The Role Of Necroptosis Irn Radiation-induced Cell Death And The Effects Of Tumor Heterogeneity On Radiosensitivity

Radiation therapy is an important treatment for pelvic and abdominal cancer.Significant progresses have been made in radiation therapy,but the challenges remain.On the one hand,normal tissue adjacent to the tumor is inevitably exposed to radiation,which leads to early inflammatory reactions,eventually causing structure and function abnormalities.On the other hand,the existence of inter-patient and intra-tumor heterogeneity result in variation to radiotherapy sensitivity,causing inherent or acquired radiotherapy resistance during radiation therapy.Ionizing radiation causes programmed cell death by damaging cellular DNA,among which apoptosis is widely studied.Necrosis has long been considered as an unprogrammed death of cells.However,this view has been reconsidered due to the latest research finding of specialized signal transduction pathway for necrosis.Necroptosis is a programmed form of necrosis,which depends on RIP1(receptor interacting protein kinase 1)and RIP3 to recruit then phosphorylate MLKL(mixed lineage kinase domain-like protein)to perform necroptosis.During the process of necroptosis,plasma membranes and organelles are permeabilized and cell contents are released into the surrounding tissues.The cell contents are regarded as damage-associated molecular pattern molecules(DAMP)and recognized by the immune system to induce an inflammatory phenotype.However,it is still unclear whether necroptosis is involved in radiation-induced cell death.Further elucidation of radiation-induced programmed cell death mechanisms can provide new treatment trategies for mitigating the side effects of radiotherapy or better radiosensitization for tumor therapy.This experiment explored whether necroptosis was involved in radiation-induced cell death.Firstly,MLKL knockout mice were subject to 7Gy total body irradiation,during which 30-day mortality rates and body weight were observed.We found that MLKL-KO mice were more sensitive to irradiation.Organ index,complete blood count,H&E staining and immunohistochemistry were performed.We found that the spleen,thymus and kidney of MLKL-KO mice were more severely damaged than wildtype after irradiation.Then,we used the CRISPR-Cas9 system to construct RIP1 or MLKL knockout B16 cell line and MLKL knockout MC38 cell line to perform colony forming assays after radiation with or without pretreated zVAD-FMK.However,knockdown of MLKL did not significantly influence colony formation rate.In vivo experiments were also performed to verify that the MLKL had no effect on the growth or radiosensitivity of allografts.Finally,gene expression profile microarray was performed to detect differentially expressed genes in MLKL-knockout B16 cell.Peli1 gene was found to be downregulated after MLKL knockout in B16 cells.And it was verified by RT-qPCR and Western blot.Based on the above observations,we infer that MLKL protein is involved in radiation-induced cell death,but its specific mechanisms still require further investigation.The heterogeneity in human breast cancer posed a challenge to effective treatments.Better understanding of tumor initiation and development will help to resolve this problem.Current models explaining intratumoral diversity are cancer stem cell,clonal evolution and cancer cell dedifferentiation and reprogramming.Herein,a new model,cancer transmission,was proposed to explain how heterogeneity was formed based on our observation in this study.We found that oncogene-transduced breast cancer cells(MCF10A.NeuT)were capable of transforming normal mammary epithelial cells(MCF10A)when these cells were co-cultured in mix.The transformed MCF10A cells exhibited cancerous properties including enhanced proliferation and migration,loss of apical-basal polarity,and depolarized acini structure associated with epithelial-mesenchymal transition(EMT).Transformed MCF10A cells display distinct EMT characteristics comparing to parental cells.Conditioned medium from oncogene-expressing cells were also sufficient to induce cancerous transformation of normal cells,raising possibilities that secreted factors from cancer cells were involved in the process.We further conducted screening of soluble factors using cytokine array and identified cytokines that had altered expression in MCF10A.NeuT cells.Furthermore,transformed cells are more resistant to radiation treatment than that of MCF10A.NeuT cells.These results extend our understanding of mechanisms how heterogeneity develops in cancer progression and the use of this new model in analyzing clinical samples will hopefully help us to design novel approaches in cancer interevention.