expression by siRNA or shRNA could inhibit CRC cell migration and invasion in vitro and in vivo, whereas HOTTIP overexpression promoted cell metastasis, as documented in the SW480 cell lines. Mechanistic analyses indicated that HOTTIP regulates CRC cell metastasis partly through the downregulation of tumor suppressor DKK1 expression. Collectively, our results suggest that tumor expression of lncRNA HOTTIP plays an important role in CRC metastasis. HOTTIP may serve as a candidate biomarker in this disease.

FOXF1 Induces Epithelial-Mesenchymal Transition in Colorectal Cancer Metastasis by Transcriptionally Activating SNAI1

Forkhead Box F1 (FOXF1) has been recently implicated in cancer progression and metastasis of lung cancer and breast cancer. However, the biological functions and underlying mechanisms of FOXF1 in the regulation of the progression of colorectal cancer (CRC) are largely unknown. We showed that FOXF1 was up-regulated in 93 paraffin-embedded archived human CRC tissue, and both high expression and nuclear location of FOXF1 were significantly associated with the aggressive characteristics and poorer survival of CRC patients. The GSEA analysis showed that the higher level of FOXF1 was positively associated with an enrichment of EMT gene signatures, and exogenous overexpression of FOXF1 induced EMT by transcriptionally activating SNAI1. Exogenous overexpression FOXF1 functionally promoted invasion and metastasis features of CRC cells, and inhibition of SNAI1 attenuates the invasive phenotype and metastatic potential of FOXF1-overexpressing CRC cells. Furthermore, the results of the tissue chip showed that the expression of FOXF1 was positively correlated with SNAI1 in CRC tissues chip. These results suggested that FOXF1 plays a critical role in CRC metastasis by inducing EMT via transcriptional activation of SNAI1, highlighting a potential new therapeutic strategy for CRC.

Mutations of key driver genes in colorectal cancer progression and metastasis.

The association between mutations of key driver genes and colorectal cancer (CRC) metastasis has been investigated by many studies. However, the results of these studies have been contradictory. Here, we perform a comprehensive analysis to screen key driver genes from the TCGA database and validate the roles of these mutations in CRC metastasis. Using bioinformatics analysis, we identified six key driver genes, namely APC, KRAS, BRAF, PIK3CA, SMAD4 and p53. Through a systematic search, 120 articles published by November 30, 2017, were included, which all showed roles for these gene mutations in CRC metastasis. A meta-analysis showed that KRAS mutations (combined OR 1.18, 95% CI 1.05-1.33) and p53 mutations (combined OR 1.49, 95% CI 1.23-1.80) were associated with CRC metastasis, including lymphatic and distant metastases. Moreover, CRC patients with a KRAS mutation (combined OR 1.29, 95% CI 1.13-1.47), p53 mutation (combined OR 1.35, 95% CI 1.06-1.72) or SMAD4 mutation (combined OR 2.04, 95% CI 1.41-2.95) were at a higher risk of distant metastasis. Subgroup analysis stratified by ethnic populations indicated that the BRAF mutation was related to CRC metastasis (combined OR 1.42, 95% CI 1.18-1.71) and distant metastasis (combined OR 1.51, 95% CI 1.20-1.91) in an Asian population. No significant association was found between mutations of APC or PIK3CA and CRC metastasis. In conclusion, mutations of KRAS, p53, SMAD4 and BRAF play significant roles in CRC metastasis and may be both potential biomarkers of CRC metastasis as well as therapeutic targets.

Exploration of the mechanism of colorectal cancer metastasis using microarray analysis.

