Mechanism Of Metabolic Protein 12-lipoxygenase Promoting Malignant Phenotype Of ESCC And Its Role In Radioresistance

Esophageal carcinoma(EC)is one of the most common malignant tumors in the world.Esophageal squamous cell carcinoma(ESCC)is the most common type of EC.Most ESCC patients were in the middle and advanced stage at the time of diagnosis,and the 5-year survival rate is only 15%-25%.For advanced ESCC,radiotherapy is one of the main treatment methods,but some patients fail radiotherapy due to low response to radiation.Even patients with the same stage and pathological type have different response to treatment.Even patients with the same pathological type and at the same stage have different response to treatment.The heterogeneity of tumor gene expression may be the main reason for the difference of treatment response.In conclusion,exploring biomarkers for early diagnosis and treatment effect prediction of ESCC,developing targeted treatment strategies and improving patient prognosis are urgent problems to be solved.Radioresistance is an important factor for local recurrence and distant metastasis of ESCC.Radioresistance is mainly produced by the following mechanisms:tumor cell reproliferation,radiation damage repair,adaptive angiogenesis and activation of radiosensitivity related signal pathways.Finding potential radiation sensitization targets and designing reasonable sensitizers are important strategies to deal with radiation resistance.Although the research of radiation sensitization has made some progress,due to the lack of radiation sensitizers for specific targets,the beneficiaries can not be effectively screened,and the current situation of clinical radiation resistance can not be significantly improved.Reprogramming of cell energy metabolism is one of the basic characteristics of tumor.Cell metabolism is the key pathway driving tumor survival,progression,drug resistance,immune escape and recurrence.Many studies have confirmed that targeting cell metabolism can improve the therapeutic response of treatment tolerant tumors.It is found that radiation irradiation of tumor tissue can not only cause the adaptive changes of TME,the production of a large number of cytokines and the changes of related signal pathways,but also regulate the metabolism of tumor cells.It is considered to be one of the potential mechanisms of tumor radiation resistance,but the specific mechanism is not clear.AA is a long-chain polyunsaturated fatty acid widely distributed in mammalian cell membrane.Lipoxygenase(LOX)pathway is the main pathway of AA metabolism.Human LOX mainly has three subtypes:5-LOX,12-LOX and 15-LOX,which can catalyze AA to form 5-hydroxyeicosatetraenoic acid(HETE),12-HETE and 15-HETE,respectively,and further convert them into leukotrienes and lipoxygenates.LOX and its metabolites are involved in a variety of physiological and pathological processes,including inflammation and tumors.In recent years,12-LOX and 12-HETE have attracted more and more attention.Through TCGA database retrieval,we found that the expression of 12-LOX in ESCC tumor tissue is significantly higher than that adjacent to cancer.However,it is still unclear what role 12-LOX plays in the development and treatment of ESCC,and whether it can be used as a therapeutic target.Based on the close relationship between cell metabolism and tumor progression and radiotherapy reactivity,this topic focused on AA metabolism related protein 12-LOX,to explore whether it can be used as a potential prognosis and efficacy prediction target of ESCC,and further explore a new strategy of radiosensitization.This topic is divided into two parts to study the role of 12-LOX in the progress of ESCC and radiotherapy,which will provide new insights into the impact of tumor cell metabolism on ESCC and provide new efficacy prediction and sensitization targets for ESCC patients.Part Ⅰ 12-LOX promotes the malignant phenotype of ESCC and its mechanismObjectives1.To explore the role and mechanism of 12-LOX in the malignant phenotype of ESCC;2.To determine whether 12-LOX can be used as a prognostic indicator of ESCC;3.To explore the effectiveness of RAD001 in inhibiting the malignant phenotype of ESCC.Methods1.Database retrieval:TCGA