臺灣博碩士論文加值系統

流行病學及動物實驗數據顯示乳癌的生長及轉移受到雌性激素濃度的影響。已發現雌性激素受體有兩種(ERα和ERβ)。在乳癌細胞中已知雌激素活化ERα 並經由基因組和非基因組途徑增加細胞增生；而活化的ERβ 主要經由非基因組途徑，利用修飾ERα的活性而降低細胞增生。於4T1腫瘤小鼠模式中，雌激素增加腫瘤轉移至肺臟。因4T1乳癌細胞並沒有ERα 的表現且雌激素不會增加其增生，目前並不清楚雌激素如何誘導4T1乳癌細胞的轉移及其機制。已知腫瘤轉移需要腫瘤細胞本身的癌化以及腫瘤微環境中發生血管新生。癌化的過程包括腫瘤細胞增生、去分化及擴散。因此本篇論文的目的是研究雌激素誘導4T1乳癌細胞於小鼠體內擴散之細胞機制。長期暴露雌激素於4T1腫瘤小鼠增加腫瘤的擴散面積，但並不會影響腫瘤的總量，若施打ERβ 拮抗劑PHTPP則可減少由雌激素增加之腫瘤擴散面積。在細胞實驗中，雌激素不影響4T1細胞的存活率及細胞週期，但會促進細胞型態的改變且增加細胞爬行的能力。更進一步測量血管新生相關蛋白的表現量也發現，雌激素使得原位癌處的血管內皮細胞生長因子A以及血管內皮生細胞長因子受體2的表現量增加，但並不會直接影響4T1乳癌細胞的血管內皮細胞生長因子A和血管內皮生細胞長因子受體2表現量。數據顯示雌激素並不會導致ERβ 由細胞質轉移至細胞核內，利用鑑別性的蛋白質譜分析細胞質內蛋白的改變，發現雌激素降低細胞骨架微管蛋白及絲切蛋白的含量，並利用西方墨點轉漬法確認雌激素降低α-微管蛋白的表現量。而太平洋紫杉醇促進微管蛋白的聚合作用但不影響微管蛋白含量。在雌激素存在下，太平洋紫杉醇降低由雌激素增加之細胞爬行能力。我們的結果顯示毋須ERα存在，雌激素藉由調控微管蛋白加速4Τ1乳癌細胞擴散。

Both epidemiological and animal studies show a positive association of breast tumor growth and metastatic spread with circulating levels of 17β-estradiol (E2). Two estrogen receptors (ERα and ERβ) in various breast tumor cells have been identified. The binding of E2 to ERα induces cell proliferation via both genomic and non-genomic pathways. However, ERβ suppresses ERα -induced cell proliferation. In tumor bearing mice, E2 increase tumor metastasis to lung in 4T1-implanted mice. Because E2 does not induce cell proliferation in 4T1 which does not contain ERα, it is not clear how estrogens increase the metastasis of 4T1. It has been well recognized that tumor metastasis requires the tumorigenesis of tumor cell and angiogenesis in the microenvironment. Tumorigenesis includes tumor proliferation, dedifferentiatioin, and migration. The purpose of the study was to investigate the cellular mechanisms of estrogen-induced 4T1 spreading in mice. Our data showed that E2 did not affect tumor mass but increased the spreading areas in 4T1-implanted mice. An ERβ antagonist, PHTPP, prevented E2-induced increases of spreading areas. E2 did not directly increase cell viability and cell cycle but induced the morphological change and migration ability of 4T1 as shown in both Wound-Healing and chemotaxis assays. We further examined the effect of E2 on angiogenesis. E2 did not directly influence the expression of VEGFA and VEGFR2 in 4T1 cell, but increased VEGF in tumor tissues. Because E2 did not cause the translocation of ERβ from cytosol to nucleus in 4T1, cellular mechanisms in 4T1 which facilitate tumor migration was then explored after quantitative proteomic analyses. Functional annotation indicated that E2 decreased the abundance of cofilin (an actin-regulated protein), α- and β- tubulin. Western blot analysis confirmed the decreased expression of α-tubulin by E2. Paclitaxel which induces the polymerization of tubulin did not affect 4T1 migration and the abundance of α and β-tubulin. In the presence of E2, paclitaxel prevented E2-induced 4T1 migration examined by both wound healing and chemotaxis assay. In conclusion, activation of ERβ may facilitate the spreading of tumor cells mianly by inducing tumor cell spreading through tubulin-dependent pathways. The significance of this study is that E2 facilitate tumor spreading by altering the composition of microtubule in ERα(-)4T1 breast tumor cells.