The aim of the present study was to investigate the mechanism of metastasis in colorectal cancer (CRC) using microRNA (miRNA) and mRNA expression profiles. The mRNA and miRNA expression profiles of the GSE2509 and GSE56350 datasets were obtained from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) were identified using the limma software package. The Database for Annotation, Visualization and Integrated Discovery was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs. The predicted target genes associated with the DEMs were identified using the miRWalk database and the enrichment analysis was conducted using the clusterProfiler package. The miRNA-gene molecular interaction network was visualized using the Cytoscape software platform. A total of 544 DEGs and 42 DEMs were identified. DEGs were annotated in 320 GO terms and 11 KEGG pathways. Overall, 366 miRNA-gene pairs were identified and the miRNA-gene network was visualized. Furthermore, the predicted target genes were mainly classified in 12 pathways. The results of the present study suggest that fibronectin type III domain-containing 3B, cysteine rich transmembrane BMP regulator 1 and forkhead box J2 may be potential therapeutic and prognostic targets of metastatic CRC. In addition, pathways in cancer, the Wnt signaling pathway and extracellular matrix-receptor interaction may play a critical role in CRC metastasis.

TRIB1 promotes colorectal cancer cell migration and invasion through activation MMP-2 via FAK/Src and ERK pathways.

Colorectal cancer (CRC) is the third most common cancer in the world and distant metastasis is the leading cause of death among CRC patients. However, the underlying mechanisms of distant metastasis remain largely unknown. Amplification of 8q24 is a common chromosomal abnormality in CRC. In the present study, a putative oncogene at 8q24, TRIB1, was characterized for its role in CRC metastasis and underlying molecular mechanisms. Higher expression of TRIB1 protein was detected in 58/83 (69.9%) of CRC tissues, compared with adjacent non-tumor tissues. Moreover, the expression of TRIB1 was significantly associated with distant metastasis (P=0.043) and advanced staging (P=0.008) in CRC tissues. TRIB1 overexpression was also correlated with poor prognosis in CRC patients as analyzed in PrognoScan database. In addition, elevated expression of TRIB1 promoted CRC cell motility and adhesive ability, while silencing of TRIB1 reduced those effects. Further study revealed that TRIB1-mediated migration and invasion of CRC cells required up-regulation of MMP-2 through the activation of FAK/Src and ERK pathway. Collectively, the results suggest that TRIB1 promotes CRC cell motility by activation MMP-2 via the FAK/Src and ERK pathways. It may provide a potential diagnostic and therapeutic target for CRC.

Gli1 promotes colorectal cancer metastasis in a Foxm1-dependent manner by activating EMT and PI3K-AKT signaling.

Colorectal cancer(CRC) is one of the most commonly diagnosed cancers in human beings and metastasis is the main death reason. Recently, Gli1 has been reported to be a key regulator of various cancer biologies and genes expressions. However, the detailed molecular mechanism of Gli1 in CRC metastasis remains largely unknown. In this study, we aimed to investigate the role of Gli1 in CRC metastasis. We used qRT-PCR, Immunohistochemistry and Western blot to test the expression levels of Gli1, Foxm1 and other target genes in the tissues and cells; Lentivirus stable transfection to change the expression levels of Gli1 and Foxm1; Wound-healing, cell invasion, migration assays and tail vein metastatic assay to test the role of Gli1 in CRC metastasis in vitro and vivo. We demonstrated that Gli1 was significantly overexpressed in colorectal cancer tissues and cells. Foxm1 level had a positive correlation with Gli1. Furthermore, we found that Gli1 promotes colorectal cancer cells metastasis in a Foxm1-dependent manner by activating EMT and PI3K-AKT signaling. Thus, we proved that Gli1 plays important role in CRC metastasis and provided a new visual field on the therapy of CRC metastasis.

DCLK1 is up-regulated and associated with metastasis and prognosis in colorectal cancer.

