The Role And Correlated Molecular Mechanisms Of β-Catenin In Invasion And Metastasis Of Colorectal Cancer

PARTⅠNuclearβ-catenin Overexpression at Invasive Front in Colorectal Cancer Is Associated with Synchronous Liver MetastasisBackground and objectiveColorectal cancer (CRC) is one of the most common cancers and the second leading cause of cancer-related death in the western world. The incidence of CRC in the world is increasing with a rate of 2%. Death usually results from uncontrolled metastatic disease. The most common organ metastasis in colorectal cancer patients is liver. Although the 5 year survival rate for patients with local CRC approaches 90%, the 5 year survival rate for metastasis patients decreases to 19%. Over 20%-40% of patients with colorectal carcinoma either have liver metastases at presentation and 50% will subsequently develop them after CRC radical operation. The metastasis rate of hepatic metastasis in clinical colorectal cancer patients is 5.5%-33.3%. The metastasis rate of hepatic metastasis in dead CRC is 45%-71%. Therefore, early detection of liver metastasis in CRC is especially important to improve patient survival rate. Several studies have investigated the risk factors influencing liver metastasis. Histopathologically, the presence of venous invasion, a deeper level of invasion, less differentiated carcinoma cells, and the presence of lymph node metastasis have been reported to be risk factors. The biological factors related to liver metastasis have been identified as Epidermal growth factor (EGF), Actin-related protein2 (Arp2), Transforming growth factor-a (TGF-a), Cluster of differentiation 44 (CD44) and Cluster of differentiation 10 (CD 10).Invasive tumor front (ITF) is defined as the 3-6 layers tumour cells at the front edge or the scattered tumour groups between tumour and host tissue or organ. It has been suggested that CRC might show cellular dedifferentiation in the invasive front area, with loss of an epithelial phenotype and a gain of a mesenchymal phenotype, which facilitates invasive and metastatic growth of originally differentiated cancer cells. The malignant progression is epithelial-mesenchymal transition. One of the key oncogenic proteins that might drive epithelial-mesenchymal transition in colorectal carcinogenesis isβ-catenin.β-catenin is a key component of the adherens junctions, necessary for homophilic cell-cell adhesions.β-catenin in cytoplasm and membrane binds with the intracellular domain of E-cadherin, which is a cell-to-cell adhesion molecule, and to play a significant role in maintaining the normal tissue architecture. In addition to the membrane associated pool,β-catenin plays a role in cell-signaling and gene transcription. In the absence of a Wnt signal, a multiprotein complex, composed of the adenomatous polyposis coli, Axin, and glycogen synthase kinase 3βpromotesβ-catenin phosphorylation and ubiquitin-mediated degradation. In the presence of a Wnt signal,β-catenin is no longer degraded and translocates to the nucleus where, interacting with lymphoid enhancing factor/T-cell factor, it promotes transcription of several target genes involved in cell proliferation. The reduced cell adhesion further sustains Wnt signaling, stimulating cell migration and metastasis formation. Previous studies indicated that the change of nuclearβ-catenin expression correlated with liver metastasis in CRC. A recent study demonstrated thatβ-catenin mRNA was markedly elevated in the CRC cells of the invasion front. Elevation of mRNA was paralleled by increased nuclear and cytoplasmicβ-catenin protein concentrations. These data suggested that increased nuclearβ-catenin may play an important role in CRC invasion and metastasis.Due to previous pathological proof, it is tempting to speculate that nuclearβ-catenin expression in CRC may represent decisive aspects in CRC invasion and metastasis. However, the correlation between nuclearβ-catenin expression in CRC and synchronous liver metastasis was not characterized up to now. Therefore, we examined nuclearβ-catenin expression in CRC and liver metastatic lesions. Meanwhile, we investigated the association between nuclearβ-catenin expression and clinicopathological data to determine its importance as a predictor of liver metastasis.MethodsClinicopathological data from 486 patients (144 cases with liver metastasis and 342 cases without liver metastasis) were reviewed.β-catenin expression in colorectal cancer and liver metastatic lesions were examined by immunohistochemistry. The association of nuclearβ-catenin overexpression in primary tumours and liver metastatic lesions was evaluated. Meanwhile, the relationship between nuclearβ-catenin overexpression and clinicopathological characteristics was analyzed. Finally, univariate analysis and logistic multivariate regression analysis were adopted to discriminate risk factors of liver metastasis.Results1.β-catenin expression was observed in all the CRC specimens. Nuclearβ-catenin overexpression at invasive front in primary tumour was observed in 103 patients with liver metastasis and 100 patients without liver metastasis (71.5% vs.29.3%; P< 0.001).2. Nuclearβ-catenin overexpression in metastatic lesions was noted in 132 patients (91.6%). Spearman rank correlation analysis demonstrated that nuclearβ-catenin expression in primary tumours had a positive correlation with that in metastatic lesions (r= 0.499, P< 0.001).3.