Involvement Of CCR7 In Metastasis Of Non-Small Cell Lung Cancer

IntroductionLung cancer is one of the most common malignant tumor.In China,Lung cancer has become the first cause of death in malignant tumors.Tumor invasion and metastasis were the critical steps in determining the aggressive phenotype of human cancers. Mortality of tumor patients principally resulted from cancer cells spread to distant organs.Clinical and pathological observations had shown that lymph node involvement was one of the earliest features of metastatic cancers.Therefore,study on the factors acting important role in the invasion and metastasis course will be contributed to providing new ideas to make out clinical therapy strategy.CC Chemokine Receptor 7(CCR7),is expressed on all naive T cells,some memory T cells,B cells and mature dendrite cells and plays a central role in lymphocyte trafficking and homing to lymph nodes.The current evidence shows that CCR7 is highly expressed in human breast cancer cells and metastases,and ligand binding to CCR7 induces chemotactic and invasive responses,including actin polymerization and pseudopodia formation.Moreover,several other tumor types express CCR7,and CCR7 plays a critical role in cancer metastasis of many organs as well as regional lymph nodes,such as breast cancer,cervical cancer,gastric cancer,esophageal squamous cell carcinoma,squamous cell carcinoma of the head and neck,melanoma cells and so on.The role of CCR7 in tumor cells invasion and metastasis becomes important day by day.However,the factors those regulate CCR7 expression and the mechanisms that how CCR7 promote tumor metastasis remains unclear.In the present study,we investigated whether hypoxia could regulate the expression of CCR7 in non-small cell lung cancer,and analysis the mechanism of CCR7 up-regulation and increased invasion ability of lung cancer cell.In addition,the effects of CCR7 on the lymphangiogenic properties of lung cancer cells were studied by investigating its effects on VEGF-D expression.This provide basis for CCR7 promote tumor cells lymph node metastasis.Materials and MethodsSample preparation:A total of 94 cases of NSCLC tissues were obtained from the January 2001 to the April 2006 at the First Affiliated Hospital of China Medical University.The tumor tissues in this study were from NSCLC patients proved by pathological diagnosis.None of the 94 patients had received radiation therapy or chemotherapy before surgery.Tissue samples to be used for immunohistochemistry were fixed in formalin and paraffin embedded.The TNM staging system of the UICC (1997) was used to classify the specimens.The study has been approved by hospital ethical committee.Immunohistochemical staining:4μm thick sections were prepared from the paraffin-embedded tissues.Immunostaining was performed by the streptavidin-peroxidase(S-P) method(MaiXin,China).The primary antibodies were anti-CCR7 at 1:200 dilution(Santa Cruz,USA),anti-HIF-1αat 1:100 dilution(BD,San Diego,CA) and anti-HIF-2αat 1:150 dilution(Novus Biological Science),respectively. The peroxidase reaction was developed with DAB staining(Maixin,China).For negative control,the primary antibodies were replaced by non-immune serum.All the immunoreactions were separately evaluated by two senior pathologists.The intensity of CCR7 immunostaining(1=weak,2=intense) and the percentage of positive tumor cells(0%=negative,1-50%=1,51-75%=2,≥76%=3) were assessed in at least 5 high power fields(×400 magnification).The tumors were finally determined as low expression:score＜3;or high expression:score≥3.The staining of HIF-1αand HIF-2αwere scored as previous study.