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研究生(外文):Yu-Shan Ting
論文名稱(外文):Small Pool Antibody Expression Screening for the Membrane-associated-proteins as Biomarkers for Cancer Prognosis
指導教授(外文):Chi-Ying F. Huang
外文關鍵詞:hepatocellular carcinomaSPAG5epithelial-mesenchymal transitionsorafenib
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質膜蛋白已被鑑定為癌細胞的治療標的和癌症的診斷標誌物。一個可靠的生物標誌可以提供臨床診斷而改善癌症治療,因此進一步的研究被視為是重要的。我們建立了一個「少量抗體庫群組表達篩選系統」,主要藉由混合的抗體群結合到LPPC (Lipo/PEI/PEG 複合物)。此系統可以用來證實新的受體或過度表現在腫瘤細胞中的膜相關蛋白,癌細胞與候選庫群抗體的結合效率可以用流式細胞儀和免疫螢光法證實。藉由此策略我們發現SPAG5 (參與細胞分裂的紡錘體相關蛋白)位於具有高度爬行能力的細胞之細胞膜中,如MCF7,Mahlavu和SKR3細胞。本人還進行細胞的質膜分離與西方點墨法確認SPAG5蛋白位於細胞膜上。儘管SPAG5在Malavu球狀細胞呈現低表現,卻有在細胞膜上觀察到。此外,Huh7R是一株抗sorafenib的Huh7細胞,帶有SPAG5的高表現、AKT訊息路徑的活化與間質細胞的特性。在異位表現SPAG5的Huh7細胞中觀察到AKT/p70 S6K路徑的活化並提高了Snail的表現。概括來說,SPAG5位於高度爬行能力的肝癌細胞和癌症球狀細胞之細胞膜,且SPAG5在Huh7細胞中的過度表現可能參與了sorafenib的抗藥性和與癌細胞轉移。這些數據顯示SPAG5與預後差的相關性,且具有潛力作為肝癌的生物標誌物。綜合來說,對於任何一種癌細胞或癌症幹細胞,「少量抗體庫群組表達篩選系統」將能成為鑑定新的受體或膜相關蛋白的有用工具,有利於癌症的診斷。
Plasma membrane protein has been identified as the therapeutic target and diagnostic biomarker of cancer cells1. The reliable biomarker discoveries are believed to provide clinical diagnostic tools to guide cancer treatment. Here, we have established a “small pool antibody expression screening” system, with a mixture of antibodies conjugated to LPPC (Lipo/PEI/PEG complex). This system could be used to identify novel receptors or membrane-associated proteins overexpressed in cancer cells. The binding efficiencies of cancer cell to candidate pools were confirmed by FACS and immunofluorescence staining. Using this strategy, we have identified SPAG5, a mitotic spindle–associated protein for cell division, localized to cell membrane in highly migratory cell lines such as MCF7, Mahlavu and SKR3. Cytoplasmic and membrane fractionation followed by Western blotting confirmed that SPAG5 protein was localized to the cell membrane. Despite low expression of SPAG5 in Malavu sphere cells, SPAG5 was still distributed to cell membrane. Moreover, Huh7R cell, a sorafenib-resistant Huh7 cell, exhibited up-regulated expression of SPAG5 and activated AKT signaling pathway coupled with mesenchymal characteristics. Ectopic expression of SPAG5 in Huh7 cells resulted in activation of AKT/p70 S6K pathways and up-regulation of Snail expression. In summary, SPAG5 is localized to highly migratory HCC cells and sphere cells. Overexpression of SPAG5 in Huh7 cells might be involved in sorafenib-resistant and metastasis. My data suggests that SPAG5 is correlated to poor prognosis and might be a potential biomarker in HCC. Taken together, we demonstrated that the “small pool antibody expression screening” could be a useful tool for uncovering a novel receptor or membrane-associated protein in cancer or cancer-stem cells to develop the clinical diagnosis of cancers.
Abstract I
Chinese abstract II
Membrane proteins as diagnostic and prognostic molecular marker in cancer 1
Characteristics of LPPC (Lipo/PEI/PEG complex) 1
Establishing LPPC/antibody complexes for screening novel receptors and membrane-associated proteins 2
Small pool antibody expression screening for identification of cancer-specific receptors and/or membrane-associated proteins 3
Characteristics of SPAG5 4
The role of SPAG5 in epithelial–mesenchymal transitionn (EMT) 5
SPAG5 as prediction marker in drug response of hepatocellular carcinoma 5
Objectives 7
Materials and Methods 8
Cell lines and cell culture 8
Spheres formation 8
Transient transfection 9
Antibodies and other reagents 9
Plasmids 10
Immunofluorescence staining 10
Western blotting 10
Cell migration assay 11
MTT assay 12
Cell fractionation 12
Cytospin centrifugation 12
Databases 13
Broad-Novartis Cacner Cell Line Ecncyclopedia 14
Genomics of Drug Sensitivity in Cancer Database 14
PrognoScan 15
The Human Protein Atlas 16
Results 17
Characterizing the subcellular localization of prioritized proteins from small pool antibody expression screening 17
SPAG5 is localized to the cell membrane of highly migrated HCC cells and associated with EMT 18
Data mining of SPAG5 in CCLE, The Human Protein Atlas and PrognoScan 19
Prediction from database for the expression of SPAG5 in drug response is unclear 20
Expression of SPAG5 is up-regulated in sorafenib resistant Huh7 cells 20
Overexpression of SPAG5 in Huh7 leads to sorafenib resistance via AKT/mTOR and MEK/MAPK signaling pathway 21
Overexpression of SPAG5 is involved in cell mobility 22
The C-terminal domains of SPAG5 play a major role in AKT signaling pathway 23
Expression of SPAG5 in cancer stem-like cells 24
Discussion 26
Figures 29
Figure 1. Verification of the subcellular localization of the positive pool Y1C by FACS and confocal microscopy. 29
Figure 2. Characterization of the expression of EMT markers, migratory ability and the subcellular localization of SPAG5 in various HCC cell lines. 31
Figure 3. SPAG5 data in CCLE, The Human Protein Atlas and PrognoScan. 35
Figure 4. The SPAG5 expression level in various cancers with different response to sorafenib, cisplatin and paclitaxel. 38
Figure 5. The characteristics of sorafenib-resistant Huh7 cell lines (Huh7R). 40
Figure 6. Elucidation of the mechanism of SPAG5 related to sorafenib resistant of HCC. 42
Figure 7. Characterization of migratory ability and EMT markers in Huh7 and Huh7 resistant cells. 44
Figure 8. Characterization of different domains of SPAG5 in Huh7 cells. 47
Figure 9. Immunofluorescence staining for SPAG5 and EGFR in Mahlavu sphere cells. 48
Figure 10. The differential expression of endogenous SPAG5 among cancer cells and cancer-stem like cells. 49
Supplementary Figures & Tables 50
Supplementary Figure 1. Schematic illustration of LPPC adsorption with antibody-targeting cancer cells. 50
Supplementary Figure 2. Schematic illustration of the antibody-based liposome for drug screening. 51
Supplementary Figure 3. Screening of liposome-mediated antibody pools to identify specific receptors in different cancer cell lines. 52
Supplementary Figure 4. Three anti-SPAG5 antibodies are used for immunofluorescence and Western blot. 54
Supplementary Table1. Verified subcellular locations of the prioritized antibodies via immunofluorescence staining. 55
References 57

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