CAP2 Promotes Gastric Cancer Metastasis By Mediating The Interaction Between Tumor Cells And Tumor-associated Macrophages

BackgroundGastric cancer(GC)is the fifth most common cancer worldwide,with a high mortality,especially in patients with advanced disease or metastasis.Most cancers are not caused by the primary tumor,but due to the metastasis,and it is important to clarify the mechanism of metastasis.The tumor microenvironment is a complex cellular environment in which macrophages are most infiltrated and involved in all stages of tumor progression.Tumor-associated macrophages are usually of the M2 subtype and play a tumor-promoting role.However,the mechanism of crosstalk between TAM and GC cells is poorly understood.In our previous study,microarray analysis was performed to compare differential gene expression in primary gastric cancer samples with or without lymph node metastasis(LNM).The results showed that CAP2 was upregulated in the gastric cancer tissue of LNM.Currently,CAP2 expression is upregulated in several human cancers.However,the biological function and underlying mechanisms of CAP2 in gastric cancer are still poorly understood.In this study,the expression and function of CAP2 in gastric cancer were evaluated,and the mechanism of CAP2-mediated interaction between tumor cells and macrophages to promote gastric cancer metastasis was elucidated.CAP2 may be a potential prognostic biomarker and therapeutic target for gastric cancer.MethodsThe expression of CAP2 in gastric cancer clinical samples and gastric cancer cells was detected by RT-qPCR,the expression of CAP2 in gastric cancer and gastric mucosa in gastric cancer with different metastatic states was detected by immunohistochemistry,and the effect of CAP2 on patient prognosis was analyzed by clinicopathological parameters,patient survival information and network database.The transcription factor of CAP2 was sought by constructing a promoter upstream truncated plasmid and the dual luciferase experiment.The binding of JUN to the CAP2 promoter region was verified using chromatin immunoprecipitation experiments and promoter region mutations.The effect of CAP2 on the migration and infiltration of gastric cancer cells was confirmed by Transwell experiment.The effect of CAP2 on the proliferation of gastric cancer cells in vitro was verified by CCK8 and EdU experiments.The effects of CAP2 on the growth and metastasis of gastric cancer cells in vivo were observed by constructing subcutaneous xenograft model and metastatic model in nude mice combined with tumor growth curves,HE staining,and small animal in vivo imaging.The binding protein of CAP2 was verified by GST-pulldown and immunoprecipitation followed by liquid mass spectrometry.The distribution of CAP2 and RACK1 in gastric cancer was observed by immunofluorescence.The truncated mutation of CAP2 protein and RACK1 protein was constructed to find the binding sites.The effect of CAP2 on IL4 and IL10 secretion was detected by enzyme-linked immunosorbent assay and ultrafiltration concentration of gastric cancer cell supernatant.The effect of CAP2 on macrophage infiltration and polarization in tumor microenvironment was explored by immunofluorescence,flow cytometry and macrophage marker expression determination.The effect of tumor-associated macrophages on gastric cancer cells was detected by co-culture,cytokine screening,and ChIP experiments.The small molecule inhibitors of CAP2 were screened by simulation and virtual docking of CAP2 structure.The effect of small molecule inhibitors of CAP2 on the growth and metastasis of gastric cancer cells was detected in vitro and in vivo.The effect of salvianolic acid B on the survival of mice was verified by monitoring animal survival.Results(1)CAP2 is upregulated in human gastric cancer tissues and is associated with a poor prognosis for patients with gastric cancerThe expression of CAP2 is gradually increased in gastric mucosa,primary gastric cancer,and gastric cancer with lymph node metastasis.CAP2 can be used as an independent diagnostic index to distinguish normal tissue,gastric cancer tissue and LNM gastric cancer tissue.Patients with higher CAP2 expression had shorter disease-free survival and overall survival.