Roles And Molecular Mechanism Of Integrin αvβ6in SDF-1/CXCR4-induced Liver Metastasis Of Colorectal Cancer

Background:Colon cancer is one of the most common gastrointestinal cancer with increasing incidence and mortality rates, which seriously endangers people’s health has become a major problem in the world. Invasion and metastasis is a major factor affecting the prognosis of colon cancer. Colon cancer displays a predilection for metastasis to liver, lung and lymph node, especially liver, which is the most common leading cause of colon cancer. common liver metastases, patients with colon cancer is the leading cause of death. The traditional view shows that, when passing liver in portal venous, colon cancer cells migrate slowly and are easy to stay and grow in this site. However, recent studies show that this phenomenon can not be explained simply by the anatomical structures and vascular and lymphatic drainage, and there should be other factors involved. But the factors and the specific mechanism is unclear.Chemokines are a class of small molecular weight of cytokines, and their receptors are seven transmembrane G protein-coupled receptors. Interaction between such receptors and their respective chemokines can induce directional migration of cells towards a gradient of chemokines (namely chemotaxis), which plays an important role in tissue development, lymphatic homing, immune and inflammation. With the deepening of research, more and more evidence shows that chemokines and their receptors are closely associated with invasion and metastasis of tumor cells. Considering many similarities shared by cancer cell metastasis and leukocyte trafficking, the binding of chemokines to their receptors can induce directional migration of tumor cells towards the organs with abundant chemokines. CXCR4is a commonly expressed chemokine receptor in different tumors. Previous studies reported that CXCR4together with its unique ligand stromal cell-derived factor-1(SDF-1or CXCL12) played a significant role in the progression and metastasis of colorectal cancer, breast cancer, prostate cancer, lung cancer, ovarian cancer and laryngeal carcinoma. SDF-1is the only ligand of CXCR4, and SDF-1/CXCR4axis plays an important role directional migration of tumor cells. Intriguingly, SDF-1is highly expressed in liver, lung, bone marrow and lymph nodes, which just represent the most common sites for CRC metastasis, which suggests the important role of SDF-1/CXCR4axis in organ-specific metastasis of colon cancer. However, the underlying mechanism is still unclear.The process of cancer metastasis consists of a series of sequential and interrelated steps, including invasion, detachment from the primary sites, intravasation, survival in the circulation and translocation to microvessels of target organs, extravasation and clonization. The definition of directional migration consists of two principles. First, organ-specific metastasis requires a complex signalling mechanism to guide CRC cells to the liver, which can be liken to a "navigation system". As a major chemokine receptor on CRC cells, CXCR4has been proved to induce chemotaxis of cells towards a gradient of SDF-1(5,17). Furthermore, high levels of SDF-1was detected in liver, lung and bone marrow, representing the most common target organs for CRC metastasis (17). This suggests that SDF-1/CXCR4axis could function as a "navigation system" for CRC cells, guiding cancer cell migration from their original site to final destination. Indeed, SDF-1/CXCR4axis has been reported to play a significant role in liver metastasis of CRC (9-11). Second, the ability of migration or motility is also required. A keystone of cell migration is the formation and regulation of adhesion contacts, especially integrin contacts, which are dynamically disassembled at the rear and reassembled at the leading edge (29,42). However, previous studies demonstrated that CXCR4itself didn’t directly contribute to adhesion of prostrate and renal carcinoma cells to extracellular matrix, but served as signal transmitters to modulate integrin α5and β3-mediated adhesion (18,19). Therefore, it is possible that SDF-1/CXCR4axis may promote CRC cell migration through cross talk with other receptors, especially the adhesion receptors.Integrins belong to superfamily of cell surface adhesion molecule, and are heterodimeric trans-membrane receptors comprising alpha and beta subunits. Integrins serve as anchoring molecules by mediating the adhesion of the cellular cytoskeleton to the extracellular matrix (ECM). They also serve as bidirectional signaling molecules by mediating outside-in and inside-out signaling. The integrin αvβ6is unique in that it is expressed exclusively in epithelial cells, and β6partners only with av forming a single heterodimer. We previously demonstrated that:integrin αvβ6was closely related with pathologic type, differentiation and stage of colon cancer and gastric