Targeting Endoplasmic Reticulum Associated Degradation(ERAD)Pathway Induces Endoplasmic Reticulum Stress And Enhances The Efficacy Of Radiation Therapy In Esophageal Squamous Cell Carcinoma

ObjectiveEsophageal squamous cell carcinoma(ESCC)arises from the epithelial cells and is one of the most common gastrointestinal malignancies in China.Due to atypical clinical symptoms,the majority of patients are at an advanced stage when diagnosed and without indications for surgical resection.Chemoradiotherapy is a standard treatment for locally advanced ESCC.However,there are still a number of patients who inevitably have tumor recurrence and/or metastasis,and the prognosis is really unfavorable.Resistance to radiation therapy(RT)leads to treatment failure and result in poor outcomes in such patients.In order to improve the efficacy of RT,it is crucial to select biomarkers indicative of radiation resistance,and applying specific small molecule inhibitors to target the signaling pathways related with radio-resistance.In addition,tumor immune escape is another important reason in the occurrence and progression of ESCC.RT induces immunogenic cell death several types of tumors.Further enhancing the immunogenicity of esophageal cancer cells is able to stimulate immune adjuvant effects,engaging the adaptive arms of the immune system and functioning like an in-situ vaccine,generating tumor specific T cells.This is another way to improve the efficacy of RT.In the present study,plasma samples prior and after RT from locally advanced ESCC patients who received RT were obtained,Mass Spectrometry(MS)was used to screen proteins that involved in radiation resistance,and verification was performed.Then,specific small molecule inhibitors were administrated to observe their effect in improving radio-sensitivity,and the related mechanism were detailed;meanwhile,the efficacy of small molecule inhibitors and RT in inducing immunogenic cell death of ESCC were examined;moreover,we evaluated the relationship between radiation resistance biomarkers and the prognosis of patients with locally advanced ESCC who underwent RT using tissue samples.Consequently,we select potential targets and elaborate the molecular mechanisms of radiation resistance in ESCC,this will increasingly affect the development of novelty radio-sensitivity strategies in the era of precision radiotherapy.Methods1.Serum was collected prior to treatment,during RT,and after RT.Plasma samples from patients of locally advanced ESCC who received RT were analyzed by MS.Then,bioinformatic analysis was performed.Moreover,selected proteins were analyzed by enzyme-linked immunosorbent assay(ELISA).2.ESCC cell lines with radiation resistance were selected and treated with endoplasmic reticulum-associated degradation(ERAD)inhibitor alone or combined with radiotherapy.Cell proliferation,colony formation,cell cycle,cell death,and radiation resistance signaling pathways were evaluated.3.ESCC cells were treated by irradiation,or in combination with ERAD inhibitor,immunogenic cell death biomarkers including calreticulin translocated to cell surface,exocellular released adenosine triphosphate(ATP)and High-mobility Group Box 1(HMGB1)were detected and analyzed.4.Patients with newly diagnosed locally advanced ESCC who were treated with RT were analyzed.Immunohistochemistry was used to detect the expression of Binding immunoglobulin protein(BIP).The correlation between overall survival and BIP was investigated.Results1.During the course of RT,both the complement pathway and endoplasmic reticulum stress involved in the regulation of radio-sensitivity;the levels of proteins in these pathways were significantly changed(p<0.05).The endoplasmic reticulum stress signaling pathway,including unfolded protein response(UPR)and ERAD pathway,were extensively associated with radiation resistance.Being vitrificated,the levels of BIP in serum were significantly elevated in patients with poor response.2.ESCC cells treated with VCP inhibitor and/or RT showed attenuated cell proliferation and colony formation and enhanced apoptosis.Moderate levels of endoplasmic reticulum stress resulted in radiation resistance.The administration of NMS-873 inhibited the ERAD pathway and enhanced cancer cell radio-sensitivity.Further investigation showed this combined strategy activated the endoplasmic reticulum stress signaling involved in UPR,and induced C/EBP-homologous protein(CHOP)mediated tumor cell apoptosis.Furthermore,ERAD inhibitor significantly increasing radiation induced G2/M phase arrest.3.RT induced immunogenic cell death in ESCC cells,especially with moderate doses;ERAD inhibitors combined with RT(?6Gy)significantly activated the autophagy pathway and triggered extracellular release of ATP;this combined strategy also induced strong endoplasmic reticulum stress and increased calreticulin cell membranes translocation;furthermore,the combination of RT and ERAD inhibitor promoted tumor cell necrosis and upregulated extracellular release of HMGB1;in short,ERAD inhibitors have a synergistic effect on immunogenic cell death induced by RT.4.Clinical analysis revealed a significant survival benefit in the low BIP expression group in patients of locally advanced ESCC who received RT(p<0.05).BIP was an independent factor in such patient population.Conclusions1.Based on plasma proteomics of ESCC patients treated with RT,it suggested that proteins related to endoplasmic reticulum stress were involved in the regulation of radio-sensitivity of esophageal squamous cell carcinoma.2.Moderate endoplasmic reticulum stress induced by RT was one of the reasons for radiation resistance;targeting ERAD pathway inhibited cancer cell proliferation and improved the radio-sensitivity of ESCC cells.3.ERAD inhibitor was able to increase the incidence of radiation induced immunogenic cell death in ESCC cells.4.In esophageal cancer patients treated with RT,BIP expression was associated with overall survival and could be served as an independent prognostic factor.