The Mechanism And Regulatory Function Of PPARγ Phosphorylation In HCC Development

Primary liver cancer is one of the most malignant cancers and is listed as the third cause of cancer-related deaths.The liver cancer is characterized by short duration,rapid development and high mortality.According to the International Cancer Research Center(IARC)statistics,about 34 million people die each year from liver cancer in China,accounting for more than 55%of worldwide liver cancer deaths.In addition,the five-year survival rate of liver cancer patients is very low,thus making it one of the most significant threats to the health of our population.HCC(hepatocellular carcinoma)is the most common type of primary liver cancer.Its pathogenesis is complex and hasn’t been elucidated completely.Hepatitis virus infection,cirrhosis,chronic liver inflammation,aflatoxin intake etc have been reported to induce HCC.The development of HCC is a multi-stage and complex process,many factors involving chromosomal abnormalities,genetic polymorphisms,gene mutations and abnormal regulation of signal transduction are vital in determining the fate of HCC.Notably,transcription factors have been confirmed to affect hepatocellular proliferation,invasion,and metastasis by numerous studies.PPARy(peroxisome proliferator-activated receptor y)is one of these important factors closely related to the development of HCC.PPARγ is a nuclear receptor and also an important drug target of metabolic diseases.Previous studies reported that PPARγ ligands inhibit a variety of tumors such as HCC,colon cancer,breast cancer,lung cancer.Regulation of PPARy activity has been recognized as a potential method in the treatment of cancers,especially HCC.Although the activity of PPARy has been confirmed to influence the development of HCC in many HCC models,the underlying mechanisms are still poorly understood.Many questions need to be answered,especially whether and how post-translational modifications of PPARy could affect the progression of HCC.Our research based on the observation that expression of PPARy Ser84 phosphorylation is increased significantly in HCC,which indicates that Ser84 phosphorylation may be associated with HCC progression.Therefore,our research for the first time based on the effects of PPARγ phosphorylation on HCC progression explores the relationship between HCC development and PPARy phosphorylation,and the possible mechanism.This thesis includes the following three contents:一、The relationship between PPARy phosphorylation and HCC progression1.Spontaneous HCC construction and PPPARy phosphorylation analysis:we induced spontaneous mice HCC by injecting chemical mutagen DEN(diethylnitrosamine)and observed/recorded mice body weight,growth,deaths in the next 12 months;By detecting biomarkers of HCC(Ki67,PCNA,AFP)in pathological section of mouse liver,we confirmed that HCC mouse model had been successfully established.Through detecting the expression of PPARy(Ser84)phosphorylation in mouse liver lysate and liver section,we found that expression of PPARy phosphorylation was higher in HCC than in adjacent normal liver tissue.Besides,PPARy transcriptional activity decreased significantly in HCC.Notably,with the progression of HCC,the level of PPARγ phosphorylation increased gradually,indicating PPARy phosphorylation associates with HCC malignant progression.2.Nude mice model construction and in vivo experiments:We constructed stable HepG2 cell line expressing WT,S84A,S84D PPARy by using ViraPowerTM Lentiviral Expression Systems and screened with Blasticidin for two weeks.Next,three cell lines were respectively inoculated into nude mice to establish in vivo ectopic implantation model.Mouse body weight,tumor volume,growth and deaths were measured regularly.We found that the tumors in PPARγS84D group grow faster than the other two groups and formed the biggest tumor.There was no difference in survival rate among these three groups.PCNA expression level was highest in S84D group,suggesting PPARy phosphorylation can accelerate HepG2 proliferation and growth,promoting malignant tumor progression.3.Analyze PPARy phosphorylation in human pathological specimens:We collected HCC samples from 17 cases in clinically diagnosed liver cancer patients.These samples were sectioned and detected for tumor markers.Taking advantage of IHC,we found that the level of phosphorylated PPARy in HCC lesion was higher than adjacent normal tissue in 12 of 17 patients(about 70%),indicating PPARy phosphorylation may promote HCC progression.Because the numbers of pathological specimens were not enough,these data were just for reference.