The Role And Possible Mechanisms Of CXCR4 In The Metastasis Of Lung Cancer

As a common malignancy, lung cancer is a leading cause of morbidity and mortality, and a gradual increase in its incidence has been recognized. Approximately 70% of lung cancer patients die from metastatic disease, even after complete resection of the primary tumor. In this situation, inhibition of metastasis of lung cancer is thought to be one of the important therapeutic strategies. To further defining the molecular mechanism involved in the process of metastasis will provide new theoretic base and experimental evidence for prevention and therapy of lung cancer.It has been reported that tumor cell migration and metastasis share many similarities to leukocyte trafficking, which is critically regulated by chemokines and their receptors. Many cancers also express an extensive network of chemokines and chemokine receptors. So the mechanisms utilized in homing of leukocytes directed by chemokines may also be applicable in the process of tumor cells metastasis. Tumor metastasis is a very complex and dynamic cell biological process, during which a number of interactions among tumor cells themselves and between tumor cells and the host tissues take place. Although extensive studies had been made in the mechanism of tumor metastasis, the regulation of which was partly understood. It suggested that different types of tumors were characterized by disregulated production of chemokines and abnormal chemokine receptor signaling and expression, which not only shared some similarities, but also had their own pattern. So determining the expression pattern of chemokine network in specific tumor would aid to elucidate the molecular mechanism of tumor metastasis. We supposed that there was a disregulated and abnormal chemokines/chemokine receptors network in lung cancer, which was associated with its invasion and metastasis. But it is unclear which molecule or cell involved this unbalanced network and how this unbalanced network affected the invasion and metastasis of lung cancer.Therefore, in this study we first analyzed the expression pattern of chemokines or chemokine receptors in lung cancer cells with high or low metastasis potential by RT-PCR and immunohistochemistry. Based on the results, we further studied the association of disregulated chemokine or chemokine receptor with the metastasis of lung cancer. Then by down- or up-regulation expression of chemokine or chemokine receptor in lung cancer cells, we studied the role and mechanism of the chemokine or chemokine receptor in metastasis of lung cancer in vitro and in vivo. Our study would provide new theoretic base and experimental evidence for therapy of metastatic lung cancer.Part I the correlation of CXCR4 expression with the metastasis potential of human lung cancer1. Expression profile of chemokine(s)/chemokine receptor(s) in human lung cancer cell lines with high or low metastasis potentialThe chemokine superfamily is divided into four subfamilies (CXC, CC, C, CX3C) based on the arrangement of their amino terminal cysteine residues. Chemokine receptors belong to the seven transmembrane spanning family of G-protein-coupled receptors, whose nomenclature is simply based on the chemokine group (CC, CXC, C or CX3C) to which its ligand (s) belong. In order to screen the chemokine receptors that may play an important role in lung carcinoma, the expression of 4 angiogenesis-related chemokines and 15 chemokine receptors was detected by RT-PCR in human lung cancer cell lines with high or low metastasis potential, which were confirmed by invasion assay, semi-quantitative RT-PCR and zymograpy. The results showed only angiogenesis chemokine GRO- a and chemokine receptor CXCR4 was detectable in human lung cancer cell lines with high or low metastasis potential, there was differential expression of CXCR4 between human lung cancer cell lines with high and low metastasis potential by semi-quantitative RT-PCR.2. Differential expression of CXCR4 between human lung cancer cell lines with high or low metastasis potentialIn order to define the expression characteristics of CXCR4 in human lung cancer cell lines with high or low metastasis potential, real-time RT-PCR and FACS further detected the expression of CXCR4. It is obvious that expression of CXCR4mRNA in 95D cells was higher than that in 95C cells (PO.05) . Flow cytometry analysis alsorevealed different levels of CXCR4 expression on 95D and 95C cells, and confirmed stronger cell surface expression of CXCR4 on 95D cells than that on 95C cells.3. Functional expression of CXCR4 on lung cancer cellsSubsequently, the functional expression of CXCR4 on 95D and 95C cells was demonstrated with calcium mobilization induced by