¹⁷ If the difference between the numbers counted by two pathologists was more than 10%,the lumens were recounted and a consensus between each observer was reached.Cell culture and hypoxic treatment:The lung cell lines A549,BE1,SK,QG,H661 and H460 were used in this study.Cells were maintained in DMEM or RPMI1640 (both from Gibco,Invitrogen Corporation) containing 10%FCS together with 100 U/ml penicillin,100 U/ml streptomycin.All cells were cultured at 37℃in an air/5% CO2 incubator.Hypoxic exposure was carried out under 1%oxygen,5%carbon dioxide,and 94%nitrogen(Galaxy R CO2 incubator,RS Biotech) at 37℃.Cells were incubated in normoxic or hypoxic condition for 4h,12h and 24h respectively.RT-PCR:Total RNA was isolated from cells in the logarithmic growth phase using TRIZOL(Invitrogen).The PCR primers were as follows:CCR7 F:5'-GAG GCT ATT GTC CCC TAA ACC -3',R:5'-TGG AGG ACA GTG AAG AAA ACG -3'.The length of PCR product was 304bp.HIF-1αF:5'-TAA GAA ACC ACC TAT GAC CTG C-3',R:5'-GTC GTG CTG AAT AAT ACC ACT C -3'.The length of PCR product was 411bp.HIF-2αF:5'-GAA AAC GAG TCC GAA GCC -3',R:5'-CCC AAA ACC AGA GCC ATT -3'.The length of PCR product was 242bp.β-actin F:5'-AAA TCG TGC GTG ACA TTA A-3',R:5'-CTC GTC ATA CTC CTG CTT G-3'.The length was: 513bp.Thirty cycles of 40 seconds denaturation at 95℃,40 seconds annealing at 53℃(HIF-1α,CCR7,β-actin) or 60℃(HIF-2α) and 40 seconds extension at 72℃were then performed.siRNA Treatment of Cells:RNA interference and siRNA preparation was performed as described previously for HIF-1α¹⁸ and HIF-2α.¹⁹ Effects of siRNA for HIF-1α(siHIF-1α) or HIF-2α(siHIF-2α) were compared with those of a random siRNA sequence.¹⁸ Cells were plated onto 10-cm² cell culture dishes and grown to 30-50%confluence before transfection.The duplexes were diluted to give a final concentration of 20nM.Transfections were performed with Lipofectamine 2000 (Invitrogen) and by following the manufacturer's instructions.Twenty-four hours later, the transfection procedure was repeated,and cells were subjected to hypoxia treatment the day after the second transfection.Stable Transfection:The plasmid pLNCX2-CCR7 was kindly provided by Sam T.Hwang(National Institutes of Health,USA).BE1 cells were transfected pLNCX2-CCR7 in the presence of Lipofectamine 2000(Invitrogen) following the manufacturer's instructions and placed under G418(500μg/ml) selection for 4 weeks. Surviving colonies were isolated and named CCR7-BE1 cells.BE1 cells were transfected empty vector as control and named pLNCX2-BE1 cells.Western blotting:Cells were serum starved for 24h and then treated as described in "Results".After treatment cells were extracted with lysis buffer(150mMNaCl,1% NP-40,0.1%SDS,2μg/ml aprotinin,1mM PMSF) for 30 min at 4℃.The supernatants were centrifuged at 12000×g for 30 min at 4℃.The supernatant containing total protein was harvested.Aliquots containing 100μg of proteins were separated on a 7.5%SDS-PAGE and transferred to PVDF membranes at 100 V for 2 h at low temperature.The membranes were blocked with 5%skimmed milk.Subsequently, mouse anti-human HIF-1α(BD,San Diego,CA) monclonal antibody was used at 1:500 dilution;mouse anti-human HIF-2α(NB100-132ss) monclonal antibody(Novus Biological Science) was used at 1:1000 dilution;goat anti-human CCR7 polyclonal antibody(Santa Cruz,USA) was used at 1:200 dilution;and anti-β-actin antibody (Santa Cruz,USA) was used at 1:400 dilution.After washing with TBST for three times,immunodetection was performed.The EC3 Imaging System(UVP Inc.) was used to catch up the specific bands,and the optical density to interest proteins andβ-actin of the same sample was calculated as relative content and expressed graphically.Cell invasion and migration assays:Cell invasion was performed using Matrigel -coated 24-well Transwell chamber(Costar).BE1 cells and CCR7-BE1 cells were preincubated in the presence/absent of PD9059 for 4h or anti-CCR7 antibody for 24h. Cells(2.5×10⁴) were seeded in the upper chamber of 8μm pore size insert in the 24-well plate and cultured in hypoxic or normoxic environment for another 24h.Cells were allowed to migrate forward to RPMI1640 containing different concentration (50,100,200,300ng/ml) of recombinant human CCL21(PEPROTECH ASIA) in the bottom chamber.The non-invasive cells on the upper membrane surface were removed with a cotton tip,and the invasive cells attached to the lower membrane surface were fixed with 4%paraformaldehyde and stained with hematoxylin.The number of invasive cells was counted under