Selective Autophagy Adaptor Protein P62 Impacts Radiation Sensitivity Of Esophageal Squamous Cell Carcinoma Through Keap1/Nrf2 Pathway

Esophageal squamous cell carcinoma(ESCC)is one of the most common malignancies in China,causing over 200,000 deaths countrywide annually.For patients with locally advanced ESCC,definitive chemoradiotherapy(CRT)was performed as an effective treatment and significantly prolonged survival of patients.Unfortunately,although CRT could initially achieve a considerable response rate over 60%,most patients would suffer recurrence within 3 years,which implies the proliferating colonies of cancer cells kept evolving during radiotherapy and a few of them survived the treatment and became the majority.However,the mechanism at molecular level within cells is far from clarified.Reactive oxygen species(ROS)plays a vital role of anticancer effect in radiotherapy.About two-thirds of DNA break damage from radiotherapy is caused indirectly,which was done by the generation of ROS,including HO· and O-2,via ionization of water molecules.Nuclear factor erythroid 2 related factor 2(Nrf2)-antioxidant response element(ARE)pathway specifically scavenges ROS within cells.Thus,it is concluded that the activation of this pathway would result in radiation resistance.Nevertheless,the mechanism underlying the activation of Nrf2/ARE pathway in ESCC remains unclear.With the development of tumor and autophagy,the interaction between them becomes increasingly clear.It has been reported that selective autophagy adaptor p62 involves in regulation of several signal pathways including Nrf2/ARE.Meanwhile,abnormal accumulation of p62 proteins caused by dysfunction of selective autophagy is common in squamous cell carcinoma of head and neck.Therefore,we concluded that elevated level of p62 could be one of reasons leading to abnormal activation of Nrf2/ARE pathway in ESCC,impairing sensitivity to radiotherapy and effect of anti cancer treatment.First section:The expression of Keapl and Nrf2 in ESCC and its impact on radiotherapyObjective:To compare the difference of Keapl and Nrf2 expression between ESCC and normal esophageal mucosae and analysis its impact on radiotherapy.Methods:Nrf2 and Keapl expressions were examined immunohistochemically in ESCC and normal esophageal mucosae biopsy specimens.All the specimens of ESCC were from patients with locally advanced ESCC who underwent CRT.The clinical information was collected retrospectively.Results:(1)Strong staining of nuclear and cytoplasmic Nrf2 and limited or absent Keapl expression was uncommon in normal tissues,whereas frequently observed in ESCC.(2)Negative correlation between Nrf2 and Keapl in normal mucosae disappeared in tumors,might reflecting loss of interaction between them in ESCC.(3)Positive Nrf2 expression in nucleus significantly associated with worse clinical response and poor progression-free survival(PFS)after CRT and was validated as an independent prognostic factor.Second section:Abnormal accumulation of p62 in ESCC could activate Nrf2 and resulted in radiotherapy resistance.Objective:To explore whether p62 could activate Nrf2,causing radiotherapy resistance and study the underlying mechanism.To analysis whether p62 expression in ESCC could impact the effect of CRT is also included.Methods:(1)We established ECA109 cell lines stably transfected by the recombinant vector comprising p62 coding sequence or blank vector.Colony formation assays under different radiation dose was performed to compare radiotherapy sensitivity between different cell lines.(2)Immunofluorescent cytochemical staining and Western blotting were conducted to compare Nrf2 expression level between different cell lines.The fluorescence intensity of ROS molecular probe and rH2AX foci formation in different cell lines after irradiation were also determined in order to analysis whether p62 could influence ROS scavenging and DNA double strain break after irradiation.(3)p62 expressions were examined immunohistochemically in ESCC biopsy specimens.All the specimens of ESCC were from patients with locally advanced ESCC who underwent CRT.The clinical information was collected retrospectively.Results:(1)Compared to ECA 109-vector,clone formation rate of ECA109 p62 after irradiation was significantly higher.(2)High expression of p62 could promote the transportation of Nrf2 to nucleus,thus inducing ROS scavenging and relieving DNA double strain breaks.(3)High expression of p62 was common in ESCC and associated with worse clinical response and poor PFS.Third section:Phosphorylation of S351 in p62 could activate Nrf2 and resulted in radiotherapy resistance.Objective:To compare the affinity between phosphorylated and non-phosphorylated p62 to Keap1.To explore the mechanism in which phosphorylation of p62 result in radiotherapy resistance.To analysis whether p62 phosphorylation in ESCC could impact the effect of CRT is also included.Methods:(1)We established ECA109 cell lines stably transfected by the recombinant vector comprising p62 S351E or S351 mutants.Colony formation assays under different radiation dose was performed to compare radiotherapy sensitivity between different cell lines.(2)Immunofluorescent cytochemical staining and Western blotting were conducted to compare Nrf2 expression level between different cell lines.The fluorescence intensity of ROS molecular probe and rH2AX foci formation in different cell lines after irradiation were also determined in order to analysis whether p62 phosphorylation could influence ROS scavenging and DNA double strain break after irradiation.(3)Co-Immunoprecipitation(Co-IP)was performed to compare the affinity between phosphorylated and non-phosphorylated p62 to Keap1 and their ability to compete with Nrf2.(4)p62 phosphorylation were examined immunohistochemically in ESCC biopsy specimens.All the specimens of ESCC were from patients with locally advanced ESCC who underwent CRT.The clinical information was collected retrospectively.Results:(1)Compared to ECA109 p62-S351A,clone formation rate of EC A109 p62-S351E after irradiation was significantly higher.(2)Phosphorylation of p62 could promote the transportation of Nrf2 to nucleus,thus inducing ROS scavenging and relieving DNA double strain breaks.(3)p62-S351E possesses the strongest affinity to Keapl,followed by wild type p62 and p62-S351A.(4)High phosphorylation of p62 was not uncommon in ESCC and associated with worse clinical response and poor PFS.