二、MEK/ERK kinase mediated PPARy phosphorylation and it’s effect on HCC progression1.Analysis of MEK/ERK kinase activity:In DEN-induced mouse HCC model,the expressions of phosphorylated MEK/ERK were analyzed through IHC and western blotting.We found that the levels of p-ERK1/2 and p-MEK increased,which was in consistent with elevated PPARy phosphorylation.Besides,the expression levels of p-ERK1/2 and p-MEK in mouse HCC model were significantly higher than normal mouse liver,which were also confirmed by human HCC samples.These data suggest that the increased PPARy phosphorylation may be due to the activation of MEK/ERK.2.Inhibition of MEK/ERK kinase activity lowers PPARy phosphorylation:To verify the above speculation,we treated four HCC cell lines SMMC7271,Hep3B,HepG2 and Hep 1-6 with MEK inhibitor PD0325901 in vitro,and wonder to know whether PPARγ phosphorylation are affected by inhibition of MEK-ERK1/2.As expected,when MEK/ERK was inhibited PPARy phosphorylation was blocked.In addition,the transcriptional activity of PPARy increased,which is in consistent with decreased PPARy phosphorylation.These data indicated that inhibition of MEK/ERK may decrease PPARy phosphorylation and in turn increase PPARy transcriptional activity.In the mean time,we analyzed proliferation,cell cycle of HCC cells and found that the proliferation of HCC was suppressed and cell cycle was arrested in G1 phase after inhibition of PPARy phosphorylation.3.Analyze the effect of MEK/ERK on PPARy phosphorylation in vivo:Based on the experiments in vitro,we wondered to know if this event took place in vivo.We firstly construct Xenografts in nude mice.When the tumor volume approached to 100mm3,mice were randomly divided into four groups.To analyze whether MEK/ERK can suppress PPARy phosphorylation and whether RSG had a synergistic effect with PD0325901.Four group mice were injected with DMSO,rosiglitazone,PD0325901,rosiglitazone+PD0325901 respectively for 28 days.The result showed that after MEK/ERK inhibition,the level of PPARy phosphorylation was significantly decreased,tumor growth was inhibited and the tumor volume was reduced.三、The mechanisn of PPARy-regulated HCC cells proliferation1.PPARy phosphorylation promotes hepatoma cells proliferation:The analysis of NimbleGen gene expression profile on PPARγWT,PPARγS84A and PPARys 4D stably transfected HepG2 cells showed that proliferation-associated differential expression genes were enriched.The analysis of Q-PCR indicated that PPARγS84D promoted the expression of proliferation-associated gene.MTT,cell colony formation assay and CFSE also confirmed that proliferation of PPARγS84D cell was significantly higher than PPARγWT and PPARγS84A.The PPARy phosphorylation altered the downstream transcription mode of hepatoma cells,which promoted the proliferation of tumor cells.2.PPARy phosphorylation inhibits hepatoma cell cycle arrest:To further elucidate the process by which PPARγS84D regulated cell growth,we performed FACS.Cell cycle distribution analysis revealed that there was a significant decrease of cell proportion in the G1 phase in presence of PPARγS84D overexpression compared with PPARyWT and PPARyS84A.These results suggested that PPARy phosphorylation attenuated cell cycle arrest.To explore the molecular mechanism of G1 phase arrest reduction,we evaluated several regulators that control the G1 checkpoint in the cell cycle.The reduced G1 phase arrest by PPARy phosphorylation was associated with reduction of CKI(p18,p21 and p27)and increase of cyclin A.Our results indicated PPARy phosphorylation attenuated cell cycle arrest by regulating the cell cycle factors.3.PPARy phosphorylation has no effect on apoptosis of hepatoma cell:The above eexperiments confirmed that PPARy phosphorylation promoted the proliferation of hepatoma cell and inhibited cell cycle arrest,which results in hepatoma cell growth.There was no difference in cell apoptosis among the stably transduced HepG2 cells.In summary,this work eatablishes a link between PPARy phosphorylation and HCC development,indicating that PPARy phosphorylation promotes HCC development.Based on these findings,we carried out further research.The results showed that MEK/ERK inhibited HCC cells proliferation by regulating PPARy phosphorylation.MEK inhibitor PD0325901 has the significant effect on HCC development.四、Analyze glycolysis related genes of HCC1.Gene microarray:Based on the research that MEK/ERK-mediated PPARy phosphorylation can influence HCC progression,We analyzed differential expression genes in PPARγWT、PPARγS84A and PPARγS84D by using gene microarray,and found that PPARy phosphorylation affected the expression of key enzymes in glycolysis significantly.Compared to PPARγS84A,the transcripts of PGK1 and PFK2 were increased in PPARγS84D HCC.Interestingly,PDK1,which inhibits TCA cycle and mitochondrion,was also increased in PPARγS84D HCC.These data were confirmed by Q-PCR.The results indicate that PPARy phosphorylation affected aerobic glycolysis of HCC.2.Measurement of glucose and lactate:According to DNA microarrays,we measured glucose uptake,lactate production of HepG2 and three stably transfected HepG2 cell lines.We found that glucose consumption of PPARγWT and PPARγS84A was significantly lower than PPARγS84D.In addition,PD0325901 treatment significantly reduced lactate production of HepG2.These data suggested that enhanced glycolysis is accompanied with PPARy phosphorylation,which explains why HCC grew faster than normal liver cell.In conclusion,this article firstly demonstrated that PPARy phosphorylation can promote the development of HCC.Based on this observation,we conducted further studies in cell and mouse,and found that MEK/ERK are key enzymes mediating PPARy phosphorylation.Inhibition of MEK/ERK strengthens HCC cell cycle arrest and inhibits cell proliferation,which indicate that PPARy phosphorylation-associated molecular node is very important in mediating HCC progression.We also found the expression patterns of downstream genes were changed after PPARy phosphorylation.These genes were enriched in aeroboc glycolytic pathway,resulted in elevated aeroboc glycolysis and thus promoted HCC growth and malignant development.