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研究生：	譚一泓 Yi-Hung Carol Tan
論文名稱：	新穎抗癌藥物及其所影響之蛋白對於肺癌治療的效性探討 Interplay between anticancer drugs and their effector proteins on therapeutics of lung cancer
指導教授：	王憶卿 Wang, Yi-Ching 謝秀梅 Hsieh, HsiuMei
學位類別：	博士 Doctor
系所名稱：	生命科學系 Department of Life Science
論文出版年：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	117
中文關鍵詞：	新穎抗癌藥物、肺癌、腫瘤、轉移、突變
英文關鍵詞：	anti-cancer drugs, OSU03013, PHA665752, c-Cbl, c-Met, mutation, migration, metastasis
論文種類：	學術論文
相關次數：	點閱：226 下載：2
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目的：標靶治療是目前癌症治療的主要研究方向，大多數的肺癌患者對於化學療法或是放射線療法都有強烈的抗藥性，因此新穎的抗癌藥物以及新的治療目標是極需被開發研究的。在此論文的第一部分研究目的，希望鑑定新穎的小分子化合物OSU03013，是否可以作為肺癌新穎抗癌藥物。OSU03013其為舊藥celecoxib的衍生物，屬於cyclooxygenase (COX)-2的抑制劑。在攝護腺癌研究中證實OSU03013透過3-phosphoinositide- dependent kinase 1 (PDK1)/AKT訊息傳導路徑抑制腫瘤生長，此外，OSU03013也使用在乳癌的研究中。因此我們也希望能定義出OSU03013在肺癌中的目標蛋白及影響的生物路徑。此論文的第二部份是探討新穎c-Met抑制劑PHA665752對肺癌細胞生長及轉移的抑制作用，以及新穎治療目標基因Cbl (Casitas B-lineage lymphoma) 於肺癌病人檢體之突變情形。c-Cbl基因位於人類染色體11q23.3的位置，c-CBL蛋白目前已被發現主要參與細胞訊息傳導路徑以及酪氨酸激酶接受器（如：c-Met 和EGFR）的負調控角色，因此，本研究推測c-Cbl的突變或許是造成c-Met和EGFR過度表現的原因之一。此外，結合PHA665752 以及c-CBL 正常蛋白的共同抑制作用，或許可以成為新穎治療肺癌的策略。
實驗方法與設計：論文的第一部分，為了探討OSU03013是否有潛力成為新穎抗癌藥物，本研究利用肺癌細胞之毒殺作用及其細胞學鑑定，之後利用二維電泳、質譜分析等蛋白質體學的方法找尋藥物的目標及影響蛋白，並分析這些蛋白/訊息傳遞路徑與細胞生長調控的關係。論文的第二部分，利用肺癌細胞之毒殺作用來探討PHA665752的抑制效果，並利用基因定序及生物功能探討的方式，在總共一百一十九位來自台灣、美國白種人及黑人肺癌病人腫瘤組織中來研究c-Cbl的基因突變圖譜，並且在非小細胞肺癌細胞株中探討c-Cbl基因突變後影響細胞生長的狀況。此外，並利用c-Met抑制劑PHA665752及c-Cbl 正常基因轉染的共同處理，在細胞及動物實驗中來探討c-Met抑制劑及c-CBL正常蛋白對癌細胞的抑制作用。
結果：第一部分的結果指出OSU03013具有高度細胞選擇性毒殺作用，而此藥物對於肺正常細胞在相同濃度處理並沒有毒殺作用，所以是一個治療肺癌的潛力藥物。在細胞學鑑定實驗中，我們發現OSU03013的細胞致死劑量在48小時的測試下約1~4 M，會造成細胞週期停滯在間期一 (Gap 1, G1 arrest) 的現象；OSU03013在肺癌細胞同時也藉由內質網壓力效應去引發細胞凋亡 (apoptosis)。在蛋白質體學的實驗中，我們發現此藥物在肺癌細胞之目標蛋白包含了cAMP-dependent protein kinase inhibitor β form (PKIB, 激酶抑制蛋白)、數種G proteins (G蛋白)、數種Heat-shock proteins (熱休克蛋白)、Antioxidant enzymes (抗氧化蛋白)、及其他調控細胞生長、代謝的蛋白；這些蛋白有許多皆以Western blot (西方點墨法) 確認。經由分子模擬的實驗顯示，OSU03013會與ATP競爭然後與cAMP-dependent protein kinase (PKA)結合，並且抑制PKA的訊息傳導路徑，推測因此抑制了肺癌細胞生長。第二部份的結果指出，c-Met普遍過度表現於肺癌細胞中，而PHA665752對癌細胞具有毒殺作用、降低c-Met的表現，並且會引發早期細胞凋亡的機制。此外，一百一十九個肺癌病人樣本中，有一個已知的基因多型性變異 (signal nucleotide polymorphism) L620F以及八個位於表現子(exon)的c-Cbl突變型，其突變機率為百分之六點七。在三十七個台灣肺癌病人樣本中，有百分之二十一點六失去異合性(Loss of heterozygosity)的情形發生；另一方面，S80N/H94Y、Q249E、W802stop突變分別專一性發現於美國白種人、台灣人、非裔美人。在A549細胞中轉染此三種突變型c-CBL蛋白會導致細胞生長率以及移動能力的增加。最後，無論是在細胞或動物實驗中，將PHA665752以及c-CBL正常蛋白單獨或共同處理癌細胞有抑制其生長的現象，亦有抑制細胞轉移的情形。
結論：本研究為首篇在肺癌細胞中偵測OSU03013藥物之抑制癌細胞潛力，並由分子及蛋白質體學的研究結果發現此藥物會經由抑制PKA訊息傳導路徑來抑制癌細胞生長，並導致GSK3的去磷酸化，進而使在肺癌細胞中通常過度表現的-catenin被分解。此外，基於c-CBL可以負調控酪氨酸激酶接受器的表現，加上PHA665752亦可抑制c-Met在細胞中的過度表現，或許將來可以發展c-Cbl之基因治療，並且配合PHA665752的共同治療成為新的癌症治療方向。

