Investigation Of The Relationship Between Phosphorylated ERK And Sorafenib Sensitivity In Hepatocellular Carcinoma And Underlying Mechanisms

BackgroundThe mortality rate of liver cancer is the second cause of cancer-related death, only secondary to lung cancer. With insidious onset, high recurrence rate, strong metastasis and common drug resistance, the prognostic of liver cancer is poor. In recent years, increased efforts were funneled to molecular targeted drugs in cancer therapy. Sorafenib was the first such drug approved by the FDA for the treatment of advanced liver cancer. However, in clinical application, the sensitivity and effectiveness of sorafenib has been confronted with great challenge. Only a minor population of HCC patients are sensitive to sorafenib and the majority of patients with liver cancer are resistant to sorafenib or even progress faster after sorafenib treatment. Further study is urgently needed to explore the molecular mechanisms of sorafenib resistance in treating of liver cancer.During sorafenib treatment, many targets are likely candidates as sensitivity-related biomarkers, including VEGFA. VEGF. aB-Crystallin, FGF3/FGF4, Nanog. EGFR/HER-3. and STAT3. But most of these biomarkers are inadequate as to determine the sensitivity of sorafenib. What’s more, for personalized therapy, effective and reliable biomarkers for sorafenib treatment in liver cancer is of great urgency.AimsThis research aims to explore the relationship between pERK level and sorafenib sensitivity in Hepatocellular Carcinoma. We compared pERK levels of HCC patients with sorafenib sensitivity and resistance. For further validation, we compared pERK levels of different HCC models and cell lines with sorafenib sensitivity and resistance. Additionally, we selected key genes related to sorafenib sensibility in liver cancer by CRISPR-Cas9 genome-wide screening using HepG2 cell line.Methods1. Clinical data were collected to compare the sensitivity of sorafenib of liver cancer patients with different pERK levels in their cancer samples.2. The sensitivity of sorafenib in hepatoma cell line with different levels of pERK in these cells was compared.3. For further validation, patient-derived xenograft models with different levels of pERK were established, and sorafenib sensitivity were investigated.4. Syngenic subcutaneous xenograft mouse models were established, and sorafenib sensitivity were investigated in mouse models with different levels of pERK.5. Orthotopic mouse models were established, and sorafenib sensitivity were investigated in mice with different levels of pERK.6. DDB1F/F; Alb-Cre^+/- mouse models were established. Level of pERK and sensitivity to sorafenib of this model were examined.7. DEN-induced HCC model was established, and level of pERK and sensitivity to sorafenib of this model were examined.8. Hematoxylin and eosin and immunohistochemical staining were used to compare pathology differences of liver cancer with different levels of pERK in patients.9. Hematoxylin and eosin and immunohistochemical staining were used to compare pathology differences between DEN-induced HCC model and DDB1F/F; Alb-Cre⁺ mouse model.10. Gene expression profile of PDXs with different pERK levels was investigated by mRNA sequencing.11. Gene expression profiles of hepatoma cell line （HepG2 and Li-0010） with different pERK levels were tested by mRNA sequencing.12. Results of RNA sequencing in patient specimens were verified by qPCR13. Key genes related to sorafenib sensitivity in liver cancer were selected by CRISPR-Cas9 genome-wide screening using HepG2 cell line.14. KEAP1 was knocked out in HepG2 cell line by sgRNA and sorafenib sensitivity was compared between wild type and KEAP1 KO HepG2 cell line.15. Level of pERK and PD1 in clinical specimens of liver cancer patients were determined by immunohistochemical and immunofluorescence staining respectively.Results1. Sorafenib was more sensitive to HCC patients expressing high levels of pERK in the tumor.2. Hepatoma cell lines with high levels of pERK （HepG2 and Hepal-6） were more sensitive to sorafenib than those with low levels of pERK （Be17404 and Hepalclc7）.3. In patient-derived xenograft model, sorafenib was more effective towards individuals with high pERK levels.4. In murine subcutaneous xenograft tumor model planted with cell lines with different levels of pERK （Hepa1-6 which was high pERK and Hepalclc7 which was low pERK）, grown xenografts express high pERK.5. In orthotopic model planted with different cell lines with different levels of pERK （HepG2 which was high pERK and Be17404 which was low pERK）, grown xenografts express high pERK.6. DDB1F F、Alb-Cre- mouse model has low level of pERK and is resistant to sorafenib.7. DEN-induced liver cancer model has high level of pERK and is sensitive to sorafenib.8. Patients with low pERK showed more obvious inflammatory cell infiltration, most of which were PD1⁺CD8⁺T cells.9. Compared with DEN-induced model, DDB1F/F;Alb-Cre^+/-mouse model showed more inflammatory cells especially with these PD1⁺CD8⁺T cells.10. According to RNA sequencing, PDx models with low pERK levels had more epithelial-mesenchymal-transition tendencies.11. According to RNA sequencing, cell lines with low level of pERK （Li-0010） had more epithelial-mesenchymal-transition tendencies than those with high level of pERK （HepG2）.12. Further evidence demonstrated that HCC with low level of pERK are better correlated with epithelial-mesenchymal-transition than those with high level of pERK.13. KEAP1 may play a crucial role in liver cancer cell sensitivity to sorafenib.14. Deletion of KEAP1 by sgRNA results in HepG2 resistant to sorafenib.15. HCC patients with low level of pERK and PD1⁺ accounts for more than half of total HCC population.Conclusions1. Sorafenib might be more effective in treating HCC patients expressing high levels of pERK in the tumors.2. HCC expressing low level of pERK exhibited more robust inflammatory infiltration, especially PD1⁺CD8⁺T cells.3. HCC expressing low levels of pERK exhibited a stronger epithelial-mesenchymal transition, which was associated with sorafenib resistance.4. KEAP1 may play a crucial role in HCC expressing high level of pERK and their sensitivity to sorafenib.