The Role And Regulatory Mechanism Of Klotho By Inhibiting CCL-2 Secretion In Senescent And Colorectal Cancer Cells

Background and aimsColorectal cancer(CRC)is one of the most common human cancers worldwide.There are up to 1.36 million new cases and nearly 0.69 million deaths from CRC each year.It is of great significance to discover key molecules and their mutual relations in CRC carcinogenesis,and understand potential mechanism of CRC.The risk of CRC is increasing with age.Recent studies provided evidence that senescent cells might promote tumour progression by altering tumour microenvironment,which was consisted of fibroblasts,endothelial cells,immune cells and extracellular matrix around tumour.Klotho is a senescence-related protein with multiple functions,which received widespread attention in anti-aging and prolonging life.Down-regulation of Klotho gene was closely associated with human diseases,including shortened life,arteriosclerosis,cardiovascular and cerebrovascular diseases,and so on.In recent years,it was found that the expression of Klotho was decreased in many tumour tissues,which indicated that Klotho was negatively correlated with cancer development.Our group have previously validated that Klotho gene was silenced or significantly down-regulated in colon cancer cells by microarray,and found that Klotho gene was epigenetically regulated by hypermethylation in the promoter regions.This study was designed to clarify the clinical significance and biological functions of Klotho in CRC,and determine the molecular mechanism of Klotho in cellular senescence and expression of senescence-associated secretory phenotype(SASP)in CRC.Materials and methods1.The expression of Klotho in CRC tissues and its clinical significance.143 cases of CRC patients were included and the tissues were immunostained with Klotho antibody.The expression of Klotho and its correlation with clinical pathological parameters and survival time in CRC were analyzed.2.The effects of Klotho on cellular senescence and CRC cell biology behaviors.Mesenchymal cells were induced by DOX in the presence or absence of human recombinant Klotho,and its effect on cellular senescence was decided by SA-?-gal staining and detection of senescence associated markers.Conditioned medium of different mesenchymal cells was collected to co-culture with CRC cells,and their effects on CRC cell biology behaviors(cell proliferation,cell migration and invasion)were evaluated.Then mesenchymal cells mixed with CRC cells were subcutaneously inoculated into the nude mice and the tumourigenicity was observed.The functions of Klotho overexpression in CRC cells were also evaluated in vitro and in vivo.3.The effects of Klotho on CCL-2 expression and its biology behaviors in CRC.We screened and validated series of SASP molecules,including CCL-2.The role of CCL-2 on colon cancer cell proliferation,cell migration and invasion was determined in vitro and in vivo.4.Klotho inhibited the secretion of CCL-2 through NF-?B signal pathway.Mesenchymal cells were induced by DOX in the presence or absence of human recombinant Klotho,colon cancer cells with Klotho overexpression or control vector,key molecules of the NF-?B signal pathway and CCL-2 were detected.Results1.Klotho was down-regulated in CRC tissues and related with the prognosis of patients.Immunohistochemical staining showed that Klotho was down-regulated in CRC tissues when compared with the adjacent non-tumour tissues.The expression of Klotho was closely correlated with age,lymph node metastasis,distant metastasis and TNM stage(P<0.05).Kaplan-Meier survival analysis indicated that higher expression of Klotho was related to better prognosis of patients.Multivariable COX regression analysis showed that high Klotho expression was an independent prognostic factor that predicts better survival in CRC patients.2.Klotho inhibited cellular senescence and CRC cell proliferation,migration and invasion.(1)Pretreatment with human recombinant Klotho could inhibit cellular senescence by SA-?-gal staining and detection of senescence associated markers.(2)Pretreatment with Klotho in mesenchymal cells inhibit CRC cell proliferation,migration and invasion in co-cultured cells.? CRC cells proliferation was prompted when co-cultured with conditioned medium from senescent mesenchymal cells,while this effect was attenuated by pretreatment with Klotho.? CRC cells migration and invasion was increased when cocultured with conditioned medium from senescent mesenchymal cells,which was inhibited by pretreatment with Klotho.? Co-injection of CRC cells with senescent mesenchymal cells accelerated mice model tumour growth,which was inhibited by pretreatment with Klotho.(3)Overexpression of Klotho suppressed CRC tumour growth,cell migration and invasion.? Overexpression of Klotho suppressed CRC cell proliferation,migration and invasion.? Overexpression of Klotho significantly suppressed exnograft CRC tumour growth.3.Klotho suppressed the expression of CCL-2 and cell proliferation,migration and invasion prompted by CCL-2.(1)Pretreatment with Klotho inhibits the expression level of CCL-2 mRNA and secreted protein.(2)The effects of CCL-2 on CRC cell proliferation and migration.? CRC cells proliferation and migration were prompted when co-cultured with CCL-2,which was suppressed by specific antagonist of its receptor CCR2.? Peritumoural injection of CCL-2 accelerated the growth of tumours in nude mice,which was suppressed by CCR2 antagonist.4.Klotho inhibited the secretion of CCL-2 through NF-?B signal pathway.(1)The NF-?B signaling pathway was activated in senescent mesenchymal cells,which was inhibited by pretreatment of Klotho or specific NF-?B inhibitors.(2)The mRNA expression and secretion of CCL-2 were significantly decreased by pretreatment of Klotho or specific NF-?B inhibitors.(3)Overexpression of Klotho suppressed the NF-?B signal pathway and secretion of CCL-2 in CRC cell.Conclusions1.The expression of Klotho was down-regulated in CRC tissues,and closely related to age,lymph node metastasis,distant metastasis,TNM stage and prognosis of patients.Higher expression of Klotho was an independent predictor of better survival.2.Overexpression of Klotho significantly suppressed CRC cell growth,migration and invasion.Pretreatment with human recombinant Klotho could also suppress above tumour behaviors through regulating cell senescence and influencing the microenvironment of CRC cells.3.Klotho could suppress the expression and secretion of CCL-2.CCL-2 promotes cell proliferation and migration and this effect was suppressed by its receptor CCR2 antagonist in CRC.4.Klotho could inhibit secretion of CCL-2 through NF-?B signal pathway both in mesenchymal cells and CRC cells.