Biological Effects Of AREG On The Growth Of Tumor Cells In Vivo

AREG(Amphiregulin)is a member of Epidermal growth factor family;AREG binds and activates the epidermal growth factor receptor(EGFR),which promotes the EGFR to form homodimer or heterodimer,leading to the phosphorylation of intracellular downstream signal,and then regulates the proliferation and differentiation of cells.In this study we investigate the role of AREG in the regulation of tumor growth.Firstly,the mouse colon cancer CT26 cells,melanoma B16 and liver tumor LPC-AKT cells with lower expression level of AREG were selected to construct AREG overexpression cell line and control cell line.The effects of AREG expression on the tumor cells growth in vitro were measured by MTT experiment,clone formation experiment and cell cycle test.The results showed that AREG overexpression have little effect on the proliferation of CT26cells,B16 cells and LPC-AKT cells under cultured condition in vitro.By constructing CT26tumor bearing mice model we found that the overexpression of AREG could significantly promote the tumor cells growth in vivo.The effects of AREG expression on the composition of immune cells in tumor tissues were studied by FACS and Real-Time PCR.The results showed that AREG overexpression can reduce the proportion of CD8~+T cells in tumor microenvironment,and could significantly down regulate the expression level of CCL5 m-RNA in tumor microenvironment.We further studied the effects of irradiation and chemotherapy on the amount of AREG produced in tumor cells by ELISA.Irradiation and chemotherapy drugs etoposide and adriamycin could significantly promote tumor cells secrete AREG.The AREG expression in Hepa1-6 cells was removed by CRISPR-Cas9 system,and then the different effects of AREG deficient Hepa1-6 cells and normal Hepa1-6 cells which were both irradiated on the growth of tumor cells in vitro and in vivo were compared by FACS and Tumor bearing mice.Under the condition of in vitro culture,radiation induced AREG expression could not effectively promote the survival of tumor cells.In vivo experiments,it was found that the irradiated AREG expression cells could significantly promote the growth of LLC lung cancer,but the AREG expression deficient cells had no such effect.In the third part,by screening anti human AREG monoclonal antibody which had been already prepared,we can obtain 5 strains of monoclonal antibody which are aimed at AREG,these antibodies did not exist cross reaction with human EGF and TGF-alpha,and 2 of them had a certain effect on neutralizing AREG signal.Conclusion:In our experiment system,we found that AREG can promote the mouse colon cancer CT26 cells growth in vivo by down regulating the expression of chemokine CCL5 in tumor microenvironment and decreasing the number of CD8+T cells.Radiation and chemotherapeutic drugs can induce tumor cells to produce AREG,AREG played an important role in the process of promoting tumor growth by irradiating cells.In addition,we obtain 2 strains of anti-human AREG monoclonal antibody with certain neutralization.This study suggests that AREG can regulate the composition of immune cells in tumor microenvironment and promote tumor growth,and the inhibition of AREG signaling may be a new therapeutic strategy for cancer.