database obtained the expression of 5/12/15-LOX in pan cancer and 12-LOX in EC,the relationship between 12-LOX expression and clinical features in ESCC,and survival curve.2.Clinical data and sample collection:the primary tumor tissues of ESCC patients were collected and the expression of 12-LOX was evaluated by IHC;Chi-square test was used to detect the relationship between 12-LOX expression and clinical features,Kaplan-Meier method and log-rank analysis were used to draw the survival curve and evaluate the effect of 12-LOX expression on OS and PFS,ROC curve and AUC were used to evaluate the predictive value of 12-LOX for OS and PFS.Univariate and multivariate analyses based on Cox proportional hazards models were used to assess whether 12-LOX expression could be used as a prognostic factor for ESCC patients.3.Construction of 12-LOX overexpression stable transformation strain with lentiviral vector:ESCC cells were infected with 12-LOX overexpression vector or control vector,and screened with puromycin to establish a cell line stably expressing 12-LOX.4.Detect the effect of 12-LOX on the proliferation of ESCC cells:EdU,Colony formation test and CCK-8 were used to detect the cell proliferation ability of each group.5.Detect the effect of 12-LOX on angiogenesis:PCR Array was used to detect the expression of angiogenesis related genes in each group;ELISA and Western blot were used to verify the secretion and protein expression of related factors;The migration ability of HUVECs was detected by transwell and Wound healing experiments,and the tube forming ability was detected by tube formation test.6.Detect the mechanism of 12-LOX promoting the malignant phenotype of ESCC:PhosPho-Kinase Array was used to analyze the expression difference of phosphokinase in each group and screen the pathway;Western blot and IHC staining verified the activation of the pathway.7.Establish subcutaneous transplanted tumor model of ESCC nude mice:construct subcutaneous transplanted tumor model of LV-Ctrl or LV-12-LOX nude mice,the tumor volume was calculated and the growth curve was drawn to evaluate the effect of 12-LOX on tumor growth;Western blot and IHC verified the activation of the pathway;IF was used to detect the co-expression of 12-LOX and CD31,and to evaluate the effect of 12-LOX on tumor angiogenesis.Results1.12-LOX is highly expressed in ESCC and is related to poor prognosis:TCGA database shows that the expression of 12-LOX in ESCC is significantly higher than that in normal tissues and EA.The expression level of 12-LOX is strongly correlated with histological type,T stage,N stage and histological grade.There is a significant difference in the survival time of patients in 12-LOX high and low expression groups.IHC staining results of tumor tissue sections of ESCC patients showed that there were differences in the expression of 12-LOX between patients,including 112 cases in the high expression group and 41 cases in the low expression group.The expression level of 12-LOX was significantly correlated with T stage and N stage.Patients in the low expression group had better OS and PFS.12-LOX can be used as a prognostic factor for ESCC patients.2.12-LOX promotes the proliferation of ESCC cells in vitro:compared with the control group,the cell proliferation ability of 12-LOX overexpression group is stronger,12-LOX specific inhibitor baicalein can inhibit the proliferation ability of cells.3.12-LOX promotes tumor angiogenesis in ESCC in vitro:compared with the control group,the secretion of angiogenesis promoting factors such as VEGF,IL-6 and IL-8 in 12-LOX overexpression group was higher.Compared with the control group,the supernatant of tumor cells in the 12-LOX overexpression group enhanced the migration and tube formation ability of HUVECs.4.PI3K/AKT/mTOR pathway was activated in 12-LOX overexpression group:significant pathway activation was detected in 12-LOX overexpression ESCC cells,12-LOX overexpression xenografts and ESCC patients.5.12-LOX plays pro-tumor effect by activating PI3K/AKT/mTOR pathway:the proliferation ability of ESCC cells and the secretion of VEGF in the PI3K inhibitor LY294002 stimulation group were significantly lower than those in the 12-LOX overexpression