its ligand-SDF-1 a . Cells were loaded with the calcium-sensitive dye fiuo-3/AM, and the changes of the intracellular calcium concentration in response to l~200ng/ml SDF-1 a were assessed. Stimulation of 95D and 95C cells with rhSDF-1 a elicited a rapid and robust increase in intracellular calcium in a time-dependent manner, but the mean fluorescence in 95D cells was stronger than that in 95C cells. We also assessed whether the expression of CXCR4 was functional in promoting cell migration. Chemotaxis assays were performed with 95D, 95C cells in the presence or absence of SDF-1 a . We observed that SDF-1 a appeared more efficacious in inducing chemotaxis of 95D cells than 95C cells. These results indicate that CXCR4 is functional on lung cancer cells.4. CXCR4 expression in human primary lung cancerIn order to elucidate the relationship between of CXCR4 expression and lung cancer, expression characteristics of CXCR4 was further confirmed in 36 specimens of human lung cancer by immunohistochemistry with antibodies specific for CXCR4. As the results shown, 34 of 36 cases showed positive staining of CXCR4 protein, although the staining intensity differed among different specimens. Staining of CXCR4 protein was identified in the cell membrane and/or cytoplasm of cancer cells and was not significantly detected in the normal lung cells taken from non-cancerous regions adjacent to lung cancer tissue. In all examined cases (n=36), higher expression of CXCR4 (+++) was apt to be detected in the tumor specimen with lymph node metastasis (n=13). The result from statistic analysis showed that there were no significant differences between CXCR4 protein expression status and histological types or differentiation degree. However, the expression of CXCR4 was significantly associated with lymph node metastasis (P<0.05). Also the differential expression of CXCR4mRNA was confirmed by semi-quantitative RT-PCR in lung cancer with or without lymph node metastasis.Part II the role of CXCR4 in the metastasis of human lung cancer 1. Construction of the recombinant plasmids pcDNA-X4 and pcDNA-ASX4In order to study the role of CXCR4 in lung cancer metastasis, we used the over-expression or antisense technique to specifically regulate CXCR4 gene expression in 95C or 95D cells, and then investigated the effect of down or up-regulation of CXCR4 expression on the metastatic behavior of tumor cells. CXCR4 full-length encoding gene, amplified by PCR and modified by restriction endonucleases EcoRI and Xbal, was subcloned into a eukaryotic expression vector pcDNA3 to construct a recombinant plasmid pcDNA-X4 that was identified by PCR or restriction endonucleases digestion. DNA sequencing showed that the opening read frame of CXCR4 encoding gene was correct and consistent with the known CXCR4 sequence. The amplified DNAs were recovered and directly cloned into pUCm-T vector in clockwise direction according to the manufacturer's instructions. Then the cDNA inserts digested from pUCm-T-X4 were subcloned into pcDNA3 in anti-clockwise direction. The identification of recombinant plasmid pcDNA-ASX4 was confirmed by PCR, double digestion with EcoRV and BamHI or DNA sequencing.2. Establishment and indentiflcation of the stable transfectantspcDNA-X4 or pcDNA-ASX4 plasmid DNA mixed with Lipofectamine2000 was transfected into 95C or 95D cells with pcDNA3 plasmid as negative control respectively. After 24~48h, 800ug/ml G418 sulfate were added. The RPMI1640/G418 was replaced ever 3 days until individual resistant clone was isolated and established in culture as individual lines. The stable transfectants were assessed CXCR4 expression on the surface by flow cytometry. CXCR4 expression on the stable transfectant 95C-X4 cells was significantly up-regulated, compared with 95C and the mock transfectant with pcDNA3 (named 95C-pC) as analyzed by FACS, while CXCR4 expression on the stable transfectant 95D-ASX4 cells was significantly down-regulated, compared with 95D and the mock transfectant with pcDNA3 (named 95D-pC)3. Effect of CXCR4 expression on invasion ability of lung cancer cells95D and 95C cells were of different degrees of metastatic potential, which confirmed by our previous study. In order to elucidate the relationship of CXCR4 expression with metastatic potential of lung cancer cells, the effect of CXCR4 differential expression on invasion ability of lung cancer cells was detected by invasion assay in vitro. As the results shown, the invasion ability of 95C-X4 enhanced 1.78 folds compared with 95C, 95C-pC cells, while the invasion ability of 95D-ASX4decreased 1.54 folds compared with 95D, 95D-pC cells. These results indicated that regulation of CXCR4 expression affected the invasion ability of lung cancer cells in