Purpose: Non-small cell lung cancer (NSCLC) is a difficult disease to treat. Targeted cancer therapies are now the main issue for cancer treatment. Most lung cancer patients have strong drug resistence whether on radiation therapy or chemotherapy. Thus, novel anti-cancer drugs or therapeutic target identification are urgently needed. In part I, a novel small molecular, OSU03013, was used for investigating whether it can be a novel anti-cancer drug for lung cancer treatment. OSU03013 is a derivative of celecoxib, which is a known cyclooxygenase (COX)-2 inhibitor, has been proved that OSU03013 can inhibit prostate cancer cells growth through the 3-phosphoinositide-dependent kinase 1 (PDK1)/AKT signaling pathway. In addition, OSU03013 has been used in breast cancer treatment. Thus, the present study identified OSU03013 with better target selectivity in lung cancer. In party II, a new potential c-Met inhibitor PHA665752 was used for anti-tumor growth and migration in vitro and in vivo. In addition, mutation spectrum of new therapeutic target Cbl (Casitas B-lineage lymphoma) in lung cancer patients was investigated. Cbl located in chromosome 11q23 and one of the Cbl family proteins, c-CBL, has been recognized as a key player in the negative regulation of signaling pathways such as receptor tyrosine kinases (RTKs) c-Met and EGFR proteins. Thus, we hypothesized that c-Cbl mutations may contribute to the oncogenic potential of c-Met and EGFR. Moreover, combined treatment with PHA665752 and c-CBL wild type (WT) protein might provide a new therapeutic strategy.
Experimental Design: In part I, to investigate whether OSU03013 can be a potential drug for lung cancer treatment, the present study investigated its cytotoxicity mechanisms by flow cytometry and phosphatidylserine (PS) staining in A549, CL1-1, and H1435 lung cancer cell lines. In addition, this study identified the affected proteins by proteomics and confirmed the selected proteins by Western blot. The interaction between OSU03013 and potential target protein was tested by molecular modeling. In part II, the inhibitive ability of PHA665752 was examined by cytotoxicity measurement. In addition, c-Cbl mutation spectrum in tumor and corresponding normal tissues from total 119 of Taiwanese, Caucasian and African American lung cancer patients was examined by polymerase chain reaction and direct sequencing. Biological function of identified c-Cbl mutations was analyzed in cell models. Moreover, combined treatment and transfection of PHA665752 and c-Cbl WT were tested for anti-tumor cells growth and migration in vitro and in vivo.
Results: The results in part I indicated that OSU03013 showed highly cell selective cytotoxicity and the IC50 was at 1~4 M in all lung cancer cell lines tested at 48 h post-treatment. OSU03013 caused cell cycle G1 phase arrest and the cells treated with OSU03013 showed PS early apoptosis via endoplasmic reticulum (ER) stress. Several proteins such as heat shock protein 27, 70, and 90, cell division cycle 2 (CDC2), -tubulin, Annexin A3, cAMP-dependent protein kinase (PKA), glycogen synthase kinase 3-beta (GSK3and -catenin were identified by proteomics and confirmed by Western blot. In addition, molecular modeling suggested that OSU03013 competes with ATP to bind to PKA. In part II, c-Met showed overexpression in lung cancer cells. The inhibitor of c-Met, PHA665752 inihibited lung cancer cells proliferation, reduced the expression of c-Met, and induced early cell apoptosis. A known single nucleotide polymorphism L620F and eight c-Cbl mutations were identified in lung cancer samples of Taiwanese, Caucasian, and African American with the mutation rate of 6.7% (8/119). In addition, 21.6% (8/37) loss of heterozygosity was found in Taiwanese lung cancer patients. The mutation S80N/H94Y, Q249E, and W802stop were specifically identified from Caucasian, Taiwanese and African American, respectively. Ectopic expression of these c-CBL mutants showed increased cell viability and migration ability in A549 lung cancer cell line. Furthermore, PHA665752 treatment and c-Cbl WT ectopical expression alone or in combination reduced tumor cell growth and migration in both cell and animal models.
Conclusions: The present study identirfied for the first time that OSU03013 inhibits PKA activity and causes dephosphorylation of GSK3 leading to -catenin degradation, which is often overexpressed in lung cancer. The molecular and proteomic results show the potential of OSU03013 as an anti cancer drug for lung cancer. Also, the results of c-Cbl study provide new evidence that c-Cbl mutations play a role in lung tumorigenesis. In addition, combined treatment of PHA665752 compound and c-CBL WT protein expression would be the new therapeutic strategy for lung cancer.