χ2 test demonstrated thatβ-catenin overexpression at invasive front in CRC is different significantly for age (P< 0.001), type of tumour (P= 0.01), tumour invasion depth (P< 0.001), lymph node metastasis (P= 0.021), liver metastasis (P < 0.001) and TNM stage (P< 0.001).4.χ2 test demonstrated that liver metastasis is accociated with age (P= 0.01), tumour size (P< 0.001), tumour cell differentiation (P< 0.001), tumour invasion depth (P < 0.001), lymph node metastasis (P< 0.001), and nuclearβ-catenin overexpression (P< 0.001).5. Logistic multivariate regression analysis indicated that the independent risk factors for synchronous liver metastasis are age (P= 0.003), tumour size (P< 0.001), tumour invasion depth (P= 0.001), tumour cell differentiation (P= 0.031), and nuclearβ-catenin overexpression (P< 0.001).Conclusions1. Overexpression of nuclearβ-catenin in CRC with liver metastasis is more evident.2. In patients with CRC liver metastasis nuclearβ-catenin expression at invasive front in CRC had a positive correlation with that in metastatic lesions.3. Nuclearβ-catenin overexpression at invasive front in CRC is different for associated with age, type of tumour, tumour invasion depth, lymph node metastasis, liver metastasis and TNM stage.4. Overexpression of nuclearβ-catenin at the invasive front is accociated with age, tumour size, tumour cell differentiation, tumour invasion depth, lymph node metastasis, and nuclearβ-catenin overexpression.5. Age, tumour size, tumour invasion depth, tumour cell differentiation, and nuclearβ-catenin overexpression are independent risk factors for synchronous liver metastasis. Overexpression of nuclearβ-catenin at invasive front in colorectal cancer is strongly associated with liver metastasis and may be a promising predictor of liver metastasis.SignificanceThis study elucidated the relationship between nuclearβ-catenin overexpression in CRC and synchronous liver metastasis based on clinical and pathological data. The clinical significance of the current study is that we provided a promising predictor for liver metastasis in CRC. PARTⅡInfluence of CXCR4/SDF-1 aixs on E-cadherin/β-catenin complex in HT29 colon cancer cells and its mechanismsBackground and objectiveThe main reason leading to death for colorectal cancer patients is liver metastasis. A great deal of studies have been done to investigate the risk factors influencing liver metastasis. The metastatic process of CRC consists of a series of individual steps, all of which are required to establish metastatic tumours. Although a number of molecules have been proved to be implicated in the metastasis of cancer cells, the precise mechanisms determining the directional migration and invasion of CRC cells into specific organs remain to be established. New evidence indicated that chemokines play a major role in this process of organ-selective metastasis.Chemokines are signaling molecules that function in myriad cell trafficking events. It is well known that chemokines and their receptors have been known to be involved in the "homing" of hematopoietic cells to specific organs as a physiological mechanism. Homing is also functional in neoplastic cells. Recent studies have indicated that tumor cells express patterns of chemokine receptors and that corresponding ligands are specifically expressed in organs to which these cancers commonly metastasize. For example, Muller et al demonstrated that breast cancer cells express the chemokine receptor CXCR4, whereas the specific ligand CXCL12, also known as stromal cell-derived factor 1(SDF-1), has been found at elevated levels in lymph nodes, lung, liver and bone marrow that represent the first metastatic sites of breast cancer. Others also speculated on the involvement of CXCR4 in the metastatic tumor growth of different types of malignancies, including CRCSDF-1 is expressed in stromal cells, including fibroblasts and endothelial cells, and interacts specifically with the seven-transmembrane, G protein-coupled receptor CXCR4. Recent studies showed that chemotaxis effect of CXCR4/SDF-1 axis is related with lymph node and liver metastasis of CRC. Although there is evidence that the CXCR4/SDF-1 signaling pathway is involved in the metastatic process of CRC, the precise molecular mechanism underlying SDF-1-induced chemotaxis effect has not been completely elucidated. E-cadherin, a transmembrane glycoprotein located at the adheren junction, mediates calcium-dependent cell-cell adhesion. C terminus of E-cadherin is linked toα-catenin and actin cytoskeleton through the association withβ-catenin. Strong cell-cell interactions result in a tight cell cluster as a community, and constrain cells from moving away. It has been shown that dysregulation of E-cadherin/β-catenin complex expression is responsible for the invasion and metastasis of CRC, indicating that CXCR4/SDF-1 axis is correlated with E-cadherin/β-catenin complex expression in invasion and metastasis of CRC.CXCR4/SDF-1 axis-mediated chemotaxis