group.LY294002 can reverse the tumor promoting effect of 12-LOX.6.RAD001 inhibited the proliferation of ESCC cells and angiogenesis promoted by 12-LOX in vitro:the proliferation ability of ESCC cells stimulated by mTOR inhibitor RAD001 was significantly lower than that of 12-LOX overexpression group.RAD001 attenuated the migration ability of HUVECs.Compared with the 12-LOX overexpression group,the tube formation ability of HUVECs in the supernatant of in RAD001+12-LOX overexpression tumor cells group decreased.7.12-LOX plays a tumor-promoting role in vivo:compared with the control group,the growth speed of xenografts in 12-LOX overexpression group is faster,and the volume and weight are larger.The expression of CD31 in 12-LOX overexpression group was higher than that in control group.12-LOX promoted the growth and tumor angiogenesis of xenografts in vivo.8.RAD001 inhibits the tumor promoting effect of 12-LOX in vivo:the growth speed and volume of transplanted tumor in RAD001+12-LOX overexpression group are lower than those in 12-LOX overexpression group.The expression of CD31 in RAD001 intervention group is lower than that in 12-LOX overexpression group.RAD001 inhibits the proliferation of ESCC cells and tumor angiogenesis induced by 12-LOX in vivo.Conclusions1.12-LOX is highly expressed in ESCC which is related to the poor prognosis of ESCC patients,and may be used as a prognostic marker of ESCC patients;2.12-LOX can promote the proliferation of ESCC cells and tumor angiogenesis by activating mTOR pathway;3.12-LOX is a potential therapeutic target for ESCC patients.RAD001 may be an effective therapeutic strategy for patients with high expression of 12-LOX.Part Ⅱ Role of 12-LOX in radioresistance of ESCCObjectives1.To explore the role of 12-LOX in ESCC radioresistance;2.To determine whether 12-LOX can be used as a predictor of efficacy in ESCC patients treated with radiotherapy alone;3.To explore the the effectiveness and potential mechanism of Baicalein as a radiosensitizer.Methods1.Establishment of radioresistant cell line(rEca109):when Eca109 cells grow to 30%-50%fusion degree,they are irradiated with 2Gy X-ray.Subculture when the density reaches 80%-100%.When the cells were in good condition,the next irradiation will be carried out until the cumulative dose reaches 60Gy.The surviving cell clones were amplified after 10Gy high-dose irradiation.2.Verify radioresistant rEca109:rEca109 cells were irradiated with a gradient dose of 0/2/4/6/8Gy.Draw the cell survival fraction curve based on colony formation experiment and calculate D0 and Dq.The xenografts models of Eca109 and rEca109 were constructed.The transplanted tumor volume was measured and calculated,and the time-volume curve was drawn.The growth rate and volume of the xenografts after irradiation were compared between the two groups.3.Detect the effects of 12-LOX and 12-HETE on the radiosensitivity of ESCC:for the cells in each treatment group,clone formation experiment was used to evaluate the cell viability,and flow cytometry was used to detect the apoptosis and evaluate the sensitivity of cells in each group to X-ray.4.Explore the potential mechanism of 12-LOX affecting radiosensitivity:TCGA database was used to search the expression correlation between 12-LOX and key genes of DDR pathway.DNA damage PCR Array was used to detect the difference in the expression of key genes in the DDR pathway between the 12-LOX overexpression group and the control group.5.Construction of radioresistant cell line stably expressing 12-LOX shRNA:the plasmid expressing 12-LOX shRNA was transfected into rEca109 cells,and the stable transformation strain expressing sh-12-LOX was obtained by puromycin screening.6.Detect the effect of knockdown 12-LOX on the radiosensitivity of radioresistant cell lines:for the cells in each treatment group,the cell viability was evaluated by colony formation assay and the apoptosis was detected by flow cytometry.7.Detect the effect of baicalein on the radiosensitivity of ESCC:the Eca109 cells were prestimulated with baicalein before irradiation.The survival and apoptosis of the cells were detected by colony formation and flow cytometry.8.To detect the effects of Baicalein combined with radiotherapy on