Tumor-Associated Macrophages Induce Colorectal Cancer Cells Invasion And Metastasis Via Galectin-1-MYO1B Pathway

BACKGROUNDColorectal cancer(CRC)is one of the commonest malignant tumor in our country.The incidence rate of CRC in China is increasing faster and the patients ' age is younger than ever during the past decades.Liver metastasis,peritoneal lymph nodes metastasis and lung metastasis are the main cause of the death of CRC patients.Tumor metastasis is decided by the change of intrinsic characteristics of cells.With the deepening research of tumor,scientists found that tumor microenvironment play an important role in the tumor formation and evolution.In many malignant tumors(for example breast cancer,gastric adenocarcinoma,ovarian cancer),tumor associated macrophages(TAMs)are the most common inflammatory cells infiltrated in tumor mesenchyma.They could facilitate tumor growth,invasion,metastasis and angiogenesis.Our previous study had confirmed that there were primarily one of macrophage subpopulation with M2 phenotype along the front edge of colorectal cancer invasion,that TAMs with M2 phenotype could promote the invasion and metastasis of colorectal cancer(CRC).In addition,we found Galectin-1 was mainly secreted by TAMs,and it was colocation with TAMs around CRC cells.As a small molecular cell effector,Galectin-1 can be transported from cytoplasm to nucleus;it participated in cell basic activity.However,the molecular mechanisms for promoting tumor metastasis are unkown.MYO1B was indentificated as one of the specific receptors for Galectin-1 on CRC cell membrane by our preliminary study.MYO1B is one member of unconventional myosin family,mainly located membrane ruffles.The gene for MYO1B,previously called MI130 or MYR1,can code for up to 6 IQ motifs which form alpha helices that bind calmodulin.The gene located in chromosome 2q12-q34.The overall length of MYO1B is 234Kb.MYO1B is the single-headed,membrane binding member of myosin superfamily that contains one heavy chain of 130KDa and multiple camodulin light chains of 17 KDa.There is no literature reported about the relationship of MYO1B and metastasis of colorectal cancer.Many studies have certificated that MYO1B partipated in cytoskeletal rearrangements and material transportation in cell.MYO1B was also reported associating with lamellipodia with the highest concentration in ruffling membranes,suggesting a role for MYO1B in cell migration.Therefore,our study aims at explore the mechanism of the correlation between MYO1B and the CRC invasion and metastasis,further to make clear the molecular mechanisms and biological significance of MYO1B in CRC invasion and metastasis.This study will not only give us a better understanding of the molecular mechanism of malignant tumor metastasis theoretically,but also provide a new view in the diagnosis and therapy of malignant tumor practically.METHODS1.The identification of relationship between Galectin-1 and MYO1BThe immunofluorescence and western blotting were used to detect the specific effector of Galectin-1 on SW620 cell membrane after rGalectin-1 coculture with 4h.Co-Immunoprecipitation,mass spectrometry analysis were uesd to identified that MYO1B was the target protein for Glectin-1.Far-western bloting was used to identified that Glectin-1 directly binded MYO1B.2.The expression of MYO1B in different colorectal cancer cell lines and cancer tissue samples.(1)The immunofluorescence was ued to detect MYOIB expression in SW480 and SW620 cell after rGalectin-1 coculture with 24h.(2)Real-time PCR and western blotting were used to detect MYOIB expression in seven kinds of colorectal cancer cell lines,SW480?SW620?HT29?Lovo?HCT116?M5?DLD1 cell respectively.Real-time PCR was used to detect MYO1B expression in 20 cases of paired fresh CRC tissues.Immunohistochemical method was used to detect MYO1B expression in primary colorectal carcinoma and corresponding normal colorectal mucosa tissues,Paraffin-embedded CRC tissue samples from 68 patients were obtained from the January 2000 and December 2010 at Nanfang Hospital,Southern Medical University.3.The relational colorectal cancer signal pathway was detected.(1)Lentiviralbb vector expressing or repressing MYO1B were packaged using the Ppackhl lenti vector packaging kit.Pseudo virus particles were subsequently used to infect CRC cells.SW480,SW620 and HT29 cell lines constitutively expressing MYO1B and SW480,SW620 and DLD1 cell lines stably suppressing MYO1B were generated.Then,the expression of MYO1B was assessed by western blotting and real-time PCR.(2)The expression of the related proteins of focal adsion signal pathway containing?-actin,FAK,Paxillin,Talin-1,Tensin2,Vinculin,Profilin-1,were assessed by western blotting.4.Statistical analysisStatistical analyses were performed using SPSS 13.0 software package.Quantitatives values of all experiments were expressed as the mean ±standard deviation(SD).Relative quantification value(2-??ct)of qRT-PCR in cells were analysed by One-way ANOVA,with the SNK,LSD or Dunnett T3 tests for multiple comparisions.Kaplan-Meier survival analysis was used to estimate cancer-specific survival,and comparisons between groups were performed with the log-rank test.Multivariate survival analyses were performed by using Cox proportional hazard models.P<0.05 was considered statistically significant.RESULTS1.The identification of relationship between Glectin-1 and MYO1BThe immunofluorescence showed that there was specific effector of Galectin-1 on SW620 cell membrane after rGalectin-1 coculture with 4h.Then,co-immunoprecipitation(CO-IP),mass spectrometry analysis were uesd to identified that MYOIB was the target protein for Galectin-1.Far-western bloting then was used to identified that Glectin-1 directly binded to MYO1B.2.The expression of MYO1B was different in seven colorectal cancer cell lines and the expression of MYO1B in cancer tissue was higher than its paired normal tissue.The expression of MYO1B was increasing in SW620 cell after rGalectin-1 coculture with them 24h.The expression of MYO1B was detected in SW480/SW620/HT29/Lovo/HCT116/M5/DLD1 cell lines by Real-time PCR and western blotting.The results showed that all of the cell lines expressed MYO1B,DLD1,SW620,HCT116 had the stronger expression,MYOIB expressed weakly in HT29.The difference has statistical significance(P<0.01).Real-time PCR was used to detect MYO1B expression in 20 cases of paired fresh CRC tissues,the result performanced that the expression of MYOIB in cancer tissues is higher than its paired normal tissues(P<0.05).Immunohistochemical method was used to detect MYO1B,CD 163 and CD68 expression in primary colorectal carcinoma and corresponding normal colorectal mucosa tissues.The result showed that the expression of MYO1B was higher than the control group and favored to locate the front edge of invasion.CD68 and CD 163 were positive in macrophages which around CRC cells with positive expression of MYO1B.3.The relational colorectal cancer signal pathway was detected.Western blotting and real-time PCR was used to detect the expression of the related proteins of focal adsion signal pathway which contains a-actin,FAK,Paxillin,Talin-1,Tensin2,Vinculin,Profilin-1.The results showed the expression of the proteins was changed along with the expression of MYO1B.Compared with control group,Cells were suppressed expressing MYOIB had lower expression of Paxillin.CONCLUSION1.MYO1B was the specific effector for Glectin-1 in CRC cell membrane,MYO1B expression in colorectal cancer tissues was higher than its paired normal colorectal mucosa tissues and favored to locate the front edge of invasion.2.Galectin-1 secreted by TAMs with M2 phenotype combined with MYO1B-one of effector in CRC cell membrane,and MYO1B interacted with RACK1,which promoting CRC invasion and metastasis through focal adsion signal pathway.