(2)JUN activates CAP2 transcriptionThe core initiation region was identified by CAP2 promoter region truncation combined with a dual luciferase experiment.RT-qPCR and western blot experiments were used to confirm that JUN could promote the expression of CAP2.ChIP analysis showed that JUN was enriched on CAP2 promoter.The correlation analysis showed that CAP2 was positively correlated with JUN.(3)CAP2 promotes the progression of gastric cancerTranswell experiments showed that the expression of CAP2 promoted the migration and invasion of gastric cancer cells.CCK-8 experiments showed that CAP2 did not affect the in vitro proliferation of gastric cancer cells in vitro.GC cells transfected with LV-shCAP2 or LVshNC are injected into the tail veins of nude mice to observe tumor metastasis.Metastasis in the LV-shCAP2 group is inhibited.Notably,knockdown CAP2 with 2’-OMe-modified RNA interference significantly reduced tumor lung metastasis.In xenograft tumor models,CAP2 knockdown inhibits local invasion of tumors.Unlike in vitro experiments,CAP2 knockdown results in a reduction in the volume and weight of xenograft tumors.(4)CAP2 binds to with RACK1 and activates the FAK/MEK/ERK axisWe performed GST pull-down and Co-IP as well as liquid chromatography-mass spectrometry to identify the ability of RACK 1 to bind to the CAP2 protein.Immunofluorescence analysis showed that CAP2 and RACK1 were co-localized in gastric cancer cell lines.CAP2 does not affect the RNA and protein levels of FAK/MEK/ERK,but enhances phosphorylation of FAK/MEK/ERK.Overexpression of CAP2 enhances the binding of RACK1 to FAK.Coexpression of CAP2 and RACK1 further enhances gastric cancer cell migration and invasion and FAK/ERK phosphorylation.Gene set enrichment analysis showed that CAP2 expression was positively correlated with adhesion foci and MAPK signaling pathway.(5)CAP2 competitively binds to domains WD5 to WD7 of RACK1 and dissociates SRCThe construction of RACK1 truncated mutant binding Co-IP experiments showed that the WD5-WD7 domain of RACK1 could bind CAP2.By constructing a CAP2 protein truncated mutant,it was shown that the N-terminal domain of CAP2 is able to bind to RACK1.Overexpression of CAP2 weakens the binding ability between RACK1 and SRC,and the expression of CAP2 enhances phosphorylation of SRC,SRC inhibitor PP2,partially reverses activation of the FAK/ERK signaling pathway caused by CAP2 overexpression.(6)Gastric cancer tissues with high CAP2 expression are rich in M2 macrophagesCAP2 was positively correlated with IL4 and IL10 expression and promoted the secretion of IL4 and IL10.Treatment of gastric cancer cells with ERK inhibitors SCH772984 reduces IL4 and IL10 expression and reverses increased ERK phosphorylation caused by CAP2 and expression of IL4 and IL10.More CD 163+cells were found in tumor tissues with high expression of CAP2.Immunofluorescence and flow cytometry show that overexpression of CAP2 in gastric cancer cells increases the polarization of CD163+/CD206+macrophages.Detection of macrophage markers suggests that CAP2 expression in gastric cancer cells promotes macrophage differentiation into an M2-like phenotype.(7)TAMs promote CAP2 expression through TGFB1-mediated activation of JUNM2 macrophages enhance the proliferation and metastasis of gastric cancer cells and promote the expression of CAP2 in gastric cancer cells.TGFB1 enhances the expression of CAP2.Induction of TGFB1 and M2 macrophages promotes activation of the TAK1/JNK/JUN signaling axis.Treatment of gastric cancer cells with TGFB1 or TAMs increases the binding of JUN to the CAP2 promoter region.(8)Salvianolic acid B is a putative molecular inhibitor of CAP2 to suppress GC progressionThe software mimics the structure,molecular docking,and cell function experiments of the CAP2 protein to screen tanshinic acid B for possible use as an inhibitor of CAP2.In vivo animal administration confirmed that salvianolic acid B inhibits tumor growth,metastasis,and M2 cell polarization.ConclusionHigh expression of CAP2 promotes the metastasis of GC cells in vitro and in vivo and causes macrophages to polarize to TAMs(M2 phenotype).Meanwhile,polarized TAMs further promoted the transcription of CAP2 in GC cells by secreting TGFB1.A positive feedback loop is formed between CAP2 and TAMs to jointly promote the metastasis of GC.