Metastasis is a primary cause of colorectal cancer (CRC)-related death, and cancer stem cells (CSCs) are thought to be majorly responsible for initiating metastatic behaviors. Doublecortin-like kinase 1 (DCLK1) was recently discovered to be a marker for gastrointestinal CSCs. Here, we aimed to explore whether DCLK1 is associated with CRC metastasis through clinical and in vitro investigations. The expression levels of DCLK1 mRNA and protein in human CRC tissues were analyzed through quantitative RT-PCR and immunohistochemistry staining, respectively. Human CRC cell line SW480 was selected to explore the effect of DCLK1 overexpression on cell migration and invasion. Besides, the associations between DCLK1 and epithelial-mesenchymal transition (EMT) were determined. Compared to normal colorectal tissues, DCLK1 expression was significantly up-regulated in human CRC tissues and correlated well with high lymphatic metastasis and poor prognosis in patients. DCLK1 expression was inversely associated with overall survival in CRC patients. Overexpression of DCLK1 in SW480 cells markedly promoted cell migration and invasion. Furthermore, we validated that DCLK1 could facilitate EMT in cancer cells by up-regulation of the mesenchymal markers Vimentin and ZEB1 and down-regulation of the epithelial marker E-cadherin in SW480 cells. DCLK1 up-regulation may play a contributory role in CRC metastasis and poor prognosis via activation of EMT. DCLK1 may serve as an independent predictor for CRC prognosis.

Dysregulation of MicroRNA-543 expression in colorectal cancer promotes tumor migration and invasion.

MicroRNAs (miRNAs) have been proven to act as key post-transcriptional regulators of gene expression, involved in the genesis and development of multiple tumor types. However, the expression and roles of miR-543 in colorectal cancer (CRC) remain unknown. Here, we found that miR-543 was constitutively up-regulated in CRC tissues and its over-expression promoted tumor cell migration and invasion. Moreover, we identified PTEN as a direct target of miR-543 using subsequent software analysis and a dual-luciferase reporter assay. In conclusion, data presented here demonstrate that miR-543 acts as a tumor promoter and plays a vital role in CRC metastasis. These findings confirm that miR-543 may potentially serve as a novel therapeutic target for treating CRC. © 2016 Wiley Periodicals, Inc.

The long non-coding RNA TUG1 indicates a poor prognosis for colorectal cancer and promotes metastasis by affecting epithelial-mesenchymal transition.

BackgroundLong intergenic non-coding RNAs (lncRNAs) are a class of non-coding RNAs that are involved in gene expression regulation. Taurine up-regulated gene 1 (TUG1) is a cancer progression related lncRNA in some tumor oncogenesis; however, its role in colorectal cancer (CRC) remains unclear. In this study, we determined the expression patterns of TUG1 in CRC patients and explored its effect on CRC cell metastasis using cultured representative CRC cell lines.MethodsThe expression levels of TUG1 in 120 CRC patients and CRC cells were determined using quantitative real-time PCR. HDACs and epithelial-mesenchymal transition (EMT)-related gene expression were determined using western blot. CRC cell metastasis was assessed by colony formation, migration assay and invasion assay.ResultsOur data showed that the levels of TUG1 were upregulated in both CRC cell lines and primary CRC clinical samples. TUG1 upregulation was closely correlated with the survival time of CRC patients. Overexpression of TUG1 in CRC cells increased their colony formation, migration, and invasion invitro and promoted their metastatic potential in vivo, whereas knockdown of TUG1 inhibited the colony formation, migration, and invasion of CRC cells invitro. It is also worth pointing out that TUG1 activated EMT-related gene expression.ConclusionOur data suggest that tumor expression of lncRNA TUG1 plays a critical role in CRC metastasis. TUG1 may have potential roles as a biomarker and/or a therapeutic target in colorectal cancer.

Rock2 stabilizes β-catenin to promote tumor invasion and metastasis in colorectal cancer

Rho-associated coiled-coil-containing protein kinase 2 (Rock2) is an effector for the small GTPase Rho and plays an important role in tumor progression and metastasis. However, the effect of Rock2 in colorectal cancer (CRC) still remains unclear. In this study, we found that Rock2 expression was markedly increased in clinical CRC tissues compared with adjacent non-cancerous tissues. High expression of Rock2 was correlated with tumor metastasis and poor prognosis in CRC. In addition, the knockdown of Rock2 suppressed the invasion and metastasis of CRC cells both in vitro and in vivo. Furthermore, we found that the β-catenin/TCF4 pathway contributed to the effects of Rock2 in CRC cells, and Rock2 stabilized β-catenin by preventing its ubiquitination and degradation. Taken together, this novel pathway for β-catenin control plays a biologically relevant role in CRC metastasis.

