The Effects Of ERK Inhibitors In Esophageal Squamous Carcinoma

Objective:Esophageal squamous carcinoma is one of the most common cancer in Asia,with two types of histology,squamous carcinoma and adenocarcinoma.There are clear differences between different histological types that affect the distribution.In China,over 280,000 people die in esophageal carcinoma every year,and the major is esophageal squamous carcinoma.For now,the treatments of advanced esophageal squamous carcinoma are still based on chemo-radiotherapy.The 5-year survival ratio is less than 30%,while the uncontrolled proliferation and metastasis of the cells might be the reason.The MAPK/ERK is proved to be a key factor for proliferation,metastasis,migration and tumor microenvironment of tumor cells.In esophageal squamous cell carcinoma,it is indirectly proved that MAPK/ERK is assocaited with its proliferation,cell cycle,apoptosis and migration.In addition,a large number of studies have revealed that MAPK/ERK might be a potential target.Many inhibitors that targeting the upper stream molecular of ERK have been developed and proved by FDA.However,obvious resistance of these inhibitors is observed after long-time treatment.The mechanism of resistance is mostly considered to be induced by the re-activation of ERK.Except of this,inhibitors of other targets have been shown to affect tumor progression via the inhibition of MAPK pathway.In esophageal squamous carcinoma,many studies have indirectly implied the efficacy of MAPK/ERK pathway blocking in tumor treatment.But no study directly confirmed that inhibition of ERK activation has good potentiality in esophageal squamous carcinoma treatment.GDC0994 is an ERK inhibitor which shows good effects in colon and pancreatic cancer.The phase I clinical trial for GDC0994 also demonstrated its good tolerability.Overall,we proposed that ERK was a potential target and closely related to the survival time of esophageal squamous carcinoma patients.In this study,we detected the expression of p-ERK by immunohistochemistry,and further performed an analysis to explore the relationship between p-ERK and survival time of esophageal squamous carcinoma patients.In addition,we explored the efficacy and mechanism of ERK inhibitors（GDC0994 and WXFL10970133）against esophageal squamous carcinoma in vivo and in vitro.Materials and Methods:Firstly,tissue microarray and clinical information of 114 patients with esophageal squamous carcinoma were collected for the Log-Rank test and Cox regression assay.The survival time of the different levels was shown in the Kaplan-Meier curve,and the differences between the two groups were compared by Log-Rank test.At the same time,the univariate or multivariate Cox regression method was used to analyze the relationship among p-ERK,age,gender,pathological stage,clinical stage,and the prognosis of patients with esophageal squamous carcinoma.Then,the value of ERK was evaluated in esophageal squamous carcinoma through in vitro and in vivo experiments.In vitro,the effects of ERK inhibitors（GDC0994/WXFL10970133）on the proliferation and migration of esophageal squamous cells was investigated by CCK-8,colony formation assay,and scratching test.Furthermore,flow cytometry analysis was carried out to evaluate the effects on the apoptosis and cell cycle distribution.In vivo,we explored the anti-tumor effects of ERK inhibitors（GDC0994/WXFL10970133）by establishing the xenograft tumor models of esophageal squamous carcinoma（KYSE150/EC9706）.The bio-safety of ERK inhibitors（GDC0994/WXFL10970133）in BALB/c nude mice was evaluated by monitoring the changes in blood biochemistry,H&E staining of the key organs,and bodyweight of mice.The expression of Ki67 and cleaved-caspase 3 was detected by immunohistochemical staining.Finally,the changes of M2 macrophage and MDSCs were analyzed by flow cytometry.Results:The high expression of p-ERK was significantly related to the poor prognosis of patients（p=0.0013）.Cox regression analysis harbored the same conclusion.