Studies Of K67 Increasing The Radiosensitivity Of Esophageal Squamous Cell Carcinoma Via Regulating P62-Keap1-Nrf2 Pathway

Esophageal cancer is one of the malignancies with high incidences and mortality rates all around the world.And in China,esophageal squamous cell carcinoma(ESCC)is the main pathological type.Because the symptoms of early ESCC lack specificities,most patients are in locally advanced or metastatic stages when they are initially diagnosed.Concurrent chemoradiotherapy(CCRT)is the standard therapy for inoperable locally advanced ESCC patients.Although CCRT shows considerable response rate,most patients would suffer recurrence or progression within three years.This means that there are a group of radioresistant tumor cells surviving from the radiotherapy and leading to treatment failure as well as poor prognosis.The molecular mechanisms underlying the radioresistance of ESCC are crucial.The targeted therapy to increase the radiosensitivity of ESCC might be a breakthrough point for the treatment of ESCC.Radiotherapy can induce DNA double strand breaks(DSBs)and dysfunction of multiple organelles directly.More importantly,the ionizing radiation beams act on H2O and produce abundant reactive oxygen species(ROS).ROS plays a critical role in anti-tumor effect of radiotherapy by oxidizing DNA,protein,lipid and so forth.Recent studies have revealed that the aberrant clearance of ROS is one of the mechanisms of radioresistance of ESCC.Besides,many radiosensitizers of ESCC increase the radiosensitivity via enhancing ROS production.As an important antioxidative pathway,nuclear factor erythroid 2 related factor 2(Nrf2)-antioxidant response element(ARE)pathway has been widely studied in the mechanisms of oncogenesis and anticancer agent tolerance.Nrf2 is an essential transcription factor and its target genes encode multiple antioxidative proteins and detoxifying enzymes.Moreover,constitutive activation of Nrf2 involves in radioresistance of multiple carcinomas including esophageal cancer.The expression of Nrf2 is mainly regulated by Kelch-like ECH-associated protein 1(Keapl).Under normal condition,Keapl combines with Nrf2 in the cytoplasm,which leads to the degradation of Nrf2 by ubiquitination.But the conformation of Keapl-Nrf2 complex would change under oxidative or ionized stress and Nrf2 escapes from the arrest of Keapl.The released Nrf2 enters nucleus and transcribes downstream antioxidative and detoxifying genes to protect the cells from oxidative or metabolic damage.The excessive activation of Keapl-Nrf2 pathway is common in tumor cells and p62,a kind of autophagy adaptor,has been reported to involve in this progression.Phosphorylated p62(p-p62)has a similar domain with Nrf2 and shows significantly higher affinity with Keap1.During selective autophagy,mTORC1 phosphorylates p62 at Ser349 and p-p62 competitively combines with Keap1,which induces the release and activation of Nrf2.Besides,the activated Nrf2 promotes the transcription of p62 to form a positive loop.Plenty of researches have demonstrated that p62-Keapl-Nrf2 pathway not only involves in the progression of oncogenesis but also mediates the anticancer agent tolerance.Certain small molecular compounds have been shown to aim at this pathway,such as K67.K67 was reported to increase the sensitivity of hepatocellular carcinoma to cisplatin and sorafenib by specifically blocking the interaction of p-p62 and Keapl.However,the role of p62-Keapl-Nrf2 pathway in ESCC is still unclear,especially in the radioresistance of ESCC.Our previous study proved that overexpression of p-p62 mediated radioresistance of ESCC and p62-Keapl-Nrf2 pathway might become a new target for radiotherapy sensitization of ESCC,Therefore,this study devoted to clarify the role of p62-Keapl-Nrf2 pathway in the response of ESCC patients to radiotherapy and explore the function and mechanism of K67 in ESCC as a radiosensitizer.Part I:The expression of p-p62 and nuclear Nrf2 in ESCC and their impact on the prognosisObjective:This part aimed to clarify the expression and correlation of p-p62 and nuclear Nrf2 in the tissue samples of ESCC.And we also investigated the relationships between p-p62 or nuclear Nrf2 and patients' clinical characteristics,responses to CCRT and long-term survival.Methods:This study included 164 cases of inoperable locally advanced ESCC(?