The critical roles of tumor-initiating cells and the lymph node stromal microenvironment in human colorectal cancer extranodal metastasis using a unique humanized orthotopic mouse model.

Colorectal cancer (CRC) is the second-most common cause of cancer-related mortality. The most important prognostic factors are lymph node (LN) involvement and extranodal metastasis. Our objective is to investigate the interactions between CD133(+)CXCR4(+) (CXC receptor 4) colorectal cancer tumor-initiating cells (Co-TICs) and the LN stromal microenvironment in human CRC extranodal metastasis. We established a unique humanized orthotopic xenograft model. Luciferase-tagged CRC cell lines and human cancer cells were injected intrarectally into nonobese diabetic/SCID mice. Mesenteric LN stromal cells, stromal cell line HK, or CXCL12 knockdown HK (HK-KD-A3) cells were coinoculated with CRC cells. Tumor growth and metastasis were monitored by bioluminescent imaging and immunohistochemistry. We found that this model mimics the human CRC metastatic pattern with CRC cell lines or patient specimens. Adding LN stromal cells promotes CRC tumor growth and extranodal metastasis (P < 0.001). Knocking down CXCL12 impaired HK cell support of CRC tumor formation and extranodal metastasis. When HK cells were added, sorted CD133(+)CXCR4(+) Co-TICs showed increased tumor formation and extranodal metastasis capacities compared to unseparated and non-Co-TIC populations. In conclusion, both Co-TIC and LN stromal factors play crucial roles in CRC metastasis through the CXCL12/CXCR4 axis. Blocking Co-TIC/LN-stromal interactions may lead to effective therapy to prevent extranodal metastasis.

Identification of critical TF-miRNA-mRNA regulation loops for colorectal cancer metastasis.

To explore the potential cause of colorectal cancer metastasis, gene expression profiles, GSE21510, and miRNA expression profiles, GSE48074, were downloaded from the Gene Expression Omnibus database. Differentially expressed genes in metastatic colorectal and non metastatic colorectal cancer compared with the normal samples were identified via the limma package in R. The differentially expressed miRNAs in colorectal cancer samples with lymph node metastasis compared with those without lymph node metastasis were screened out by the some method. Differentially expressed genes that were upregulated in colorectal cancer samples with distant metastasis in comparison to that in samples without distant metastasis and normal samples were considered to play important roles in colorectal cancer metastasis. Functional enrichment analysis of these genes was conducted using the Database for Annotation, Visualization, and Integrated Discovery v6.7. Biological processes related to cell differentiation and cell proliferation were significantly enriched. TF (transcription factor)-miRNA-mRNA regulation loops were constructed by using the starBase and ChIPBase databases. Finally, six critical regulation loops were screened out. They were composed of two TFs, two miRNAs, and three mRNAs. Some of these TFs, mRNAs, or miRNAs have previously been identified as critical targets in colorectal cancer metastasis. Additionally, several new targets were identified in our study, which may be helpful to improve metastatic colorectal cancer treatment.

FOXC2 promotes colorectal cancer metastasis by directly targeting MET.

