BRD4 Inhibition Is Synthetic Lethal With DHFR Inhibition In Pancreatic Cancer And The Mechanism

Part I BRD4 Inhibition Is Synthetic Lethal With DHFR Inhibition in pancreatic cancerObjectives: To study whether BRD4 Inhibition Is Synthetic Lethal With DHFR Inhibition in pancreatic cancer cell and xenograft models.Methods:(1)By analyzing mRNA-Seq data in TCGA database,we compared the difference of expression of DHFR and BRD4 in pancreatic cancer tissues and adjacent normal tissues,and revealed the correlation of DHFR,BRD4 and MYC.(2)Immunohistochemistry assay was performed to detect the difference of DHFR protein in pancreatic cancer tissues and adjacent normal tissues.(3)The effect of JQ-1 and MTX on cell proliferation and cell death was determined by cell proliferation assay,clonogenic assay,cell death assay,LDH release assay and mitochondrial membrane potential assay in vitro and nude mice model in vivo.Results:(1)The mRNA level of DHFR,BRD4 and MYC was higher in pancreatic cancer tissues than that in adjacent normal tissues,according to data in TCGA database.Patients with higher level of DHFR and MYC lived shorter,compared with those with lower level.mRNA level of DHFR correlates with that of BRD4 in pancreatic cancer tissues.(2)Protein level of DHFR was higher in pancreatic cancer tissues than that in normal tissues,according to results from IHC assay.(3)The combination of MTX and JQ-1 significantly decreased cell proliferation and increased cell death rate in pancreatic cancer cells.Conclusions: This study revealed that expression of DHFR,BRD4 and MYC was higher in pancreatic cancer tissues,and DHFR correlates with BRD4.BRD4 inhibition(by JQ-1)is synthetic lethal with DHFR inhibition(by MTX)both in vitro and in vivo.Part II the application of synthetic lethality in radiotherapy for pancreatic cancerObjectives: To study the application of synthetic lethality,caused by DHFR inhibition and BRD4 inhibition,in radiotherapy for pancreatic cancer.Methods:(1)clonogenic assay,cell cycle and cell death assay were performed to investigate the influence of MTX and JQ-1 on radiosensitivity of pancreatic cancer cells.(2)Western Blot and immunocytochemistry assay were used to evaluate the effect of MTX and JQ-1 on DNA damage response and DNA damage repair.(3)After DHFR knockdown and overexpression,Western Blot and immunocytochemistry assay were used in order to uncover the role of DHFR during DNA damage response and DNA damage repair.Results:(1)JQ-1 mediated radioresistance,while MTX increased radiosensitivity in pancreatic cancer cells.The combination of MTX and JQ-1 significantly increased radiosensitivity.(2)JQ-1 could increase the level of ?H2AX and significantly upregulated ?H2AX upon IR treatment.MTX could increase the level of ?H2AX.(3)DHFR level in pancreatic cancer cells was upregulated after MTX treatment.Overexpression of DHFR could increase ?H2AX in nucleus even without IR.But DHFR overexpression did not induce ?H2AX foci in nucleus.Knockdown of DHFR resulted in delay of DNA damage repair.Conclusions: this study revealed that synthetic lethality induced after double inhibition of DHFR and BRD4 could increase the radiosensitivity of pancreatic cancer cells.JQ-1 could make H2 AX easier to be phosphorylated after IR,thus decreasing radiosensitivity of pancreatic cancer cells.DHFR might participate in the process of DNA damage response and lack of DHFR might lead to delay of DNA repair.Part III Investigation of the mechanism of synthetic lethality induced by DHFR and BRD4 inhibitionObjectives: To study mechanism synthetic lethality,caused by DHFR inhibition and BRD4 inhibition,and the type of cell death.Methods:(1)Gene expression data with or without JQ-1 treatment from GEO database was re-analyzed by bioinformatics tools.Differentially expressed genes were delivered to KEGG/GO pathway analyze,in order to better understand the biological processes influenced by JQ-1(2)the expression of genes involved in purine,pyrimidine and folate synthesis pathway,in pancreatic cancer and normal tissues,were evaluated according to TCGA database.(3)Real time PCR was used to determine the change of genes involved in purine,pyrimidine and folate synthesis pathway,after JQ-1 and MTX treatment.(4)Western Blot and immunocytochemistry were used to detect cell death associated proteins and death inhibitors were used to uncover the type of cell death.Results:(1)Results from gene expression data indicated that JQ-1 could regulate purine and pyrimidine synthesis pathway,while DHFR is the rate-limiting enzyme in folate metabolism.Thus we proposed a hypothesis that purine,pyrimidine and folate synthesis may be the common target of MTX and JQ-1,which exerted synthetic lethality in pancreatic cancer cells.(2)Results from TCGA database revealed that purine,pyrimidine and folate synthesis pathways were activated in pancreatic cancer,which may lead to poor prognosis.(3)The m RNA level of TYMS,GART,DHFR and MTR was discovered to be upregulated after MTX treatment,which was downregulated after JQ-1 treatment.(4)Necroptosis may be one of the main types of cell death during synthetic lethality and autophagy may also play an important role.Conclusions: Results from this study indicated that purine,pyrimidine and folate pathway were influenced after MTX and JQ-1 treatment,which may be the reason that leads to synthetic lethality.Necroptosis may be one of the main types of cell death during synthetic lethality and autophagy may also play an important role.