vitro.4. Effect of CXCR4 expression on chemotaxis and chemoinvasion of lung cancer cells induced by SDF-1 aTo further elucidate the biological functions of SDF-1 a /CXCR4 system in lung cancer, in vitro chemotaxis and chemoinvasion assay were performed to determine the effect of CXCR4 on lung cancer cell migration toward SDF-1 a . As the results showed that up-regulation of CXCR4 expression enhanced the cell migration of 95C-X4 cells to SDF-1 a when compared with either 95C or 95C-pC cells(P<0.05), while down-regulation of CXCR4 significantly impaired the migratory response of 95D-ASX4 cells to SDF-1 a when compared with either 95D or 95D-pC ceIls(/><0.05). SDF-1 a activation also resulted in an increased artificial ECM invasion of 95D, 95D-pC or 95C-X4 cells and weakly increased invasion of 95D-ASX4, 95C, or 95C- pC cells, compared to unstimulated controls. All these migratory response could be specifically interfered by a neutralizing anti-CXCR4 antibody, which suggested that these migratory responses were dependent on CXCR4/ SDF-1 a interaction.5. Effect of CXCR4 expression on metastasis in vivo1) Establishment of spontaneous metastasis models of lung cancer in nude miceHaving established that lung cancer cells express functionally active CXCR4, we evaluated the contribution of CXCR4/ SDF-1 a interactions to metastasis in vivo by using spontaneous metastasis models of lung cancer in nude mice. The model of spontaneous metastasis models of lung cancer in nude mice was successfully copied as previously described. 10 weeks later, none of mice with 95C cells exhibited metastasis in lung or lymph node, while 3 of 4 mice injected with 95D cells exhibited metastasis in lung or lymph node, this suggested that 95Cand 95D cells exhibited different metastatic potential in vivo.2) Migratory response of lung cancer cells to organ-derived protein of nude mice95D but not 95C cells had the ability to spontaneously metastasize to lung, lymph node of nude mice when tumor cells were injected into subcutaneous of nude mice. In order to elucidate whether CXCR4 participated this process, we compared the homology between human and mice SDF-1 by running the BLAST program from NCBFs web site, they were significantly homologous as 99%. Then the tissue extractfrom lung, lymph node of nude mice was prepared as previous and tested for chemotatic activity. The results showed that protein extracts of lung and lymph node of nude mice induced chemotatic responses in cancer cells, and the migration of 95C-X4 cells to nude mice organ-derived protein was significantly enhanced, while the migration of 95D-ASX4 cells in response to nude mice organ-derived protein was significantly reduced after blocking the SDF-1 a /CXCR4 interactions by down-regulation of CXCR4 (P<0.05, PO.05). All these migratory response could be specifically interfered by a neutralizing anti-CXCR4 antibody. 3) The association of CXCR4 expression with the metastasis potential of lung cancer cell in vivoTo evaluated the contribution of CXCR4/ SDF-1 a interactions to metastasis in vivo, 95C, 95C-pC, 95C-X4 95D, 95D-pC or 95D-ASX4 cells were impanted into subcutaneous of nude mice, respectively. 10 weeks later, The tumor formation rate of 95C, 95C-pC or 95C-X4 cells in nude mice was 15%, 50%or 80%. There was no difference in tumorgenesis but significant difference in the average weight of tumor between 95C-X4 cells and 95C or 95C-pC group (PO.05). The solid tumors were formed in all 95D, 95D-pC or 95D-ASX4 group (one died of infection), there was no difference in tumorgenesis but significant difference in the average weight of tumor between 95D-ASX4 and 95D or 95D-pC group (P<0.05). Moreover, they also exhibited different degree of metastatic potential in vivo, the incidences of spontaneous metastasis were significantly lower for the 95C and 95C-pC cells than for 95C-X4 cells (incidences: 0 of 4, 0 of 4, 2 of 5). Conversely, the incidences of spontaneous metastasis were significantly greater for the 95D and 95D-pC cells than for 95D-ASX4 cells (incidences: 3 of 4, 3of 4, 2 of 5).Part III the possible mechanisms of CXCR4 in the metastasis ofhuman lung cancer1. Expression of CXCR4 mRNA in 95D cells in different culturesExpression of CXCR4 mRNA was determined in 95D cells in different cultures ( 2%NBS, 10%NBS, 10%NBS+PMA), in which cells were resting, modestly activated and fully activated. The results by semi-quantitive RT-PCR showed that expression of CXCR4 mRNA was highest under the condition of 10%NBS+PMA. CXCR4 mRNA increased in 95D activated by PMA within the first 30 minutes afterstimulation, reaching the summit at 1 hours, then decreased after 12~24 hours. These results suggested that expression of CXCR4 was associated with activation