Metastasis is the major cause of death in colorectal cancer (CRC). Although multiple genes have been identified to be responsible for the development of CRC, the molecular changes that enable CRC cells to undergo early local invasion and to form distant metastatic colonies still remain largely unknown. Herein, we investigated the role of Forkhead box protein C2 (FOXC2) and explored the underlying mechanisms in invasion and metastasis of CRC. We show that both high FOXC2 expression and nuclear localization of FOXC2 are significantly correlated with advanced TNM (T=primary tumor; N=regional lymph nodes; M=distant metastasis) stages. FOXC2 enhanced the invasive abilities of CRC cells in vitro and promoted local invasion and distant metastasis in an orthotopic mouse metastatic model of CRC. Microarray analysis revealed that overexpression of FOXC2 increased the proto-oncogene MET tyrosine kinase expression and activated the hepatocyte growth factor (HGF)-MET signaling pathway. Furthermore, luciferase reporter assays and chromatin immunoprecipitation assays revealed that FOXC2 directly associated with MET promoter to increase the transcriptional activity of MET. Inhibition of MET attenuates the invasive phenotype and metastatic potential of FOXC2-overexpressing CRC cells, indicating that MET is a major mediator of FOXC2-promoted metastasis. In addition, FOXC2 expression was positively correlated with MET expression in CRC tissue samples. Our findings suggest that FOXC2 has a crucial role in CRC metastasis by regulating HGF-MET signaling via inducing MET expression, highlighting FOXC2 as a potential therapeutic target for preventing or reducing metastasis in CRC.

CCR6 Is a Prognostic Marker for Overall Survival in Patients with Colorectal Cancer, and Its Overexpression Enhances Metastasis In Vivo

The chemokine receptor CCR6 has been recently shown to be associated with colorectal cancer (CRC) progression. However, the direct evidence for whether CCR6 in tumors is a prognostic marker for the survival of patients with CRC and whether it plays a critical role in CRC metastasis in vivo is lacking. Here we show that the levels of CCR6 were upregulated in CRC cell lines and primary CRC clinical samples. CCR6 upregulation was closely correlated with disease stages and the survival time of CRC patients. Knockdown of CCR6 inhibited the migration of CRC cells in vitro. Overexpression of CCR6 in CRC cells increased their proliferation, migration, and colony formation in vitro and promoted their metastatic potential in vivo. CCR6 activated Akt signaling, upregulated metastasis genes and downregulated metastasis suppressor genes. Selective targeting of CCR6 in tumors dramatically inhibited the growth of CRC in mice. Thus, the tumor expression of CCR6 plays a critical role in CRC metastasis, upregulated CCR6 predicts poor survival in CRC patients, and targeting CCR6 expression in tumors may be a potential therapeutic strategy for CRC.

Hypoxia-Inducible MiR-210 Is an Independent Prognostic Factor and Contributes to Metastasis in Colorectal Cancer

MicroRNA-210 (miR-210), the master hypoxamir, plays pleiotropic roles in certain cancers; however, its role in the development of human colorectal cancer remains unclear. Herein, we report that miR-210 is frequently up-regulated in colorectal cancer tissues, with high miR-210 expression significantly correlating with large tumor size, lymph node metastasis, advanced clinical stage and poor prognosis. Functionally, miR-210 overexpression promotes the migration and invasion of colorectal cancer cells. Furthermore, miR-210 can be induced by hypoxia and mediates the hypoxia-induced metastasis of colorectal cancer cells. In addition, vacuole membrane protein 1 (VMP1) is identified as the direct and functional target of miR-210. Thus, miR-210 is a useful biomarker for hypoxic tumor cells and a prognostic factor that plays an essential role in colorectal cancer metastasis.

Microarray analysis reveals that high mobility group A1 is involved in colorectal cancer metastasis

