This proposal aims to detect hepatocellular carcinoma (HCC) at a treatable stage by non-invasive methods. To this end, we propose to quantify site specific glycoforms of liver secreted N-glycoproteins (LNP) associated with the progression of liver disease to HCC. We and others have shown that changes in protein glycosylation accompany the development of HCC. Major re-distribution of protein N-glycoforms occurs at the premalignant stage of liver cirrhosis but our latest studies show that specific glycoforms of a glycopeptide of haptoglobin increase exclusively in HCC. We demonstrate that these minor glycoforms of haptoglobin are detectable in serum and have the potential to improve detection of HCC compared to the serologic gold standard, alpha fetoprotein (AFP). This supports our hypothesis that minor site specific glycoforms of abundant liver secreted N-glycoproteins provide HCC specific marker candidates detectable in the circulation. We propose to complete identification of the HCC specific N-glycans;we have already evidence that the glycans are characterized by multiple fucosylations and increased branching. We propose to identify glycopeptides of additional proteins carrying these HCC specific N-glycan modifications by newly optimized mass spectrometric methods. This is done because haptoglobin is downregulated in a fraction of cirrhotics and other proteins may be needed for optimal detection. We expand our informatic analysis to classify all polymorphic and mutant human N- glycoproteins with newly created or abolished N-glycosylation sites. These protein variants are over- represented in the human genome, represent prime candidates for association with diseases, and we expect that they have direct impact on carcinogenesis. We believe that a publically available resource of these variant proteins will stimulate glycoproteomic cancer prevention research. The proposed examination of HCC specific N-glycoforms is feasible because we have already created a repository of samples of HCC patients and cirrhotic controls. This repository, and additional outstanding QC resources, is essential for the isolation of the HCC specific minor glycoforms of liver secreted N-glycoproteins;these glycoforms are not detectable in disease free subjects. By the end of the study, we will have quantified the selected site specific protein glycoforms by targeted LC-MS selective reaction monitoring (SRM) methods. These methods will quantify the HCC associated site specific glycopeptides in serum with an unprecedented accuracy. We do this because we believe that quantification of specific glycoforms of specific glycoprotein peptides offers the highest diagnostic accuracy. Defining clinically applicable cancer markers has potentially far-reaching consequences for disease management and patient health. Our study is expected to generate new hypotheses on the functional impact of protein glycosylation on the development of cancer and to stimulate an entirely new line of cancer prevention and detection research.

Public Health Relevance

The incidence of liver cancer in the United States is steadily increasing. Early detection of cancer improves patient survival. Our study seeks to identify new biomarkers for early detection of liver cancer, which would have a direct impact on the disease management and outcome.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01CA168926-02
Application #
8545750
Study Section
Special Emphasis Panel (ZCA1-SRLB-4 (M1))
Program Officer
Rinaudo, Jo Ann S
Project Start
2012-09-14
Project End
2017-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
2
Fiscal Year
2013
Total Cost
$353,401
Indirect Cost
$109,155
Name
Georgetown University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
Yuan, Wei; Benicky, Julius; Wei, Renhuizi et al. (2018) Quantitative Analysis of Sex-Hormone-Binding Globulin Glycosylation in Liver Diseases by Liquid Chromatography-Mass Spectrometry Parallel Reaction Monitoring. J Proteome Res 17:2755-2766
Kozlik, Petr; Goldman, Radoslav; Sanda, Miloslav (2018) Hydrophilic interaction liquid chromatography in the separation of glycopeptides and their isomers. Anal Bioanal Chem 410:5001-5008
Fan, Yu; Hu, Yu; Yan, Cheng et al. (2018) Loss and gain of N-linked glycosylation sequons due to single-nucleotide variation in cancer. Sci Rep 8:4322
Ma, Junfeng; Sanda, Miloslav; Wei, Renhuizi et al. (2018) Quantitative analysis of core fucosylation of serum proteins in liver diseases by LC-MS-MRM. J Proteomics 189:67-74
Sanda, Miloslav; Zhang, Lihua; Edwards, Nathan J et al. (2017) Site-specific analysis of changes in the glycosylation of proteins in liver cirrhosis using data-independent workflow with soft fragmentation. Anal Bioanal Chem 409:619-627
Kozlik, Petr; Sanda, Miloslav; Goldman, Radoslav (2017) Nano reversed phase versus nano hydrophilic interaction liquid chromatography on a chip in the analysis of hemopexin glycopeptides. J Chromatogr A 1519:152-155
Darebna, Petra; Novak, Petr; Kucera, Radek et al. (2017) Changes in the expression of N- and O-glycopeptides in patients with colorectal cancer and hepatocellular carcinoma quantified by full-MS scan FT-ICR and multiple reaction monitoring. J Proteomics 153:44-52
Kozlik, Petr; Goldman, Radoslav; Sanda, Miloslav (2017) Study of structure-dependent chromatographic behavior of glycopeptides using reversed phase nanoLC. Electrophoresis 38:2193-2199
Wang, Mengjun; Sanda, Miloslav; Comunale, Mary Ann et al. (2017) Changes in the Glycosylation of Kininogen and the Development of a Kininogen-Based Algorithm for the Early Detection of HCC. Cancer Epidemiol Biomarkers Prev 26:795-803
Flowers, Sarah A; Zhou, Xin; Wu, Jing et al. (2016) Expression of the extracellular sulfatase SULF2 is associated with squamous cell carcinoma of the head and neck. Oncotarget 7:43177-43187

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