Abstract I
中文摘要 III
誌謝 V
Index VII
List of tables XI
List of figures XII

Chapter I. Introduction 1

I. Development of breast tumor 1
Epidemiological studies of breast cancer 1
Cell cycle progression and tumor proliferation growth 1
Epithelial–mesenchymal transition 2
Migration and tumor spreading 3
VEGF-induced angiogenesis and tumor expansion 4

II. Estrogen and breast tumor formation 4
Estrogen and reproductive function 4
Contribution of estrogen to breast tumor growth and metastasis 5

III. Current studies of ER isoforms (ERs) in tumorigenesis and angiogenesis 5
The effect of ERs on proliferation 6
The effect of ERs on EMT 7
The effect of ERs on migration 7
The effect of ERs on angiogenesis 8
Chapter II. Motive of this study 9
Controversial role of ERα and ERβ in breast tumor formation 9
Unsolved issues of estrogens on breast tumor formation 9
The purpose and design of this study 9

Chapter III. Materials and Methods 11
I. Establishment of tumor-bearing mice with E2 capsule implantation 11
1. Animals 11
2. Cell culture 11
3. Preparation of E2 capsule 12
4. Implantation of E2 capsule into tumor-bearing mice 13
5. In vivo image 14

II. Functional assays in 4T1 cell 15
1. MTT assay 15
2. Analysis of cell cycle by flow cytometry 15
3. Counting of elongated cell 16
4. Wound healing assay 16
5. Boyden chamber assay 17
6. Immunofluorescence staining 17

III. Sample preparation 18
1. Tumor tissue homogenate 18
2. Cell lysate 18
3. Subcellular fraction 18

IV. Western blot and Proteomic analysis 19
1. Western blot analysis 19
2. In solution digestion 20
3. Stable isotope dimethyl labeling 20
4. Mass spectrometry and protein identification 22
5. Quantitative analysis of dimethyl labeled peptides 24

V. Data analysis and statistical evaluation 24

Chapter IV. Results 25
1. The effect of E2 on the tumor area and tumor mass in the primary tumor site 25
2. The effect of E2 on the tumor mass in lungs 26
3. The effect of E2 on cell proliferation 26
4. The effect of E2 on EMT 26
5. The effect of E2 on cell migration 27
6. The expression of VEGFA and VEGFR2 in primary tumor site 27
7. The expression of VEGFA and VEGFR2 in 4T1 27
8. Subcellular localization and translocation of ERβ in 4T1 27
9. Quantitative proteome of cytosolic proteins in 4T1 28
10. Proposed hypothesis of estrogen-induced changes in cell phenotypes 29
11. To test the hypothesis that the increased production of tubulin isoforms enhances estrogen-induced migration in 4T1 29

Chapter V. Discussion 31
Summary of this study 31
E2-increased tumor spreading in mice 32
Acceleration of tumorigenesis by long-term treatment with E2 34
Increase of angiogenesis-related factors by long-term treatment of E2 37
E2-induced alterations in various cytoskeleton proteins in 4T1 38
Facilitation of cell migration by estrogen through tubulin-dependent pathways 40
Significance of this study 41

Chapter VI. Reference 43
Chapter VII. Tables 51
Chapter VIII. Figures 70
Curriculim Vitae 92

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