inverted microscope(200×).Data presented are representative of three individual wells.Cell migration was assayed using a similar approach without Matrigel coating,and cells in migration chamber were incubated under normoxic(N) or hypoxic(H) conditions for 8h.Statistical analysis:The SPSS 13.0 software was applied to complete data processing.X²-test was applied to analyze the correlation between the expressions of HIF-1α,HIF-2α,CCR7 and clinicopathological characteristics for the results of immunohistochemistry;Correlation between HIF-1α,HIF-2αand CCR7 expressions was analysed by Spearman's test;Independent-samples t-test was used to evaluate the differences of optical density(OD) values or numbers of migrated cells between groups with various treatments.All data were represented as mean±SD of three independent experiments.Results were considered statistically significant when the p-value was less than 0.05.Results1.1 Correlation of CCR7 with HIF-1αand HIF-2αexpression:The predominant staining pattern of CCR7 was cytoplasmic and /or plasma membranes in 94 cases NSCLC tissues.The CCR7 protein was detected in part of the endothelial cells.The high expression rates of CCR7 in the 94 cases were 75.53%(71/94).Both HIF-1αand HIF-2αprotein showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells,HIF-1αand HIF-2αprotein were detected in part of the tumoural vessels and turnout-infiltrating inflammatory cells,while normal lung components showed negative staining.The high expression rates of HIF-1αand HIF-2αin the 94 cases were 54.25% (51/94) and 70.21%(66/94),respectively.Statistical analysis showed that CCR7 expression was correlated positively with the expression of HIF-1α(r=0.315,P=0.002) and HIF-2α(r=0.247,P=0.017).CCR7 expression was significantly associated with lymph node metastasis(P=0.001) and clinical pathological stage(P=0.027).Both HIF-1αand HIF-2αexpression were significantly associated with lymph node metastasis(P=0.001),(P=0.015) and clinical pathological stage(P=0.011),(P=0.015).All the six cell lines we tested were positive for CCR7,HIF-1αand HIF-2αmRNA,and cell lines expressing higher levels of CCR7 also displayed greater abundance of HIF-1αand HIF-2α.The above data clearly demonstrate that the correlation of CCR7 with HIF-1αand HIF-2αexpression was commonly observed in the different human lung cancer cells.1.2 Hypoxia promotes CCR7 expression by HIF-1αand HIF-2αin BE1 cells:We exposed BE1 cells under hypoxia conditions(physical hypoxia,chemical hypoxia),the expression of CCR7 mRNA and protein were strongly elevated,and meanwhile the expression of HIF-1αand HIF-2αwas up-regulated.To investigate the role of HIF-1αand HIF-2αin the hypoxic induction of CCR7,we established siRNA for the inhibition of HIF-1αand HIF-2αin BE1 cells.Then,cells were incubated under hypoxia conditions.The results showed that CCR7 mRNA and protein levels were reduced more significantly in siHIF-1αthan siHIF-2αtansfected cells(P＜0.05).1.3 HIF-1αand HIF-2αregulate CCL21-induced chemotaxis,migration and invasion of BE1 cells:Migration and invasion activity of BE1 cells were analysized by Transwell assay.Chemotaxis toward different concentrations of CCL21 was greatly changes in a dose-dependent manner.Invaded cells achieved the most at the concentration of 200ng/ml CCL21 in BE1 cells.The number of migration and invasive cells was increased under hypoxia compared with normoxia,decreased after down-regulation of HIF-1αor HIF-2α,which was more significant in siHIF-1αtransfected cells.1.4 ERK_1/2 activation is required for CCR7-mediated migration and invasion of BE1 cells:we transfected BE1 cells with vectors carrying CCR7(CCR7-BElcells) or empty vector,and observed the transfection efficiency by RT-PCR and western blotting methods.The results showed that CCR7-BE1 cells expressed more CCR7 both at mRNA and protein levels;simultaneously the expression of p-ERK_1/2 protein was increased.Transwell migration assays showed that the number of cells in control group and empty vector group migrate through the microporous membrane