Tumor-Derived Prelp Activates Integrin β3-FAK-AKT-Integrin β3 Positive Feedback Loop To Promote Lymph Node Metastasis In Colorectal Cancer

Objective: Colorectal cancer(CRC)has the third highest rate of new morbidity and mortality around the world.Nowadays,surgical excision is the most important treatment for CRC,but patients die as a consequence of the progression and metastasis after surgery.Lymph node metastasis is the main pathway of distant metastasis in colorectal cancer and is also a key factor in predicting patient prognosis.For most tumors,the lymphatic system provide a pathway for tumor cell dissemination and lymphatic spread is often an early event.For early stage patients of colorectal cancer without lymph node metastasis,the 5-year survival rate is 80-90%,whereas for patients with advanced stage colorectal cancer,the 5-year survival rate drops to 25-60%.Lymphatic dissemination is not well known for CRC.To our knowledge,tumor associated lymphangiogenesis acts as a promoter in lymphatic metastasis,aiding to promote cancer cell recruitment to lymph nodes.lymphatic vessels are constructed by a single layer of endothelial cells,lacking intermittent basement membrane,pericytes or smooth muscle cells,which makes them easier for tumor cells intravasation.Increasing evidence has shown that lymphangiogenesis facilitate lymphatic metastasis in the development of colorectal cancer.Unfortunately,the specific molecular mechanisms governing this tumor progression have not yet been completely understood,and lacks of new specific prognostic biomarkers and therapeutic targets.Therefore,it is essential to search a key marker for CRC lymph node prognostic and therapy in order to provide a valuable strategy for cancer therapy.Methods: 1.TCGA data were used to explore genes with high expression and poor prognosis in lymph node metastasis tissues of colorectal cancer patients;2.GO enrichment analysis was used to study the possible functional pathways of genes.3.GSE17536 dataset was used to verify the gene expression and prognostic effects in the enrichment pathway;4.Immunohistochemistry was used to detect the expression of PRELP protein in colorectal cancer tissues.5.Immunohistochemical method was used to detect the density of lymphatic microvessels stained with LYVE1,a marker of lymphoendothelial cells,in colorectal cancer tissues,and to study their correlation.6.Western blot assay was used to detect the expression of PRELP in each cell line and the transfection efficiency of si RNA and overexpressed plasmid.7.Experiments related to migration and invasion: The effect of PRELP on migration and invasion of colorectal cancer cells was observed by wound healing and transwell experiments.8.ELISA was used to detect the protein level of PRELP in conditioned medium derived from colorectal cancer cells.9.Lymphangiogenesis related experiments: the effect of PRELP on lymphangiogenesis was studied by lymphangiogenesis and transwell experiment.10.The nude mice were injected into the plantar with colorectal cancer cells to construct lymph node metastasis model,and the regulation of PRELP on lymph node metastasis of colorectal cancer cells was studied in vivo.11.Co-immunoprecipitation and western blot were used to detect the effect of PRELP on the downstream pathway.12.Statistical analysis: SPSS version 26 software was used for statistical analysis,and Graph Pad Prism version 6 software was used to make graphs.T test and one-way ANOVA were used to analyze the differences between two groups and multiple groups.Results: 1.To search for genes highly expressed in lymph node metastasis of colorectal cancer and associated with poor prognosis.Through bioinformatics analysis,TCGA data were used to identify genes that were highly expressed and associated with poor prognosis in lymph node metastasis tissues.GO enrichment analysis was performed for 75 genes,and the genes in the pathway were verified by GSE17536 data set.Results showed that PRELP and PCDHB2 were positively correlated with lymph node metastasis and poor prognosis.2.PRELP is highly expressed in the tissues of patients with lymph node metastasis of colorectal cancer,and is an independent predictor of lymph node metastasis.We collected patient tissue samples,and immunohistochemical results showed that PRELP was highly expressed in the tissues of patients with lymph node metastasis compared with the tissues without lymph node metastasis.Correlation analysis of PRELP and LYVE-1expression showed