本論文主要探討在非轉型的老鼠細胞NIH/3T3模式中，肝癌衍生生長因子(Hepatoma derived growth factor; HDGF)誘發細胞移行的機轉。實驗顯示，纖維母細胞在HDGF刺激下能有效地促進細胞的移行能力及增加細胞骨架重組的現象，也會誘發細胞訊息蛋白質-磷酸肌醇3-激酶(PI3K)的表現，促進同源性磷酸酶-張力蛋白(PTEN)與蛋白激B (Akt) 磷酸化及增加小型G蛋白鳥嘌呤三磷酸酶(Rho GTPases): RhoA, Rac1 及Cdc42的活性。另外，腺病毒攜帶PTEN基因傳送到NIH/3T3-HDGF細胞株後會抑制HDGF所誘發的PI3K/Akt/Rho GTPases訊息路徑。同樣地， PI3K抑制劑-LY294002亦可阻擾HDGF所誘導的細胞移行及細胞骨架的重組及RhoA, Rac1 及Cdc42蛋白質的活化。這些結果證實HDGF乃透過活化PI3K/Akt /Rho GTPases 訊息路徑及細胞骨架重組進而誘發細胞移行的進行。 另外，本論文也發現HDGF在乳癌組織的表現相較正常組織表現量高，且乳癌細胞核內的HDGF labelling index (LI)與腫瘤分化程度,腫瘤分期以及增值指數呈正相關;而與乳癌病人的存活率呈負相關。我們的結果也顯示HDGF過度表現亦與淋巴結轉移(TNM)有相關並可作為對腫瘤復發的預後因子。此外，本論文也探討HDGF如何誘導上皮-間質細胞形變的發生。
實驗發現，癌化程度高的細胞株-MDA-MB-231細胞，其HDGF的表現量高於非轉型的細胞株-MCF-7 細胞。MDA-MB-231細胞在表現細胞侵犯能力及形成細胞群落的能力上優於MCF-7細胞。無論以腺病毒攜帶HDGF基因過量表現或外加HDGF蛋白質皆會刺激細胞的侵犯性與增加細胞群落的形成，也會使得鈣黏著素E (E-cadherin)下降及中間絲蛋白(Vimentin)的增加。相反的，無論shHDGF RNA干擾序列傳送、HDGF抗體中和實驗或BITC處理也發現，抑制MDA-MB-231細胞的HDGF表現會使得細胞的E-cadherin增加而Vimentin下降，因而抑制其侵犯能力與上皮-間質細胞形變的發生。
總結我們以上的研究得知，HDGF可利用活化細胞的PI3K/Akt /Rho GTPases訊息傳遞路徑來刺激細胞骨架的重組，因而改變細胞移行能力。而此結果，也誘發細胞的上皮-間質細胞形變現象發生並促使細胞的侵犯能力提高，因而促使癌細胞的轉移。所以我們研究結果，不僅提供HDGF在誘發細胞移行及轉移的新見解，而且也證實HDGF可作為乳癌預後指標的可行性。

關鍵詞:肝癌衍生生長因子,磷酸肌醇3-激酶,蛋白激酶B,上皮-間質細胞形變,鈣黏著素E, 中間絲蛋白

In this study, we investigated the mechanisms of HDGF on cell migration in non-transformed NIH/3T3 cells. HDGF promoted the migration and the formation of dorsal ruffles and podosome rosettes. Besides, HDGF supply increased the PI3K expression, Akt phosphorylation and PTEN phosphorylation as well as stimulated the RhoA, Rac1, and Cdc42 activities. Furthermore, Adenoviral gene transfer of PTEN attenuated migration and PI3K/Akt/Rho GTPases signaling in HDGF-overexpressing transfectants. Pharmaceutical intervention using the PI3K inhibitor, LY294002, potently reversed HDGF-stimulated cell migration, dorsal ruffles formation and podosome formation as well as the RhoA, Rac1, and Cdc42 activities. Thus, HDGF elicits the activation of PI3K/Akt /Rho GTPases signaling cascade and promotes cytoskeleton remodeling to stimulate cellular migration. Moreover, we investigate the expression profile of HDGF during breast carcinogenesis. Immunohistochemical studies revealed elevated HDGF expression in human breast cancer. Nuclear HDGF labelling index was positively correlated with tumour grade, stage and proliferation index, but negatively correlated with survival rate in breast cancer patients. Our data also showed that HDGF over-expression was associated with lymph node metastasis and represented an independent prognostic factor for tumor recurrence. Furthermore, Immunoblot study revealed that elevated HDGF expression significantly higher in breast cancer cells (MDA-MB-231 cells) than that in non-transformed breast cells (MCF-7 cells). Consistently, higher invasive potency and colony formation also observed in MDA-MB-231 cells than in MCF-7 cells. Adenovirus-mediated HDGF over-expression and exogenous HDGF treatment stimulated the invasiveness and colony formation as well as E-cadherin down-regulation and Vimentin up-regulation. Conversely, either HDGF knockdown by RNA interference, HDGF antibody neutralization or BITC-induced EMT suppression in MDA-MB-231 cells attenuated the malignant behavior and elicited EMT reversal by enhancing E-cadherin expression while depleting Vimentin expression.
In summary, HDGF elicits the activation of PI3K/Akt/Rho GTPases signaling cascade, thereby promoting cytoskeleton remodeling to stimulate cellular migration. Moreover, the formation of podosome rosettes is correlated with cell invasion, the podosome-stimulating capability of HDGF is consistent with HDGF regulates the metastasis of breast cancer through modulating of epithelial-mesenchymal transition. Therefore, our results provide not only novel insights into the role of the HDGF in cell migration and tumor metastasis, but also validate a novel prognostic indicator for breast cancer.

Key words: Hepatoma-derived growth factor, PI3K, Akt, epithelal-mesenchemal transition, E-cadherin, Vimentin

Abstract in Chinese …………………………………… 2
Abstract in English ...………………………………….. 4
Abbreviation …………………………………… 8
Chapter 1 Background
1-1 Introduction ...…………………………………. 9 1-2 Specific Aims …..……………………………… 13
1-3 Figures and Legends …..……………………………… 14
Chapter 2 Delineation of the Mechanism Underlying HDGF Induced Migration in Non-transformed NIH/3T3 cells
2-1 Introduction .…………………………………. 16
2-2 Materials and Methods ……………..……………………. 22
2-3 Results ….…………….………………… 28
2-4 Discussion ....……………………………….. 35
2-5 Figures and Legends ……….……………………….... 39
Chapter 3 Investigation on the Expression Profiles of HDGF in Breast Cancer Progression
3-1 Introduction …………………………………. 56
3-2 Materials and Methods ……………..…………………… 61
3-3 Results .....……………………………… 63
3-4 Discussion ..………………………………… 65
3-5 Tables .…….…………………………… 67
3-6 Figures and Legends .…..…………..………………… 72

Chapter 4 Clarification on the role of HDGF in Epithelial- Mesenchymal transition of breast cancer cells
4-1 Introduction ..………………………………… 76
4-2 Materials and Methods …...……………………………… 81
4-3 Results ….……………………………… 85
4-4 Discussion .………………………………… 89
4-5 Figures and Legends ….……………………………… 92
Chapter 5 Conclusion ………………………………… 108
Reference ..……………………………… 110 Publications and Conferences ………………………………… 122

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