cancer, and could function as an independent indicator for prognosis; there was a direct binding between integrin αvβ6and ERK, and colon cancer cells could induce secretion of uPA, MMP-9and MMP-2and promote degradation of ECM via the direct binding of αvβ6-ERK, which rendered colon cancer cells more invasive. Recently we have verified that avP6can promote colon cancer cell migration in a mechanism of integrin trafficking.Thus, SDF-1/CXCR4axis-induced invasion and metastasis of colon cancer is the result of interaction of multiple factors, and current research on the mechanism of this process was restricted to SDF-1/CXCR4axis itself and its downstream signaling pathways. However, the research on the interaction of SDF-1/CXCR4axis with other molecules, especially adhesion molecules. Indeed, there are none studies about the crosstalk between SDF-1/CXCR4axis and integrin αvβ6.This study aims to investigate the expression profiles of integrin avP6and CXCR4in colon cancer specimens and colon cancer cell lines and the relationship between their expression and clinicopathological features of colon cancer, and also to analyse the correlation between expression of integrin αvβ6and CXCR4. In addition, we further investigate the potential role and underlying mechanism of αvβ6in SDF-1/CXCR4-mediated CRC cell migration. This study will present a novel mechanism for SDF-1/CXCR4-induced organ-specific metastasis and shed light on effective therapeutic approaches for liver metastasis of CRC.Part ⅠExpression of αvβ6and CXCR4in colon cancer tissue and colon cancer cell lines with different metastatic potentialObjectiveTo explore the effect of the expression of integrin αvβ6and CXCR4on livr metastasis of colon cancer, and to investigate the correlation between the the expression of integrin αvβ6and CXCR4MethodsColon cancer patients who underwent surgery by the same surgical team at the Department of General Surgery of Qilu hospital (Shandong University, china) were selected consecutively from2005to2008; Patients were regularly followed up and clinical data was collected, including gender, age, tumor size, pathological type, degree of differentiation, TNM stage, surgical methods as well as organic and lymphonodus metastasis; Then immunohistochemistry was carried out to detect the expression of αvβ6and CXCR4and the final immunohistochemistry score was also calculated; Expression of CXCR4and αvβ6in colon cancer tissues and associated liver metastatic tissues and their impact on the prognosis were statistically analysed; Furthermore, Mann-Whitney test and Spearman rank correlation analysis were both used to investigate the correlation between the expression of CXCR4and αvβ6. Highly metastatic colon cancer cell lines HT-29and WiDr and non-metastatic cell line Caco-2were used in this study; Reverse transcription PCR (RT-PCR) was used to detect the mRNA expression of β6subunit and CXCR4in these cell lines; Western blot was used to detect the protein expression of β6subunit and CXCR4; Flow cytometry was used to detect the surface expression of integrin avP6and CXCR4.Results159colon cancer patients meeting the requirements were finally included in the study. And159cases of primary colon cancer tissue and21cases of liver metastatic tissue were collected. Immunohistochemistry analysis of159primary CRC samples demonstrated that CXCR4was positive in107tumors (67.3%) and αvβ6was positive in73tumors (45.9%), but there were no or equivocal staining of CXCR4and αvβ6in normal colon tissues. In the21liver metastatic samples,19cases (90.5%) showed positive CXCR4staining and16(76.2%) cases showed positive αvβ6staining, but none to equivocal staining of CXCR4and αvβ6were observed in normal liver specimens; Expression of CXCR4was significantly associated with lymph node metastasis (p<0.05), TNM stage (p<0.05) and liver metastasis (p<0.01); Expression of αvβ6was significantly associated with lymph node metastasis (p<0.05), TNM stage (p<0.05), lymphovascular invasion (p<0.05) and liver metastasis (p<0.01); Notably, high αvβ6and high CXCR4expressions, when compared with other combinations, were significantly associated with lymph node metastasis (p<0.05) and liver metastasis (p<0.01); Mann-Whitney test and Spearman rank correlation analysis demonstrated that expression of αvβ6was positively correlated with expression of CXCR4. The relative mRNA and protein levels of06subunit (in affiliation only with the αv subunit) and CXCR4were high in metastatic cell lines HT-29and WiDr, while non-metastatic cell line Caco-2exhibited very low levels of both CXCR4and β6. With respect to cell surface presentation, flow cytometry showed that high membrane levels of both integrin αvβ6and CXCR4were detected in HT-29and WiDr cells, while lower levels of CXCR4and hardly any αvβ6in Caco-2ConclusionExpression of integrin αvβ6and CXCR4was significantly associated with liver metastasis of colon cancer. Moreover, expression of αvβ6is