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Biomarker discovery applicable to lung cancer diagnosis and prognosis

  • 김정은 (대학원 의학과 분자·세포·발생생물학전공)

초록/요약

Lung cancer is one of the deadliest and commonly diagnosed neoplasms. Early diagnosis of this disease is critical for improving clinical outcome and prognosis. Because the early stages of lung cancer often produce no symptoms, it is necessary to identify biomarkers for early detection, prognostic evaluation, and recurrence monitoring of the cancer. To identify potential lung cancer biomarkers, we analyzed the differential protein secretion from transformed bronchial epithelial cells (1198 and 1170-I) as compared to immortalized normal bronchial epithelial cells (BEAS-2B) and non-transformed cells (1799), all of which are derived from BEAS-2B and represent multistage bronchial epithelial carcinogenesis. The proteins recovered from the conditioned media of the cells were separated on two-dimensional gels. There was little difference between the secretome of the BEAS-2B and 1799 cells, whereas the patterns between the transformed 1198 and 1170-I cells and non-transformed 1799 cells were significantly different. Using mass spectrometry and database search, we identified 20 proteins including protein gene product 9.5 (PGP9.5), translationally controlled tumor protein (TCTP), tissue inhibitors of metalloproteinases-2 (TIMP-2), and triosephosphate isomerase (TPI), that were either increased or decreased simultaneously in conditioned media of both 1198 and 1170-I cells. Furthermore, levels of PGP9.5, TCTP, TIMP-2, and TPI were significantly increased not only in the conditioned media of both transformed cell lines when compared to those of BEAS-2B and 1799 cells, but also in plasmas and tissues from lung cancer patients when compared to those in normal controls. We suggest the PGP9.5, TCTP, TIMP-2, and TPI as promising candidates for lung cancer serum biomarkers.

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초록/요약

Lung cancer is the leading cause of cancer death, largely because it is often diagnosed at a late stage. Therefore, specific and sensitive biomarkers are required to improve survival rates. During the early stages of cancer, tumor associated antigen (TAA) expression on tumor cells, or in the circulation, is very low. However, the immune system can still recognize these antigens. Anti-tumor antibodies are frequently observed in the sera of patients with different cancers, and may be potential tumor markers. To identify anti-tumor antibodies highly associated with lung cancer, pooled plasma from lung cancer patients (n = 15; three females and twelve males, aged 55？75 years) and healthy controls (n = 10; five females, five males, aged 55？72 years) was used to examine immunoreactivity to proteins which were prepared from seven lung cancer tissues and separated by 2D-PAGE. Thirty-one protein spots were identified among the proteins detected by pooled plasma from lung cancer patients. To verify the results of 2D western blotting, the immunoreactivity of individual sera against purified recombinant proteins was examined, along with the level of TAA expression. The present study showed that detecting levels of anti-tumor antibodies and TAA in serum may be useful for lung cancer screening.

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목차

Abbreviations------------------------------------------------------------------------- 1

Part I. Identification of potential lung cancer biomarkers using a lung cancer cell line.
Abstract ------------------------------------------------------------------------------- 3
Introduction -------------------------------------------------------------------------- 5
Materials and Methods ----------------------------------------------------------- 9
Table 1. Clinical features of lung cancer patients who donated plasmas
Results ---------------------------------------------------------------------------------18
Figure 1. Optimization of medium conditioning.
Figure 2. Representative 2-dimensional reference map of 1170-I-conditioned medium and change in secretion pattern across 1799, 1198 and 1170-I cells.
Table 2. Protein identification of 20 differentially secreted proteins with recurring ≥4 times out of the 5 pairs of samples.
Figure 3. Immunoblot analysis of differentially secreted proteins in conditioned media and whole cell lysates.
Figure 4. Immunoblot analysis of differentially secreted proteins in lung cancer tissues and adjacent normal lung tissues.
Figure 5. Plasma levels of PGP9.5, PRX6, TCTO, TIMP-2, and TPI in lung cancer patients and healthy controls.
Discussion --------------------------------------------------------------------------- 37
References --------------------------------------------------------------------------- 46

Part II. Identification of lung cancer biomarkers using patients？ sera
Abstract ------------------------------------------------------------------------------ 58
Introduction ------------------------------------------------------------------------ 60
Materials and Methods --------------------------------------------------------- 64
Results -------------------------------------------------------------------------------- 71
Figure 1. Screening of anti-tumor antibodies against lung cancer-associated proteins in pooled serum from patients with lung cancer.
Figure 2. Representative reference map showing putative TAAs.
Table 1. Protein identification of 31 proteins highly recognizable by patients pooled serum.
Figure 3. Cloning and purification of recombinant proteins.
Figure 4. Determination of optimal ELISA conditions and anti-tumor antibody levels.
Table 2. Clinical features of donated sera.
Figure 5. Levels of TAAs in individual sera of lung cancer patients and normal controls.
Figure 6. Expression levels of TAAs in lung cancer tissues.
Discussion -------------------------------------------------------------------------- 107
References-------------------------------------------------------------------------- 113
국문요약 --------------------------------------------------------------------------- 125

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