-?stage,UICC2002 cTNM).We collected the tumor tissue samples by esophageal endoscopy.All the patients received CCRT.Immunohistochemical staining was used to detect the expression of p-p62 and nuclear Nrf2.The results(Q score)were analyzed by 2 pathologists independently.And the median of Q scores was considered as the cut-off value.The clinical characteristics were collected from patients' medical records.The treatment response was determined by the Response Evaluation Criteria in Solid Tumors.Progression-free survival(PFS)was defined as the time between the start of active treatment and date of disease progression.Overall survival(OS)was defined as the time between the start of active treatment and date of death.The relationships between p-p62 or nuclear Nrf2 and patients'clinical characteristics and treatment response were analyzed by X2 test.The correlation between the expression of p-p62 and nuclear Nrf2 were analyzed by Spearman test.PFS and OS curves were constructed by the Kaplan-Meier method.Differences in survival were compared by using the log-rank test for univariate analysis.And Cox-proportional hazards regression was performed for multivariate analysis.Results:The expression levels of p-p62 and nuclear Nrf2 were fluctuant among patients.There was no significant relationship between p-p62 or nuclear Nrf2 and patients'clinical characteristics,such as age,sex,PS score,smoking history,location of primary tumor and clinical TNM stage.Compared to patients with low expression of p-p62,patients with high expression of p-p62 showed lower objective response rate(ORR).Similarly,patients with high expression of nuclear Nrf2 exhibited lower ORR compared to those with low expression of nuclear Nrf2.The expression of p-p62 was positively correlated with that of nuclear Nrf2.Moreover,the correlation coefficient between them was higher among patients showing no response to CCRT.Univariate analysis revealed that higher expression of p-p62 or nuclear Nrf2 was significantly associated with poorer PFS and OS.Besides,PS score,N stage and treatment response were also associated with PFS.And the factors associated with OS also included sex,PS score,location of primary tumor and treatment response.Multivariate analysis indicated that the expression of nuclear Nrf2 and treatment response were independent prognostic factors for PFS.And sex,treatment response,expression of p-p62 and nuclear Nrf2 were independent prognostic factors for OS.Conclusion:The expression of p-p62 is positively correlated with nuclear Nrf2,especially in ESCC patients showing no response to CCRT.Higher expression of p-p62 and nuclear Nrf2 are associated with lower ORR as well as poorer prognosis,which indicates that p62-Keapl-Nrf2 pathway might play an essential role in the radioresistance of ESCC.Part II:The role of K67 in the radiosensitization of ESCC and its mechanismObjective:Based on the results of previous part,we hypothesized that the small molecular compound K67 which blocked the interaction of p-p62 and Keapl could increase the radiosensitivity of ESCC.This part mainly explored the role of K67 in the radiosensitization of ESCC and its mechanism.Methods:Western blot was used to detect the changes of expression of p62,p-p62 and Nrf2 in ESCC cell lines after radiation.The cytotoxicity of K67 was determined by CCK-8 assay.The ESCC cell lines were treated with different dose of radiation and K67.The ability of colony formation of cells was detected by colony formation assay.The effect of K67 on cell apoptosis was investigated by flow cytometry and western blot was used to detect the expression of cleaved-PARP.We utilized DCFH-DA fluorescence probe to detect the production of ROS.And we stained y-H2AX via immunofluorescence technique in order to reflect the DSBs.The cell cycle distribution was determined by PI staining.To further investigate the mechanism of K67,immunofluorescence and western blot were applied to detect the changes of location and expression level of Nrf2.The impact of K67 on the interaction of Keap1 with p-p62 and Nrf2 in ESCC cells was determined by co-immunoprecipitation(CoIP).Results:Among four kinds of ESCC cell lines,the expression of p-p62 and nuclear Nrf2 were upregulated after radiation only in TE-1 cells,which implied that there might be p62-Keapl-Nrf2 axis in TE-1 cells and made TE-1 cells an ideal cell lines for research.And we chose KYSE30 cell lines as control cells.The results of CCK-8 assay indicated that K67 showed no cytotoxicity to ESCC cells.In TE-1 cells,K67 could inhibit the ability of colony formation after radiation and promote the cell apoptosis induced by radiation.And the expression of cleaved-PARP was elevated in radiation plus K67 group compared to radiation alone group.Furthermore,K67 significantly increased the production of ROS and DSBs in TE-1 cells after radiation.However,K67 showed no impact on KYSE30 cells.The cell cycle arrest induced by radiation was not affected by K67 both in TE-1 cells and KYSE30 cells.The results of immunofluorescence staining and western blot proved that K67 could downregulate the expression level of nuclear Nrf2 induced by radiation in TE-1 cells but showed no impact on KYSE30 cells.The results of CoIP revealed that K67 dramatically inhibited the interaction between p-p62 and Keap1.Meanwhile,it promoted the interaction between Nrf2 and Keapl because of the elevated level of p62-free Keapl.Conclusion:K67 increases the radiosensitivity of ESCC and this effect depends on the regulation of p62-Keapl-Nrf2 pathway.More specifically,K67 overcomes the radioresistance of ESCC via directly blocking the interaction of Keapl and p-p62 and promoting the interaction between Nrf2 and Keapl.