effect is crucial in organ-selective metastasis and E-cadherin/β-catenin plays an important role in colorectal tumorigenesis. Relatively little is known about the role E-cadherin/β-catenin plays in CXCR4/SDF-1 axis-mediated tumour cells' invasion and metastasis. Based on our previous study, this study was to observe whether CXCR4/SDF-1 axis can alter E-cadherin/β-catenin expression in HT29 colon cancer cell line. In addition, the E-cadherin/β-catenin mRNA expression level was measured and the phosphorylation of PI3K/AKT andβ-catenin was examined to provide insights into the mechanism underlying the change in E-cadherin/β-catenin expression.Methods1. MTT method was used to evaluate the influence of SDF-1 on HT29 cells proliferation.2. Transwell invasion assay was adopted to tested the effect of SDF-1 on HT29 cells' invasive ability.3. The effect of SDF-1 on E-cadherin/β-catenin expression was examined by immunocytochemistry.4. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was adopted to examine E-cadherin/β-catenin mRNA level.5. The phosphorylation of PI3K/AKT andβ-catenin induced by SDF-1 was tested by western blotting. Results1. The viability of HT29 cells in any experiment groups was not different from that in control group 48 h after incubated with SDF-1. The cells grew much faster with a proliferation rate of 129% and 135%, respectively (P=0.034和P=0.026) 72 h after incubated with SDF-1 at the concentrations of 20 ng/mL and 40ng/mL. AMD3100 plus SDF-1 inhibited the cell growth. AMD3100 alone had no effect on cell proliferation.2. After 24 h incubation, SDF-1 markedly enhanced the migration ability of HT29 cells compared with the control group at 10 ng/mL (149±13.3 vs 92.3±12.4; P = 0.041),20 ng/mL (161±13.5 vs 92.3±12.4; P= 0.023) and 40 ng/mL (187.5±14 vs 92.3±12.4; P< 0.001). AMD3100 inhibited the migration of HT29 cells. AMD3100 alone had no effect on cell migration.3. A significant decrease in E-cadherin expression in HT29 cells was found in 20 and 40 ng/mL groups after 48 h (P= 0.044 and P< 0.001, respectively). HT29 cells treated with AMD3100 prior to administration of SDF-1 did not show a decrease in E-cadherin expression compared with the SDF-1 groups. AMD3100 alone had no effect on E-cadherin expression. RT-PCR analysis demonstrated that E-cadherin mRNA decreased after 48 h incubation with SDF-1 at 20 and 40 ng/mL (P= 0.033 and P< 0.001, respectively). The administration of AMD3100 inhibited the decrease in E-cadherin mRNA. AMD3100 alone had no influence on E-cadherin mRNA.4. After incubation with SDF-1 (20 and 40 ng/mL) for 24 h,β-catenin expression in HT29 cells decreased. But the difference had no statistical significance.β-catenin was downregulated significantly after 48 h at 20 and 40 ng/mL (P= 0.031 and P < 0.001, respectively). Even thoughβ-catenin decreased slightly in cells treated with AMD3100, the difference was not significant. AMD3100 alone had no influence on P-catenin expression. RT-PCR analysis demonstrated thatβ-catenin mRNA decreased after 24 h incubation with SDF-1. But the difference had no statistical significance.β-catenin mRNA downregulated significantly after 48 h at 20 and 40 ng/mL (P= 0.037 and P< 0.001, respectively). The down-regulation ofβ-catenin mRNA was inhibited by AMD3100. AMD3100 alone did not influence expression of P-catenin mRNA.5. SDF-1 induced an increase in phosphorylation of PI3K/AKT and P-catenin after 1 minute. Activation ofβ-catenin was evident at 5 min and the phosphorylation of PI3K/AKT was significant at 15 min. The peak of P-catenin phosphorylation was observed at 30 min. Then, evident phosphorylation of P-catenin lasted for 2 d. The peak phosphorylation of PI3K/AKT occurred at 15-60 min, lasted up to 2 h, and then slowly declined. HT29 cells were treatment with SDF-1 at 0,5,10, 20,40 and 100 ng/mL for 30 min which showed that the administration of SDF-1 for 30 min induced a dose-dependent increase in PI3K/AKT and P-catenin phosphorylation. The phosphorylation of PI3K/AKT andβ-catenin was observed at a minimal concentration of 5 ng/mL SDF-1 and reached maximal level at 100 ng/mL.6. AMD3100 inhibited the phosphorylation of PI3K/AKT andβ-catenin. Further study demonstrated that administration of LY294002 prior to SDF-1 also prevented the phosphorylation of PI3K/AKT andβ-catenin.Conclusions1.SDF-1 enhanced viability of HT29 colon cancer cells;2. SDF-1 promoted migration of HT29 colon cancer cells;3. CXCR4/SDF-1 axis is involved in down-regulation of E-cadherin/β-catenin in HT29 colon cancer cells;4. Phosphorylation of PI3K/AKT andβ-catenin and down-regulation of E-cadherin/β-catenin mRNA are involed in SDF-1-induced down-regulation of E-cadherin/β-catenin expression;5. Inhibition of PI3K/AKT prevented phosphorylation of SDF-1-inducedβ-catenin and P-catenin may be a down-stream effector of PI3K/AKT.Significance To the best of our knowledge, this study is the first that analyzed the relationshipbetween CXCR4/SDF-1 axis and E-cadherin/p-catenin complex. We provided a possible explanation for CXCR4/SDF-1 axis-induced invasion and metastasis in HT29 colon cancer cells. Moreover, we also provide insights into the mechanisms responsible for the change in E-cadherin/p-catenin.