Study of Lung cancer Biomarkers:Identification and validation of two protein biomarkers from different sources : 혈액유래 단백질 바이오마커의 검증방법의 확립과 폐암조직 유래 단백질 바이오마커의 발굴과 그 기전연구

다국어 초록 (Multilingual Abstract)

Quantification is an essential step in biomarker development. Multiple reaction monitoring (MRM) is a new modified mass spectrometry-based quantification technology that does not require antibody development. Serum amyloid A (SAA) is a positive acute-phase protein identified as a lung cancer biomarker in our previous study. Acute SAA exists in two isoforms with highly similar (92%) amino acid sequences. Until now, studies of SAA have been unable to distinguish between SAA1 and SAA2. To overcome the unavailability of a SAA2-specific antibody, we developed MRM methodology for the verification of SAA1 and SAA2 in clinical crude serum samples from 99 healthy controls and 100 lung adenocarcinoma patients. Differential measurement of SAA1 and SAA2 was made possible for the first time with the developed isotype-specific MRM method. Most healthy control samples had small or no MS/MS peaks of the targeted peptides otherwise, higher peak areas with 10- to 34-fold increase over controls were detected in lung cancer samples. In addition, our SAA1 MRM data demonstrated good agreement with the SAA1 enzyme-linked immunosorbent assay (ELISA) data. Finally, successful quantification of SAA2 in crude serum by MRM, for the first time, shows that SAA2 can be a good biomarker for the detection of lung cancers.

다국어 초록 (Multilingual Abstract)

As lung cancer is showing the highest mortality in cancer related death, convenient and rapid molecular diagnostic methods might be helpful for the lung cancer diagnosis and its treatment. Protein biomarkers could be lead to the development of molecular diagnostics. Biomarkers from distal fluids generally have high cross reactivity to many similar diseases. To discover lung cancer tissue specific protein biomarkers, secreted proteins from primary cultured lung cancer and adjacent normal tissues from patients were subjected to proteomic analysis. QSOX1 (Quescin sulfhydryl oxidase), one of the proteins in the increased protein list, is selected as a biomarker candidate. Increase of QSOX1 in lung cancer was verified through western blot analysis of about 60 patients’ tissue lysates. Unique higher expression of QSOX1 in lung cancer compared to other solid cancers was confirmed by immunohistochemistry. Lewis lung adenocarcinoma mouse model experiment proved the QSOX1 increase in the induced lung cancer tissues of mice. QSOX1 is known as a secreted protein, therefore, QSOX1 level in the albumin and IgG depleted serum was measured by MRM (multiple reaction monitoring). Significant increase was detected in lung cancer patients compared to healthy control. In conclusion, QSOX1 might be a lung cancer tissue derived specific biomarker and in combination use with other blood biomarkers, it is expected to increase specificity of biomarkers in diagnostics.

목차 (Table of Contents)

• LIST OF FIGURES.......................................................................................i
• BACKGROUND ..........................................................................................1
• 1. Lung cancer ......................................................................................1
• 2. Biomarkers: Definition and clinical application ..............................2
• 3. Proteomics & lung cancer Biomarker research ...............................3
• CHAPTER Ⅰ. Large-scale validation of two isotype lung cancer biomarkers, Serum amyloid A 1/2 by multiple reaction monitoring in crude sera.
• ABSTRACT...................................................................................................8
• INTRODUCTION......................................................................................10
• MATERIALS AND METHOD..................................................................13
• 1. Sample collection........................................................................13
• 2. Material and sample preparation for mass spectrometry…........13
• 3. Instrumentation and MS analysis................................................14
• 4. Data analysis...............................................................................15
• 5. ELISA.........................................................................................16
• 6. Statistical analysis.........................................................................16
• RESULTS....................................................................................................17
• 1. Overall strategy for the experiment and selection of MRM.........17
• 2. Standard curve and transition determination..............................19
• 3. MRM analysis of SAA1 and SAA2 in crude serum.....................20
• 4. ELISA verification of samples and comparison to MRM experimental results......................................................................25
• 5. Comparison between two isoforms of SAA, SAA1 and SAA2..........................................................................................27
• FIGURES AND LEGENDS.......................................................................29
• TABLES.......................................................................................................43
• DISCUSSION .............................................................................................47
• CHAPTER Ⅱ. Quescin sulfhydryl oxidase 1 (QSOX1), lung cancer cell secreted protein, is a potent lung cancer specific biomarker and promotes lung cancer progression and metastasis
• ABSTRACT.................................................................................................53
• INTRODUCTION......................................................................................55
• MATERIALS AND METHOD..................................................................58
• 1. Human tissues and serum samples..............................................58.
• 2. Cell culture……………………………......................................58
• 3. Secretome collection and protein precipitation..........................59
• 4. Proteomic analysis of secretome protein....................................60
• 5. Western Blot analysis..................................................................61
• 6. Immnohistochemistry.................................................................62
• 7. Sample preparation and MS methods for MRM.........................62
• 8. RNA isolation and real-time PCR...............................................64
• 9. In vivo metastasis assay in mouse model...................................64
• 10. Knock-down of QSOX1 and in vitro analysis..........................65
• 11. Statistical analysis.....................................................................66
• RESULTS....................................................................................................68
• 1. Strategy for Lung cancer biomarker discovery from primary lung cancer cell secretome....................................................................68
• 2. LC-ESI-MS/MS analysis of secretome and biomarker candidate discovery.......................................................................................68
• 3. Elevation of QSOX1 in lung cancer tissues compared to adjacent normal tissues...............................................................................70
• 4. Validation of QSOX1 in serum of lung cancer patients by MRM analysis.......................................................................................70
• 5. Increased expression of QSOX1 in lung cancer cells...................72
• 6. Verification of QSOX1 increase in lung cancer and metastasized tissues in LLC mouse model.......................................................74
• 7. Knock down of QSOX1 decreases lung cancer metastasis...........75
• FIGURES AND LEGENDS.......................................................................77
• TABLES.......................................................................................................91
• DISCUSSION..............................................................................................96
• REFERENCES.........................................................................................102
• ABSTRACTS IN KOREAN.....................................................................110