(HR_low/high=0.53,p=0.002;HR_low/high=0.309,p<0.001).In addition,the age,gender,p T staging,and pathological staging were related to the prognosis of patients(HR_age=0.534,p=0.022;HR_gender=2.289,p=0.008;HR_{p T staging}=0.394,p=0.009;HRpathological staging=2.289,p=0.004).ERK inhibition（GDC0994）exhibited a good therapeutic effect on esophageal squamous carcinoma.In vitro,GDC0994 significantly inhibited the proliferation of esophageal cancer cells by CCK-8(half inhibitory concentration of 48h/72h,IC50_EC9706=18.73/18.06μM,IC50_KYSE150=31.22/16.81μM).GDC0994 obviously inhibited the colony formation of esophageal squamous carcinoma cells（KYSE70,KYSE150,and EC9706）.Further studies revealed that GDC0994 inhibited the proliferation of KYSE150 and EC9706 cells by inducing G2/M phase arrest and apoptosis,and the cell cycle arrest was companied with CDK1/Cyclin B down-regulation and p21/Cyclin E up-regulation.In addition,ERK inhibition（GDC0994）significantly inhibited the migration of KYSE150 and EC9706 cells（p<0.001）.In vivo,GDC0994 significantly inhibited the growth of EC9706/KYSE150 xenograft tumors.In the EC9706 tumor model,the tumor weight of the vehicle and GDC0994（50 mg/kg）was 0.78±0.28 g and 0.25±0.16 g,respectively（p<0.05）.The tumor volume of the vehicleand GDC0994（50 mg/kg）was 1245.05±357.29 mm³ and 315.69±119.89 mm³,respectively（p<0.001）.In the KYSE150 xenograft tumor model,the tumor weight of vehicle and GDC0994（50 mg/kg）was 0.74±0.11 g and 0.29±0.05 g,respectively（p<0.001）.The tumor volume of vehicle and GDC0994（50 mg/kg）was 613.95±133.61 mm³ and 236.48±152.89 mm³,respectively（p<0.001）.The new developed ERK inhibitor,WXFL10970133,also exhibited a good therapeutic effect on esophageal squamous carcinoma.In vitro,WXFL10970133was more efficacy than GDC0994 on the proliferation of esophageal cancer cells.In addition,it obviously inhibited the colony formation of EC9706 and KYSE150 cells.The flow cytometry results showed that WXFL10970133 induced significant G2/M phase arrest and apoptosis of EC9706 and KYSE150 cells with CDK1/Cyclin B down-regulation and p21/Cyclin E up-regulation.WXFL10970133 also significantly inhibited the migration of EC9706 and KYSE150 cells.In the EC9706 xenograft tumor model,the tumor weight of vehicle was 0.73±0.14 g,GDC0994（50 mg/kg）was 0.45±0.07 g（p<0.001）,and WXFL10970133（15 mg/kg）was 0.38±0.05 g（p<0.001）,the tumor volume of vehicle was 1069.70±177.66 mm³,GDC0994（50mg/kg）was 658.02±120.68 mm³（p<0.001）,and WXFL10970133（15 mg/kg）was408.88±56.83 mm³（p<0.001）.Body weight monitoring showed that a slight weight loss was shown in both WXFL10970133 and GDC0994.No significant differences was declared by biochemical indexes among vehicle,WXFL10970133,and GDC0994.A few inflammatory cells were infiltrated in the lungs of WXFL10970133 treated mice.The high expression of Ki67 and low expression of cleaved-caspase 3 were observed in WXFL10970133 and GDC0994.In the KYSE150 subcutaneous tumor model,the tumor weight of vehicle was 0.25±0.09g,GDC0994（50 mg/kg）was 0.06±0.06 g（p<0.01）,and WXFL10970133（15mg/kg）was 0.09±0.07 g（p<0.05）.The tumor volume of vehicle was 452.46±53.72 mm³,GDC0994（50 mg/kg）was 117.75±83.85 mm³（p<0.001）,and WXFL10970133（15 mg/kg）was 217.03±158.68 mm³（p<0.001）.No significant differences were observed among vehicle,WXFL10970133,and GDC0994 through weight monitoring and biochemical indexs.Obvious inflammation was also observed in the lung of WXFL10970133 treated mice.The high expression of Ki67and low expression of cleaved-caspase 3 were also shown in WXFL10970133 and GDC0994.M2 macrophages and MDSCs of WXFL10970133 and GDC0994 were significantly reduced.Exactly,the M2 macrophages of vehicle was 10.47±4.04%,GDC0994（50 mg/kg）was 1.39±0.89%（p<0.05）,and WXFL10970133（15mg/kg）was 1.95±0.72%（p<0.01）.The MDSCs of vehicle was 22.8±5.11%,GDC0994（50 mg/kg）was 10.35±2.97%（p<0.05）,and WXFL10970133（15mg/kg）was 11.92±2.77%（p<0.01）.Conclusion:Our study found that significant differences were observed on the survival time among the different groups that had different levels of p-ERK expression.In addition,ERK might be a potential target for esophageal squamous carcinoma.In vitro,ERK inhibitors（GDC0994/WXFL10970133）inhibited the proliferation of esophageal squamous carcinoma cells through G2/M arrest and apoptosis.Meanwhile,ERK inhibitors（GDC0994/WXFL10970133）inhibited the migration of EC9706 and KYSE150 cells.In vivo,ERK inhibitors（GDC0994/WXFL10970133）significantly inhibited the growth of EC9706 and KYSE150 xenograft tumors with good biological safety.It also revealed that GDC0994 and WXFL10970133decreased the expression of Ki67,and increased the expression of cleaved-caspase 3,and decreased the M2 macrophages and MDSCs infiltrations in tumor microenvironment.In conclusion,our research not only proved that p-ERK might be a good prognostic marker for esophageal squamous carcinoma,and developed a new ERK inhibitor,WXFL10970133,and explored the efficacy and mechanism of ERK targeting in esophageal squamous carcinoma.