a significant positive correlation.Analysis of clinicopathological parameters suggested that PRELP was an independent predictor of lymph node metastasis.3.PRELP can promote the migration and invasion of colorectal cancer cells.Colorectal cancer cells RKO and HCT15 were subjected to PRELP knockdown and overexpression treatment.Western blot was used to verify knockdown and overexpression efficiency,and wound healing and transwell test were used to detect changes in cell migration and invasion ability.The results showed that the migration and invasion ability of both kinds of cells decreased after PRELP knockout,and increased after overexpression.4.PRELP secreted by tumor cells can promote lymphangiogenesis.We collected conditioned medium of knockdown and overexpression treated colorectal cancer cells,and analyzed the expression level of PRELP in conditioned medium by ELISA.Tubular formation of lymphatic endothelial cells and transwell assay were used to verify the effect of PRELP on lymphangiogenesis.ELISA results showed that the expression of PRELP in conditioned culture medium changed with knockdown or overexpression of PRELP in colorectal cancer cells.Tubule formation and transwell experiments related to h LECs showed that tubule formation and migration of h LECs cells decreased significantly in conditioned medium after knockout of PRELP,but increased significantly after overexpression of PRELP.5.PRELP can significantly promote lymph node metastasis in nude mice.To study the promoting effect of PRELP on lymph node metastasis in mice,we screened stable HCT15 cells with overexpression of PRELP,and established a mouse popliteal lymph node metastasis model.Immunohistochemical results verified the efficiency of overexpression of PRELP in tumor-forming sites in vivo.In vivo experiments showed that overexpression of PRELP significantly increased the volume of popliteal lymph nodes in mice,and the number of metastatic lymph nodes was significantly increased after overexpression.6.PRELP can affect integrin α/β expression and activate downstream FAK/AKT pathway.In order to study the mechanism of PRELP promoting tumor cell migration and invasion,we first performed GSEA enrichment analysis,and the results showed that PRELP expression was related to focal adhesion and actin skeleton regulatory pathway.A variety of integrin family genes were obtained by the intersection of genes in the two pathways.Correlation analysis of integrin and PRELP expression was conducted by using TCGA and GSE17536 data.ITGA5,ITGA7,ITGAV and ITGB3 were selected for expression analysis.Western blot results showed that PRELP knockdown downregulated the expression of integrin α5,integrin α7,integrin αV and integrin β3,whereas the expression of multiple integrins was up-regulated after PRELP overexpression.7.PRELP can interacted with integrin β3 to promote the metastasis of colorectal cancer cells.Co-IP assay showed that PRELP could combine with integrin β3 but not integrin α5to promote cancer.8.PRELP activates integrin β3-FAK-AKT-integrin β3 positive feedback loop to promote colorectal cancer metastasis.Western blot results showed that the CRC cells incubated with LY294002(AKT inhibitor)significantly reduced rh PRELP stimulated the level of integrin expression.Transwell experiment results showed that the carcinogenic effect of PRELP was weakened after the addition of LY294002.9.The interaction of PRELP and integrin β3 activates the integrin β3-FAK-AKT-integrin β3positive feedback loop to promote lymphangiogenesis.Co-IP results showed that PRELP still binds integrin β3 in h LECs cells and activates the downstream FAK-AKT pathway to promote lymphangiogenesis.The results of tube formation experiment and transwell experiment showed that the ability of PRELP to promote tube formation and migration of h LECs was weakened after the addition of LY294002.Conclusion: 1.Bioinformatics methods was performed using TCGA and GEO dataset to screen PRELP as a biomarker of lymph node metastasis;2.PRELP is highly expressed in the tissues of patients with lymph node metastasis and can be used as an independent predictor of lymph node metastasis.3.PRELP promotes the migration and invasion of colorectal cancer cells and lymphangiogenesis in vivo and in vitro;4.PRELP can activate integrin β3-FAK-AKT-integrin β3 positive feedback loop to promote colorectal cancer cell metastasis.5.PRELP can promote lymphangiogenesis through integrin β3-FAK-AKTintegrin β3 positive feedback loop.