of lung cancer cells.2. Regulation of CXCR4mRNA expression by hypoxiaThe outgrowth of tumor often faced up with lackness of blood and oxygen, then they would be degenerative, necrotic. So CXCR4 expression was analyzed between lung cancer tissue away from necrosis and near necrosis, as the results from immunohistochemistry showed, high levels of CXCR4 expression was found in lung cancer tissue near necrosis rather than region away from necrosis. These data suggested that hypoxia might regulate CXCR4 expression. To further determine the regulation mechanism of CXCR4 expression, CXCR4 transcripts were analyzed by semi-quantitive RT-PCR when 95D cells were in the presence of hypoxia. The results showed that CXCR4 mRNA was strongly induced by hypoxia, it started up-regulation within 2h, and maintained until 6h. These results suggested that hypoxia might be a regulatory factor of CXCR4expression.3. Regulation of MMP-2 activity by CXCR4/SDF-1Tumor cell invasion through the ECM and entry into the circulation involve cellular detachment, motility through ECM and basement membrane degradation which are processes depending on a variety of enzymes that include metalloproteinases (MMPs). The metastatic potential of tumor cells was positively associated with MMP activity. There was significantly difference in MMP-2 activity between 95D and 95C cells according to our previous results, so the effect of CXCR4 expression regulation on MMP-2 activity was measured by zymography. The results showed that there were lower MMP-2 activity in 95C, 95C-pC and 95C-X4 cells in the absence of SDF-1 a , compared with 95D group. There were no difference among each group in the absence of SDF-1 a , while in the presence of SDF-1 a , Higher MMP-2 activity were detected in 95C-X4 cells than in 95 C and 95C-pC cells, and in 95D and 95D-pC cells than that in 95D-ASX4 cells. These results indicated that regulation of CXCR4 expression was associated with MMP-2 activity in lung cancer cells.4. Regulation of adhesion to vascular endothelial cells by CXCR4/SDF-1In order to extravasate to a target tissue, tumor cells must firmly arrest on vascular endothelium and then transmigrate. We further assessed if CXCR4/SDF-1 a interaction could induce the adhesion of CXCR4+ cancer cells to microvascularendothelial cells. Calcein AM labeled cancer cells were incubated with ECV-304 in the absence or presence exogenously added SDF-1 a . 95C, 95C-pC cells showed low appreciable adhesion to ECV-304 cells without exogenous SDF-1 a , which were 11.2 ±1.38%, 10.57 + 0.89 %#3 16.75 + 1.25%, compared with 95D group, which the adhesion to ECV-304 was 22.35+1.31%, 22.22+1.52 %orl 7.77 ±0.90%. In the presence of exogenously added SDF-1 a , 95C-X4 cells showed the more enhanced adhesion (43.21 + 1.89%) to ECV-304 cells, compared with 95C or 95C-pC cells (27.32+1.07%, 28.32±0.92%). The exogenously added SDF-1 a was also induce higher adhesion of CXCR4-expressing 95D or 95D-pC cells to ECV-304 cells than 95D-ASX4 cells, which were 50.92 ±2.63%, 50.27 ±0.98% or 22.41 + 1.21%, respectively. As further demonstrated by the inhibition studies, this adhesion is partially dependent on CXCR4/ SDF-1 a interaction, because this increase adhesion was reduced by a specific neutralizing CXCR4 mAbs.5. Regulation of GRO- a expression by CXCR4/SDF-1Our previous results showed that lung cancer cells also expressed an angiogenesis chemokine GRO- a , which are often detected at elevated levels in patient serum and tumor microenvironment during malignant progression. Overexpression of GRO- a has been shown to promote tumor growth and metastasis. Chemokines/chemokine receptors exerted their effects in a network pattern. Here we demonstrated a striking increase in the expression of GRO- a by following exposure of 95D cells to SDF-1 a within lh, maintaining until 6h. CXCR4 antibody could neutralized this effect, so stimulation of GRO- a production may be a possible pathway involved in the regulation of tumor metastasis, which was dependent on SDF-1 a ligation of CXCR4.6. Actin polymerization in human lung cancer cells induced by SDF-1 aCytoskeletal reorganization is a prerequisite for motility and migration. To evaluate the ability of SDF-1 to induce changes in the actin cytoskeleton of lung cancer cells, we examined for changes in the content and reorganization of filamentous actin (F-actin) in response to SDF-1 a with FACS and confocal microcopy. As the results shown, SDF-1 a induced up-regulation of F-actin content. Cells were serum starved overnight and displayed a disorganized cytoskeleton, SDF-1 a caused a cytoskeletal rearrangement with stress fiber formation due to CXCR4 activation.7. Phosphorylation of ERK1/2 in human lung cancer cells induced SDF-1 a...