were(20.57±4.2) and(21.7±3.2).The number of the cells(51.29±4.89,P=0.000) that migrate through microporous membrane increased significantly in CCR7-BE1 group.In the invasion assay,the number of cells in control group and empty vector group that invade through Matrigel were(25.43±3.99) and(26±3.96).In CCR7-BE1 cells group the number of the cells that invade through Matrigel increased significantly(74.57±4.69,P=0.000). P-ERK_1/2 protein expression and the number of invasion cells decreased significantly in the presence of either PD98059 or anti-CCR7 antibody.The results showed that CCR7-mediated migration and invasion of BE1 cells dependent on ERK_1/2 activation.2.1 CCR7 Up-Regulated VEGF-D in NSCLC Cell Lines:treatment of A549 cells with human CCL21 increase the expression' of VEGF-D mRNA and protein.Treatment of A549 cells with CCR7 antibody significantly attenuated the endogenous VEGF-D protein level.Of the three cell lines tested,we found that CCL21 increased the expression of VEGF-D,and blocking CCR7 decreased the expression of VEGF-D,indicating that CCL21 could regulate VEGF-D via CCR7.2.2 Involvement of ERK_1/2 and Akt in CCR7-Mediated VEGF-D Up-Regulation: we detected p-ERK_1/2,ERK_1/2,p-Akt,Akt protein expressions in A549 cells.Western blotting showed that incubation of A549 cells with CCL21 increased the expressions of p-ERK_1/2 and p-Akt,while exhibiting no effect on the total protein levels of ERK_1/2 or Akt.Furthermore,Specific blocking CCR7 expression by anti-CCR7 Ab inhibited the expression of p-ERK_1/2 and p- Akt.Furthermore,the VEGF-D mRNA and protein expression in A549 cells were decreased significantly after incubation with PD98059 or LY294002,respectively.However,in A549 cells,LY294002 did not inhibit ERK_1/2 phosphorylation,and PD98059 did not affect phosphorylation of Akt under basal condition.2.3 CCR7 Expression Correlate with VEGF-D Level,Lymphatic Vessel Density, Clinical Stages,Lymph Node Metastasis,and Patient Survival in NSCLC: Immunohistochemical analysis of 90 NSCLC specimens revealed that the VEGF-D expression was significantly associated with the expression of CCR7(r =0.455,P＜0.001).Quantification of the D2-40-positive vessels in the tumors indicated that tumor with high expression of CCR7 had a higher LVD than in low CCR7 expressing tumors (28.09±10.57 for CCR7 high expression tumors,n=63,and 20.58±7.6 for CCR7 low expression tumors,n=27,P=0.001).Tumors with VEGF-D high expression had a higher LVD than in low VEGF-D expressing tumors 30.14±9.87 for VEGF-D high expression tumors,n=54,and 19.38±7.22 for VEGF-D low expression tumors,n=36, P＜0.001).Patients with CCR7 high expression were more likely than low expression patients to have advanced disease(P=0.003) and lymph node metastasis(P=0.004). VEGF-D high expression patients were more likely than low expression patients to have advanced disease(P=0.005) and lymph node metastasis(P=0.001).For CCR7 low expression patients had a statistically significantly longer survival than CCR7 high expression patients(P=0.036).The median survival time for patients with tumors with high VEGF-D was 16±3.8 months;95%confidence interval 8.6-23.4 months, significantly shorter than that for patients with tumors with low VEGF-D expression (P=0.009).Conclusions1,CCR7 expression was correlated positively with the expressions of HIF-1αand HIF-2αin 94 cases NSCLC tissues.Each of them was significantly associated with lymph node metastasis and clinical pathological stage.2,Hypoxia- HIF-1α,2α-CCR7-ERK1/2 pathway is likely to regulate the invasion and migration of lung cancer cells under hypoxic tissue microenvironments.HIF-1α, HIF-2αand CCR7 activation might play a role in promoting a more malignant phenotype of NSCLC and represents novel therapeutic strategies.3,Acause-effect link between CCR7 over-expression and tumor lymphangiogenesis through VEGF-D activity was demonstrated in lung cancer.CCR7 could regulate VEGF-D expression through ERK1/2 and Akt signaling pathway.Our findings open newer therapeutic modalities against lymphangiogenesis and lymphatic borne。...