Gastric Cancer Serum Biomarker Discovery And Arsenic Trioxide Target Identification Using Protein Microarray Technology

With the implementation and completion of “Human Genome Project”, it has entered into the era of systems biology for life science. Proteomics is a rising branch of the systems biology methodology. It eventually holds the key for the treatment and cure of complex diseases such as cancer by studing the proteomic variations of biological systems in different physiological and pathological states and the network of networks, in order to discover the function of proteins and the transformation law of cells. Featured as highthroughput, miniaturized, and capable of parallel analysis, protein microarrays have already become an important technology platform for systems biology,which would systematically identify and compare the composition and transformation law of proteins during the diagnosis and treatment of cancer to seek biomarkers and novel therapy targets. In this thesis, focusing on diagnosis and treatment of gastric cancer, we performed proteome microarray strategy to search for new serum biomarkers and novel therapeutical targets of gastric cancer.Firstly, we’ve developed a human proteome microarray technology(carries 16,368 human proteins) based strategy for screening serum biomarkers for early diagnosis and prognosis of gastric cancer. GC serum autoantibodies were discovered and validated using serum samples from independent patient cohorts encompassing 1,401 participants divided into three groups, i.e., healthy, GC high-risk and GC patients. To discover biomarkers for GC, the human proteome microarray was first applied to screen specific autoantibodies in a total of 87 serum samples from GC patients and healthy controls. Potential biomarkers were identified via a statistical analysis protocol. Targeted protein microarrays with only the potential biomarkers were constructed and used to validate the candidate biomarkers using 914 samples. To provide further validation, the abundance of autoantibodies specific to the biomarker candidates was analyzed using enzyme-linked immunosorbent assays(ELISA). Receiver operating characteristic(ROC) curves were generated to evaluate the diagnostic accuracy of the serum biomarkers. The final panel of biomarkers consisting of COPS2, CTSF, NT5 E and TERF1 provides high diagnostic power, with 95% sensitivity and 92% specificity. Furthermore, after the protein microarray results were correlated with the follow-up data of patient survival, three proteins in the panel, i.e., COPS2, CTSF, NT5 E and TEFR1 also have potential to serve as independent predictors of overall GC patient survival.Secondly, using the same human proteome microarray, we’ve developed a proteome microarray based strategy for identification of ATO binding proteins on a systems level, and successfully identified 360 arsenic binding proteins, which is about 18 times of the number that of the known ATO binding proteins in human prior to this study. These proteins represent a wide range of biological functions. Of the arsenic binding proteins, a high confident motif with vicinal thiol was identified for the first time. Unexpectedly, enzymes of glycolysis were highly enriched, including HK2, a tumor specific protein. Further biochemical studies showed that HK2 is indeed the target of ATO and the activity of HK2 could be inhibited upon the addition of ATO both in vitro and in vivo. The strategy that we developed could be readily adopted for the study of other compound. The identified ATO binding proteins could serve as a valuable resource for future MOA study, and the developing of novel synergistic therapeutic strategies for treating solid tumors.Thirdly, we’ve applied ultrahigh performance liquid chromatography/tandem mass spectrometry(UPLC/MS/MS) and Gas Chromatography/Mass Spectrometry(GC/MS) platforms to characterize metabolic adaptation and mechanism of action associated with drug treatment of human gastric carcinoma cell line SGC-7901. Since arsenic binds to the majority of the enzymes in the glycolysis pathway, we hypothesized that arsenic effects through disturb the activity of those enzymes, and cause the typic phenotype of growth inhibition and apoptosis when tumor cells are treated with arsenic. Thus dynamical changes of the corresponding inter-metabolites could be expected. To test this hypothesis, SGC-7901 from gastric cancer originated from solid tumor, which showed clear response upon ATO treatment, was chosen and treated with for 2 μM arsenic trixode. To prepare cell samples for metabolomics analysis, SGC-7901 cells were harvested after treated with arsenic for 6, 12 and 24 hrs. Vehicle controls were also carried out without arsenic treatment. We found that most of the metaboliteswere concentrated in the glycolytic pathway, i.e., 6-phosphate glucose, glyceraldehyde 3-phosphoric acid, pyruvic acid and lactic acid. The levels of them were significantly lower in the drug-treated groups in SGC-7901 cells at 12 and 24 hour time points. In addition, the levels of glycerate, a metabolite derived from glycine metabolism that feeds into glycolysis, were significantly elevated in SGC-7901 cells over the vehicle control. These alterations suggest that glycolysis was inhibited by ATO treatment.In summary, proteomics and metabolomics platforms have been applied for sceening new serum biomarkers and novel therapeutical targets for gastric cancer. In addition, based on the success of identification of new biomarkers and direct targeting proteins for ATO, we developed a proteome microarray based strategy for identification of cancer biomarker and micromolecule binding proteins on a systems level.