significantly correlated with that of CXCR4. They were highly expressed in highly metastatic colon cancer cells and lowly expressed in non-metastatic colon cancer cells, and patients with both high expression of αvβ6and CXCR4are more prone to develop liver metastasis.SignificanceThis section confirms the close relationship between the expression of integrin αvβ6and CXCR4and the liver metastasis of colon cancer, which lays a foundation for further study of the mechanisms of αvβ6and CXCR4in liver metastasis of colon cancer. Part II Effect of SDF-1/CXCR4on the expression of integrin αvβ6and other integrinsObjectiveTo investigate the effect of SDF-1/CXCR4on the expression of αvβ6and other integrins and to explore the potential mechanism.MethodsReal-time quatitative PCR and western blot were used to detect the effect of SDF-1/CXCR4on the expression of av, β1, β3, β5and β6subunits in HT-29and WiDr cells; Flow cytometry was used to detect the effect of SDF-1/CXCR4on the surface expression of αvβ6; Western blot was used to detect the effect of SDF-1/CXCR4on ERK1/2phosphorylation, and the effect of ERK specific inhibitor U0126on the expression of αvβ6and the directional migration of HT-29cells was also observed; Western blot and confocal microscopy were used to detect the effect of SDF-1/CXCR4on the activation of Ets-1, and the effect of Ets-1specific siRNA on the expression of αvβ6and the directional migration of HT-29cells was also observed. ResultsReal-time quatitative PCR and western blot demonstrate that SDF-1/CXCR4could induce significant upregulation of β6subunit and slight upregulation of β1subunit, but has no effect on the expression of αv,β3and (35; Flow cytometry demonstrates that SDF-1/CXCR4could significantly increase the surface expression of integrin αvβ6; SDF-1/CXCR4could promote phosphorylation of ERK1/2, and ERK specific inhibitor U0126could significantly inhibit SDF-1/CXCR4-induced upregulation of αvβ6and directional migration of HT-29cells.ConclusionSDF-1/CXCR4could upregulate expresion of αvβ6via ERK-Ets-1pathway.Significance This section demonstrates the effect of SDF-1/CXCR4on the expression of αvβ6and the underlying mechanism, which further confirms the important role of αvβ6in SDF-1/CXCR4-induced directional migration. Part Ⅲ Role of αvβ6in SDF-1/CXCR4axis-induced directional migration of colon cancer cellsObjectiveTo verify the role of integrin αvβ6in SDF-1/CXCR4axis-induced directional migration of colon cancer cells.MethodsSerum-free medium containing200ng/ml SDF-la was used as a chemoattractant in the lower chamber, and the ability of directional migration on fibronectin were detected in HT-29and WiDr cells; Cells were pretreated with αvβ6-functional blocking antibody10D5,αvβ6specific siRNA and CXCR4specific inhibitor AMD3100, and effect of these interventions on directional migration of colon cancer cells was detected. Non-metastatic Caco-2cells were transfected with human CXCR4-expressed plasmid (pcDNA-CXCR4); Green fluorescence protein-expressed plasmid (pcDNA-GFP) was used as a control to demonstrate transfection efficiency, and western blot and flow cytometry were used to detect overexpression effect of CXCR4; pcDNA-CXCR4transfected Caco-2cells were incubated with SDF-la (200ng/ml), and real-time PCR, western blot and flow cytometry were used to detect effect of SDF-1on avP6expression; Migration assay were used to detect effect of CXCR4overexpression on directional migration ability of Caco-2cells, and to observe role of avP6in this process by interfering αvβ6.ResultsCompared with control group (without chemoattractant SDF-1in the lower chamber), SDF-1could significantly increase the directional migration on fibronectin of HT-29and WiDr cells;10D5, αvβ6specific siRNA and AMD3100could significantly inhibit SDF-1-induced increase in directional migration. Transfection efficiency was more than80%in our experiment, and pcDNA-CXCR4could significantly overexpress CXCR4in Caco-2cells; Furthermore pcDNA-CXCR4transfected Caco-2cells were incubated with SDF-la and, as a result, SDF-la induced a significant increase in mRNA and protein levels of β6integrin which was evidently attenuated by CXCR4inhibitor AMD3100; SDF-la dramatically increased the migration of pcDNA-CXCR4but not mock vector transfected Caco-2cells, Moreover inhibition of αvβ6by neutralizing antibody10D5significantly alleviated SDF-1a-induced migration of pcDNA-CXCR4transfected Caco-2cells.ConclusionSDF-1could significantly increase the ability of directional migration of HT-29and WiDr cells, and avP6is essential for this process. Caco-2cells were rendered migrating after overexpression of CXCR4, treatment with SDF-la and subsequent upregulation of integrin αvβ6.Significance This sectin verifies the important role of integrin αvβ6in SDF-1/CXCR4-induced directional migration and further confirms that SDF-1/CXCR4promotes directional metastasis of colon cancer cells through upregulation of αvβ6.