Tumor size indicates the extent of cell proliferation in most cases of colorectal cancer (CRC), although there are some advanced small tumors with metastases. Lymph node metastasis is a significant factor that greatly impacts disease prognosis in CRC cases. The underlying factors that cause lymph node metastasis in CRC cells are not fully understood. We investigated the mechanism that might induce CRC metastasis by focusing on smaller sized (<2 cm) invasive tumors. We carried out gene expression array analysis for CRC cases; group 1 consisted of 6 cases with tumors <2 cm with metastases, and group 2 consisted of 65 cases with tumors >2 cm without metastases. Results were validated using gene expression array data from an additional 77 cases and another bulk case set of 172 cases. Gene ontology and pathway analysis using microarray data revealed that anti-apoptotic activity had a crucial role in CRC metastasis. High mobility group A1 (HMGA1) was identified as a biomarker for poor prognosis and metastasis formation. HMGA1 expression levels were higher in lymph node-positive cases than in lymph node-negative cases, even in subgroup analysis of submucosal invasive cases. The present study strongly supports the clinical significance of HMGA1 expression as a predictive indicator of lymph node metastasis in CRC cases, even in submucosal invasive cases which could be cured by local resection.

Abstract C32: The critical roles of tumor-initiating cell and lymph node stromal microenvironment in human colorectal cancer extranodal metastasis

Abstract In the U.S., colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer-related mortality, with an estimated incidence of 143,000 cases and 51,000 deaths per year. The outcome of CRC patients depends on multiple factors, such as, staging (depth of tumor invasion into colon or rectum wall), lymph node (LN) involvement, local recurrence, and extra-nodal metastasis. Despite optimal oncologic treatment including surgery, chemotherapy and/or radiation therapy, up to 50% of the American Joint Committee on Cancer stage II and III patients will develop extra-nodal metastasis. The most common metastatic sites are the liver or lung. These metastatic cancer cells have acquired a chemoresistant phenotype and developed genetic alterations. Recent studies from our group and others have proposed that metastasis or recurrence is closely associated with the presence of CRC-cancer stem cells or -tumor initiating cells (Co-TIC) expressing CD133 and the chemokine (C-X-C motif) receptor 4 (CXCR4), and their interaction with the LN microenvironment. Our hypothesis is that LN stromal cells prime, support, educate, and stimulate Co-TIC through soluble factors such as the chemokine (C-X-C motif) ligand 12 (CXCL12, ligand to CXCR4), and CD133+CXCR4+ Co-TIC are responsible for drug resistance and extra-nodal metastasis. In this study, we investigated the critical roles of Co-TIC and LN stromal microenvironment in human CRC extra-nodal metastasis using our unique humanized orthortopic xenograft model. The luciferase-tagged colon cancer cell lines and CRC cancer cells isolated from consented patients were injected intra-rectal (IR) to NOD/SCID mice. The LN stromal cell lines HK and CXCL12 knockdown HK (HK-KD-A3) cells were co-inoculated with CRC cells. In addition, Co-TIC were FACS sorted based on positivity of cell surface markers CD326, CD133 and CXCR4. Tumor growth and extra-nodal metastasis were followed by luciferase activity as detected by the IVIS imaging system and quantitative PCR for human DNA in mice livers and lungs. The IR model closely mimicked the expected pattern of CRC spread by showing progressive CRC tumor growth under rectum mucosa with liver and lung metastasis in NOD/SCID mice both injected with CRC cell lines and patient cancer cells. The presence of HK cells or pre-incubation of cancer cells with HK cell conditioned media promoted CRC cell distant metastasis. CXCL12 knockdown HK-KD-A3 cells showed significantly impaired HK cell support of CRC primary tumor formation and more importantly distant organ metastasis. In the presence of HK cells, CD133+CXCR4+ Co-TIC showed increased tumor formation and extra-nodal metastasis in comparison to un-separated and double negative cancer cell populations. Our results indicate both Co-TIC and LN stromal factors play crucial roles in CRC metastasis through CXCL12/CXCR4 axis. Blocking the signaling in Co-TIC/LN stromal interaction may lead to an effective therapy to prevent CRC extra-nodal metastasis, a major cause of cancer morbidity and mortality. In addition, the IR model would serve as a launching pad for evaluating the mechanism of CRC metastasis and testing novel targeted therapies. Citation Format: Tamara Myers, Izi Obokhare, Chelsea Grimes, Xin Zhang, Jennifer Silinsky, Jose Cordova, Heather Green, David Margolin, Li Li. The critical roles of tumor-initiating cell and lymph node stromal microenvironment in human colorectal cancer extranodal metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C32.

