Mutual Activation Between Ca²⁺-activated Cl^- Channel Anoctamin 1 And Annexin A1 Promotes Colorectal Cancer Metastasis

Objective:Colorectal cancer is a disease with a high incidence worldwide.It is one of the three most common cancers in the world and has the second highest mortality rate among all tumors.Anoctamin 1（Ano1）calcium-activated chlorine channels are highly expressed in a variety of tumors and are closely associated with poor prognosis of patients,which is expected to become a novel prognostic marker of tumors.Studies have shown that Ano1 is highly expressed in colorectal cancer and can promote the biological behaviors of colorectal cancer cell proliferation,migration and invasion.Studies have shown that high Ano1 expression is associated with lymph node metastasis of colorectal cancer,and tumor metastasis is the main cause of death in colorectal cancer patients.Therefore,studying the molecular mechanism of Ano1 promoting colorectal cancer metastasis can provide a new idea for the treatment of colorectal cancer metastasis.The purpose of this study was to explore the mechanisms,by which Ano1promotes the invasion and metastasis of colorectal cancer,and to provide theoretical basis for the development of Ano1-targeted drugs for the treatment of colorectal cancer metastasis.Methods:1.TCGA and immunohistochemical methods were used to detect the expression difference of Ano1 in colorectal patients and the influence of high and low Ano1 expression on lymph node metastasis and survival of colorectal cancer patients.Stable high and low expression of Ano1 cell lines were prepared by lentivirus transfection technique.Wound healing assay and Transwell assay were used to verify the migration and invasion ability of colorectal cancer cells with different Ano1expression levels.A lung metastasis mouse model was used to investigate whether Ano1 promoted CRC metastasis by tail injection of colorectal cancer cells.In vitro imaging was used to detect the size of metastatic tumors of colorectal cancer cells with different Ano1 expression levels.2.Based on transcriptomic analysis to identify key genes by difference analysis and pathway enrichment analysis in SW620 cells treated with lentiviral vectors containing scrambled sh RNAs or Ano1-sh RNAs.q RT-PCR and Western blot were used to detect the expression of ANXA1 in colorectal cancer cells.q RT-PCR and Western blot were used to detect the m RNA and protein expression in colorectal cancer cells treated with lentiviral vectors and Ano1-containing lentiviral vectors or lentiviral vectors containing scrambled sh RNAs and Ano1-sh RNAs,respectively.After transfection with STAT3-overexpressing vector and its control Vector plasmid,the expression of ANXA1 protein was detected by Western blot.The combination of Ano1 and Src in colorectal cancer cells was detected by co-immunoprecipitation.The P887L/P890L-Ano1 mutant was constructed by site-directed mutagenesis and transfected into HEK293 cells.The binding of P887L/P890L-Ano1 mutant to Src was detected by co-immunoprecipitation.Western blot was used to detect the expression of downstream pathway proteins and ANXA1 after transfection of P887L/P890L-Ano1 mutant or treatment with Src inhibitor Src inhibitor I or STAT3 inhibitor JSI-124.3.Co-immunoprecipitation was used to detect the binding of endogenous ANXA1 and Ano1 in colorectal cancer cells.Whole-cell patch clamp was used to record the Ano1calcium-activated chloride current under different Ca²⁺（0.18μM,0.36μM,1μM,25μM）concentration in SW620 cells treated with Scrambled-sh RNAs or ANXA1-sh RNAs plasmid.4.The site-directed mutagenesis was used to constructed the E654A-Ano1 mutant,which exhibited low channel activity due to disrupted Ca²⁺-binding site.The migration and invasion abilities of colorectal cancer cells treated with Ano1 inhibitor Ani9 or transfected with E654A-Ano1 mutant were determined by wound healing assay and Transwell migration and invasion assay.Using Lipofectamine 2000 transfection technology,ANXA1-sh RNAs or ANXA1 plasmids or their control plasmids were transfected into stable colorectal cancer cells with high and low Ano1 expression respectively.The changes of migration and invasion ability of colorectal cancer cells after transfection were detected by wound healing assay and Transwell migration and invasion assay.Results:1.Ano1 m RNA expression was significantly higher in colorectal cancer tissues than in normal tissues.High Ano1 expression was significantly associated with lymph node metastasis and shorter overall survival in colorectal cancer patients.Ano1expression was relatively high in SW620 cells,but relatively low in RKO cells.Ano1overexpression promoted migration and invasion in RKO cells and Ano1 knockdown reduced migration and invasion in SW620 cells.The size of metastatic tumors,measured by the intensity of the green fluorescence of SW620 cells treated with EGFP-expressing lentiviral vectors,was significantly decreased in mice injected with Ano1-sh RNAs compared with control mice.2.Transcriptome analysis identified ANXA1 as one of the genes related to cell migration regulated by Ano1.ANXA1 knockdown reduced migration and invasion in colorectal cancer cells.ANXA1 m RNA and protein expression were relatively low in RKO cells and Ano1 overexpression promoted ANXA1 m RNA and protein expression and the expression of p-STAT3/STAT3 in RKO cells.ANXA1 m RNA and protein expression were relatively high in SW620 cells and Ano1 knockdown reduced the expression of ANXA1 m RNA and protein and the expression of p-STAT3/STAT3.STAT3 overexpression promoted ANXA1 expression in RKO cells.STAT3 inhibitor JSI-124 significantly blocked Ano1 overexpression-induced ANXA1 expression in RKO cells.Ano1 directly interacted with Src proteins in SW620 cells;the interaction between Ano1 and Src was blocked by the P887L/P890L-Ano1 mutant.In RKO cells,P887L/P890L-Ano1 mutant reduced WT Ano1-induced STAT3 phosphorylation and ANXA1 expression.Src inhibitor I inhibited STAT3 phosphorylation and ANXA1expression induced by Ano1 overexpression in RKO cells.3.Endogenous Ano1 co-immunoprecipitated with ANXA1 in SW620 cells.Whole-cell patch clamp was used to record whole-cell Cl^-currents activated by different concentrations of Ca²⁺（0.18μM,0.36μM,1μM,25μM）in SW620 cells treated with scrambled sh RNAs or ANXA1-sh RNAs.The current intensity decreased significantly and calcium sensitivity decreased in a voltage dependent manner after transfection with ANXA1-sh RNA plasmid.4.Ano1-specific inhibitor Ani9 inhibited the migration and invasion in SW620 cells compared with the control group.Overexpression of E654A-Ano1 mutant abolished the promoting effect of WT-Ano1 on migration and invasion in RKO cells.In Ano1-overexpressing RKO cells or vector control cells,transfection of ANXA1-sh RNA plasmid significantly inhibited the ability of Ano1 to promote cell migration and invasion.In Ano1-sh RNA-treated SW620 cells or its scrambled control cells,transfection of ANXA1 plasmid promoted the migration and invasion in SW620 cells,while Ano1 Knockdown significantly reduced the migration and invasion abilities of ANXA1-induced in SW620 cells.Conclusion:1.Ano1 calcium-activated chloride channel can promote the invasion and metastasis of colorectal cancer cells and shorter overall survival of colorectal cancer patients;2.Ano1 activated Src by direct interaction with Src,and subsequently increased ANXA1 expression via the Src/STAT3 signaling pathway;3.ANXA1activated Ano1 calcium-activated chloride channels by voltage-dependently via direct interaction with Ano1 channels;4.Ano1 and ANXA1 are mutually dependent in the regulation of migration and invasion in colorectal cancer cells.