DNA damage:Eca109 cells were pre-stimulated with baicalein before irradiation.The expression of y-H2AX in each group was detected by IF and compare the degree of DNA damage.9.Explore the potential mechanism of baicalein affecting the radiosensitivity of ESCC:CSCs PCR array was used to detected the expression of CSCs related genes between two groups,and detected the effect of baicalein on ESCC tumor stem cells.10.Data collection of clinical specimens:specimens and clinical case data of patients with advanced ESCC were collected.The expression of 12-LOX were evaluated by IHC.The P value was calculated by chi-square test to detect the correlation between the curative effect and the expression of 12-LOX;Kaplan-Meier curves were used to evaluate the effects of 12-LOX on OS and PFS.Results1.Establishment of radiation resistant cell line reca109:under gradient dose X-ray irradiation,the survival fraction,D0 and Dq of rEca109 were significantly higher than those of Eca109.The growth speed and volume of transplanted tumor in rEca 109 irradiation group were significantly higher than those in Eca109 irradiation group.2.12-LOX and 12-HETE increased the radioresistance of ESCC cells:under 6Gy irradiation,the number of colony in 12-LOX overexpression and 12-HETE stimulation group was significantly higher than that in the control group and the rate of annexin V positive cells was significantly lower than that in the control group.12-LOX and 12-HETE can promote the survival of ESCC cells under irradiation and participate in radioresistance.3.Down-regulation of 12-LOX increased the radiosensitivity of rEca 109:under 6Gy irradiation,the number of colony in sh-12-LOX-rEca109 group was significantly lower than that in rEca109 irradiation group,and the Annexin V+cell rate was significantly higher than that in rEca109 irradiation group.These results indicated that knocking down 12-LOX could weaken the resistance of rEca109 to X-ray.4.12-LOX was related to the expression of a variety of DDR genes:TCGA database retrieval results show that 12-LOX is related to the expression of a variety of DDR key genes,including DNA damage repair related genes,cell cycle related genes,apoptosis related genes and ATM/ATR pathway related genes.5.Overexpression of 12-LOX leaded to changes in DDR gene expression:The expression of multiple DDR-related genes in 12-LOX overexpressed ESCC cells changed.The mRNA of RAD9A,CDC25A,BBC3,XRCC3,GADD45A and other genes increased significantly,while RAD1,CDC25C,ATRX and other genes decreased significantly.These results indicated that 12-LOX significantly affected the expression of DDR-related genes in ESCC cells.6.Baicalein combined with radiotherapy reduced the survival of ESCC cells:the rate of annexin V+cells in the radiation+baicalein group was significantly higher than that in the irradiation group,and the number of colony was significantly less than that in the irradiation group.The above results suggest that baicalein had a radiosensitization effect on ESCC.7.Baicalein combined with radiotherapy increased DNA damage in ESCC cells:IF staining showed that the expression of y-H2AX was higher and the degree of DNA damage was higher in the radiation combined with baicalein group,suggested that baicalein can increase DNA damage caused by X-ray.8.Baicalein affected the expression of CSCs-related genes in rEca109:the expression of multiple CSCs-related genes changed after baicalein stimulation.The differently expressed genes were mainly enriched in Notch pathway,suggesting that baicalein may affect the stem cell characteristics of rEca109 through Notch pathway.9.12-LOX affected the efficacy and survival of ESCC patients treated with radiotherapy alone:the DCR of 12-LOX low expression group was significantly higher and patients with low 12-LOX expression had better OS and PFS.Conclusions1.X-ray irradiation promoted the expression of 12-LOX,and 12-LOX was related to radioresistance of ESCC cells;2.12-LOX is related to the radiotherapy response of ESCC patients treated with radiotherapy alone and may be a potential radiosensitization target;3.Baicalein combined with radiotherapy can increase the DNA damage of ESCC cells caused by radiation and may be used as a radiosensitizer.