Chinese Abstract-------------------------------------    1
English Abstract-------------------------------------    4
Staudy Basis-----------------------------------------    7
Specific Aims----------------------------------------    9

Part I.    Molecular Mechanisms of Cytotoxicity and
        Proteomics Approach for Potential Anti-
        Cancer Drug Osu03013 in Lung Cancer:
        involvement of ER response and the
        PKA/-catenin pathway-----------------------    12

Introduction-----------------------------------------    13
1.    Target therapy in cancer---------------------    13
2.    Small molecular: Potential ant-cancer
        drug-OSU03013--------------------------------    13
3.    Proteomic studies: Genome wild target
        proteins approach----------------------------    14
4.    The apoptosis mechanisms---------------------    15
4.1.    Caspases (cycteine-dependent aspartate
        specific proteases)--------------------------    16
4.2.    Pathways of apoptosis------------------------    16
4.2.1.    Extrinsic pathway - The death receptor
        mediated caspase activation pathway----------    17
4.2.2.    Intrinsic pathway - The cytochrome c
        initiated caspase activation pathway---------    17
4.2.3.    Endoplasmic reticulum (ER) Pathway-----------    18

Materials and Methods--------------------------------    20
1.    Cell culture---------------------------------    20
2.    Cytotoxicity measurement---------------------    20
3.    Flow cytometry-------------------------------    20
4.    Early apoptosis detection/
        Phosphatidylserine (PS) staining-------------    21
5.    AKT activity assay --------------------------    21
6.    Two-dimensional gel electrophoresis and
        image analysis and in-gel digestion and
        MALDI-TOF-MS and MALDI-TOF-MS/MS-------------    22
7.    Western blot---------------------------------    23
8.    Molecular modeling---------------------------    24