Osteopontin-Enhanced Hepatic Metastasis of Colorectal Cancer Cells

Liver metastasis is a major cause of mortality from colorectal cancer (CRC). However, mechanisms underlying this process are largely unknown. Osteopontin (OPN) is a secreted phosphorylated glycoprotein that is involved in tumor migration and metastasis. The role of OPN in cancer is currently unclear. In this study, OPN mRNA was examined in tissues from CRC, adjacent normal mucosa, and liver metastatic lesions using quantitative real-time PCR analysis. The protein expression of OPN and its receptors (integrin αv and CD44 v6) was detected by using an immunohistochemical (IHC) method. The role of OPN in liver metastasis was studied in established colon cancer Colo-205 and SW-480 cell lines transfected with sense- or antisense-OPN eukaryotic expression plasmids by flow cytometry and cell adhesion assay. Florescence redistribution after photobleaching (FRAP) was used to study gap functional intercellular communication (GJIC) among OPN-transfected cells. It was found that OPN was highly expressed in metastatic hepatic lesions from CRC compared to primary CRC tissue and adjacent normal mucosa. The expression of OPN mRNA in tumor tissues was significantly related with the CRC stages. OPN expression was also detected in normal hepatocytes surrounding CRC metastatic lesions. Two known receptors of OPN, integrin αv and CD44v6 proteins, were strongly expressed in hepatocytes from normal liver. CRC cells with forced OPN expression exhibited increased heterotypic adhesion with endothelial cells and weakened intercellular communication. OPN plays a significant role in CRC metastasis to liver through interaction with its receptors in hepatocytes, decreased homotypic adhesion, and enhanced heterotypic adhesion.

Silencing of miR-1-1 and miR-133a-2 cluster expression by DNA hypermethylation in colorectal cancer

MicroRNAs are small non-coding RNA molecules that play important roles in the multistep process of colorectal carcinoma (CRC) development. The present study evaluated the relationship between miR-1-1 and miR-133a-2 expression and DNA methylation, and its putative biological role in CRC. The results indicated that DNA methylation regulated the expression of the miR-1-1 and miR-133a-2 cluster in CRC cell lines. Expression of miR-1 and miR-133a was further evaluated in 64 paired tissue samples (CRC tumor and adjacent normal mucosa) using the stem-loop real-time polymerase chain reaction. The miR-1-133a cluster displayed significantly lower expression in CRC tissue compared to adjacent normal mucosa (P<0.001). The results also indicated frequent hypermethylation of the CpG islands upstream of miR-1-133a (54.6%). Liver metastatic tissues exhibited significantly lower miR-1 (P<0.001) and miR-133a (P<0.001) expression compared to adjacent normal mucosa. Expression of the miR-1-133a cluster inversely correlated with TAGLN2 in the tumor specimens. In conclusion, epigenetic repression of the miR-1-133a cluster may play a critical role in colorectal cancer metastasis by silencing TAGLN2.

GCF2/LRRFIP1 promotes colorectal cancer metastasis and liver invasion through integrin-dependent RhoA activation

The precise relationship between GCF2 expression and carcinogenesis has not yet been established. To clarify the metastatic potential of GCF2 in colorectal cancer, HT-29 cells stably suppressing GCF2 expression were injected into the spleens of severe combined immunodeficient (SCID) mice. GCF2 suppression reduced the number of metastatic foci in the liver and reduced fibronectin-induced cell adhesion, migration, and invasion. Downstream from the integrin signaling pathways, GCF2 regulates RhoA interaction with the RGS domain of Leukemia associated RhoGEF (LARG). Altogether, our results suggest that GCF2 plays an important role in colorectal cancer metastasis by regulating RhoA-induced cell adhesion, migration, and invasion.