臺灣博碩士論文加值系統

p21活化激酶（PAKs）是一個受到Rho GTPases家族調控的激酶，參與了細胞骨架重排，細胞流動，細胞增生，凋亡和轉化等相關的生物功能。根據基因序列，結構同源性和活化的機制，將六個PAK分為兩大類，第一類為PAK 1-3（I組）和第二類為PAK 4-6（II組）。已經在很多不同類型的人類腫瘤中檢測到PAK的異常表達，其中包括乳癌，大腸癌，卵巢癌，膀胱癌，胰臟癌和腦癌，並且PAKs的過度表達與腫瘤的生成過程和抗藥性都有非常大的關聯。相較於其他的PAK成員，PAK3如何調節癌細胞功能中的作用機制仍然不清楚。在我們的研究中發現並證明，PAK3可以調控其中幾種胰腺癌的細胞株並且透過Akt激酶來調節和活化Akt-GSK3β-β-catenin此路徑的訊號傳遞。
特別地，當我們抑制PAK3或過度表達顯性抑制突變PAK3時會抑制Ser473-Akt和GSK3β的磷酸化，從而導致β-catenin的降解。相反地，當PAK3過表達則導致激活Akt的訊號傳遞並且造成β-catenin的表達增加。但是這些情況，在我們進行PAK1，PAK2或PAK4的沉默和/或過表達時並未觀察到相同的訊號傳遞變化，這些訊號傳遞讓我們聯想到PAK3可能是作為控制這些胰臟癌細胞惡性表型和胰臟癌幹細胞的關鍵調控因子。
因此，PAK3的減少可以有效地抑制了tumorsphere的形成，ALDH活性以及胰臟癌幹細胞表面生物標記的表達。此外，我們在AsPC-1細胞株中使用穩定的剔除並降低PAK3的表現來證明PAK3可以抑制體內腫瘤的形成和異種移植腫瘤生長的功能。最後，這些發現告訴我們PAK3可能可以作為治療胰臟癌時一個有潛力的標靶，這部分值得未來更進一步的研究。

The p21-actiated kinases (PAKs) are important effectors of the Rho GTPases and have been implicated in cytoskeletal remodeling, cell motility, cell proliferation, apoptosis, and transformation. Based on the sequence, structure homology, and activation mechanism, six PAKs are classified into two groups, PAK 1-3 (group I) and PAK 4-6 (group II). Aberrant expression of PAKs have been detected in many types of human tumors, including breast, colon, ovarian, bladder, pancreas, and brain, and the overexpression of PAKs is linked to tumor progression and drug resistance. Relative to several other p21-activated kinase (PAK) family members, the role of PAK3 in regulating cancer cell functions remains unclear. Our study obtained evidence that PAK3 regulates the Akt-GSK3β-β-catenin signaling by acting as Ser473-Akt kinase in several pancreatic cancer cell lines. Specifically, knockdown of PAK3 or overexpression of dominant-negative PAK3 inhibited the phosphorylation of Ser473-Akt and GSK3β, resulting in the proteasomal degradation of β-catenin. Conversely, overexpression of PAK3 led to activation of Akt signaling and increased β-catenin expression. These changes, however, were not noted with the silencing and/or overexpression of PAK1, PAK2, or PAK4, facts which underlie the impetus of PAK3 as a key effector in governing malignant phenotypes in these pancreatic cancer cells, including cancer stem cell (CSC) expansion. Accordingly, PAK3 depletion effectively suppresses tumorsphere formation, ALDH activity, and the expression of CSC surface markers. Moreover, we used a stable knockdown clone of AsPC-1 cells to demonstrate the in vivo efficacy of PAK3 inhibition in suppressing tumorigenesis and xenograft tumor growth. Together, these findings suggest the potential role of PAK3 as a target for pancreatic cancer therapy, something which warrants further investigations.

國立臺灣大學博士學位論文口試委員會審定書 i
謝誌 ii
中文摘要 iii
Abstract iv
Abbreviations vi
Contents viii
List of Figures xi
List of Tables xvi
Chapter 1. Introduction and Research Background 1
1.1 Pancreatic cancer 1
1.2 The structure and activation mechanism of PAKs 2
1.3 Biological functions of PAKs 4
1.4 PAK activation and amplification in cancer 5
1.5 PAKs regulation of cell signals in human Cancer 5
1.5.1 The Function of PAK1 in cancer 6
1.5.2 The Function of PAK2 in cancer 7
1.5.3 The Function of PAK3 in cancer 8
1.5.4 The Function of PAK4 in cancer 8
1.5.5 The Function of PAK5 in cancer 9
1.5.6 The Function of PAK6 in cancer 9
1.6 Wnt/β-catenin signaling in pancreatic cancer 10
1.7 Pancreatic cancer stem cells and PAKs’ role in stemness 11
1.8 Specific aims and experimental designs 12
Chapter 2. Materials and Methods 23
2.1 Cell culture and reagents 23
2.2 shRNA lentivirus preparation and isolation of stable cell lines 24
2.3 Co-immunoprecipitation (Co-IP) 24
2.4 Immunocytochemical analysis 24
2.5 Tumorsphere formation assays 25
2.6 Immunoblotting 25
2.7 RT -PCR analysis 25
2.8 Soft agar colony formation assays 26
2.9 Colony formation assays 26
2.10 Cell proliferation assays 26
2.11 Plasmid construction and transient transfection 26
2.12 ALDH staining 27
2.13 Migration assays 27
2.14 Animal models 27
2.15 Statistics analysis 28
Chapter 3. Results 29
3.1 Evidence that PAK3 is involved in the regulation of pancreatic cancer cell proliferation 29
3.2 Evidence that PAK3 regulates β-catenin protein stability in pancreatic cancer cells 30
3.3 PAK3 regulates β-catenin stability via Akt/GSK3β signaling 32
3.4 Evidence that PAK3 interacts with Akt and PAK3 facilitates Ser473-Akt phosphorylation 33
3.5 Role of PAK3 in regulating of CSC-like properties in pancreatic cancer cells 35
3.6 In vivo efficacy of PAK3 knockdown in suppressing tumorigenicity and xenograft tumor growth in nude mice 36
3.7 Correlation of PAK3 and Ser-473 Akt phosphorylation in the course of pancreatic tumorigenesis in transgenic KPfl/flC mice 37
3.8 Analysis of the PAK3 for Metastasis and drug resistance in Pancreatic Cancer cell lines 37
Chapter 4. Discussion 64
Chapter 5. Perspective 69
Chapter 6. References 71
Appendix 81

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