Results----------------------------------------------    26
1.    Cytotoxicity of OSU03013 to lung cancer
        cell lines A549, CL1-1, and H1435, and
        to normal lung cell line MRC5----------------    26
2.    OSU03013 induced endoplasmic reticulum (ER)
        stress in lung   cancer cell lines A549,
        CL1-1, and H1435-----------------------------    26
3.    OSU03013 induces G1 arrest and early
        apoptosis------------------------------------    27
4.    Affected proteins of treatment with OSU03013
        identified by 2-dimension electrophoresis/
        MALDI-Q-TOF----------------------------------    27
5.    Molecular modeling of OSU03013 to the cAMP-
        dependent protein kinase (PKA) and the
        activity assay by Western blot of its down
        stream proteins------------------------------    28

Discussion-------------------------------------------    29
References-------------------------------------------    34

Part II. c-Met inhibitor PHA665752 and c-Cbl
         Ubiquitination as lung cancer therapeutic
         targets in cell and animal models-----------    40

Introduction-----------------------------------------    41
1.    Tyrosine Kinase receptors (RTKs)-------------    41
1.1.    Hepatocyte growth factor receptor
        (HGFR/c-Met)---------------------------------    42
1.2.    Epidermal growth factor receptor (EGFR)------    42
2    RTKs as a arget of cancer therapy------------    43
2.1    Monoclonal antibody--------------------------    43
2.2    Tyrosine kinase inhibitors-------------------    44
2.3    Small molecular: Potential anti-cancer
        drug-PHA665752-------------------------------    45
3.    Ubiquitination-------------------------------    45
3.1.    CBL family proteins and structures-----------    46
3.2.    c-CBL protein downregulates protein
        tyrosine kinases-----------------------------    47
3.3.    Loss of function of CBL protein in human
        cancer---------------------------------------    48

Materials and Methods--------------------------------    49
1.    Tissue samples-------------------------------    49
2.    Cell culture---------------------------------    49
3.    Cell proliferation rate measurement----------    50
4.    c-Cbl gene mutational analysis---------------    50
5.    Plasmid constructs and site-directed
        mutagenesis----------------------------------    50
6.    Loss of heterozygosity (LOH) analysis--------    51
7.    Transfection of c-Cbl constructs-------------    52
8.    c-Cbl knockdown------------------------------    52
9.    Early apoptosis detection/
        Phosphatidylersine (PS) staining-------------    53
10.    Wound healing assay--------------------------    53
11.    Transwell assay------------------------------    54
12.    Western blot---------------------------------    54
13.    Flow cytometry-------------------------------    55
14.    Ubiquitin ligase activity--------------------    55
15.    Tumor growth and metastasis analyses
        in vivo--------------------------------------    56
16.    Tissue Western blot--------------------------    56
17.    Statistical analysis-------------------------    57

Results----------------------------------------------    58
1.    c-Cbl gene mutations in lung cancer----------    58
2.    c-Cbl mutations in different ethnic groups---    59
3.    Mutations in c-Met and EGFR can be
        co-associated with c-Cbl alterations in
        Taiwanese lung cancer patients---------------    59
4.    Cellular functions of c-Cbl alterations
        in the context of lung tumorigenesis---------    60
4.1.    E3 activity is intact in the mutant c-CBL
        proteins-------------------------------------    60
4.2.    Effect on lung cancer cell viability---------    60
4.3.    Effect on cell cycle-------------------------    61
4.4.    Effect on cell motility----------------------    62
4.5.    c-Cbl knockdown increases cell viability-----    62
5.    c-Cbl wild type transfection alone and
        combined with PHA665752 treatment------------    63
5.1.    In hibition of cell proliferation and
        phosphor-c-Met expression of PHA665752
        to lung cancer cell lines A549 and H226br ---    63
5.2.    c-Cbl WT transfection enhances PHA665752
        cytotoxicity potential ----------------------    63
5.3.    PHA665752 treatment but not c-CBL WT
        ectopic expression induces early apoptosis---    64
5.4.    Effect on cell motility----------------------    64
5.5.    c-Cbl WT transfection alone and combined
        PHA665752 treatment effectively inhibites
        cell growth in vivo--------------------------    65
5.6.    c-Cbl WT transfection and combined
        PHA665752 treatment effectively inhibites
        metastasis in vivo---------------------------    66

Discussion-------------------------------------------    67
References-------------------------------------------    72
Tables-----------------------------------------------    80
Figures----------------------------------------------    89
Appendix---------------------------------------------    117
                                

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