十年之前胰臟癌的五年存活率不到5%，十年過去了胰臟癌的五年存活率依然不到8%。儘管近年來有非常多的團隊、成就非常多的科學努力但我們僅增加了3%的五年存活率。而造成此現象的最大問題在於，胰臟癌的早期症狀不明顯及難以偵測，待有胰臟癌相關症狀被診斷出來時往往已經是晚期並且轉移到其他器官。對於胰臟癌如何發展成高轉移及轉移性前的研究變成胰臟癌治療及延長存活目前最重要的研究課題 - 為何後期的胰臟癌會開始侵犯到其他組織並且發生遠端轉移？許多的研究指出腫瘤組織及其周圍的腫瘤微觀環境在腫瘤的形成過程中會大量表現TGF-β，而這些TGF-β可能是影響癌症轉移的關鍵。已經有許多癌症轉移的研究指出，TGF-β會參與腫瘤的形成過程，並且促使腫瘤轉移的發生。有趣的是在胰臟癌中，TGF-β的下游訊息傳遞中SMAD4有高達50%的突變率，這些訊息透露出胰臟癌的發生以及進程與TGF-β訊息傳遞的極大關聯性。我的博士論文分為兩部分，第一部分是研究Krueppel樣因子10（KLF10），也稱為TGFβ誘導的早期生長反應蛋白1（TIEG-1）對胰腺發育的影響及探討及對於胰臟癌之影響。一開始我們證明胰腺上皮中有條件的KLF10缺失對胰腺發育及生理學沒有可辨別的影響。然而與突變KRASG12D基因搭配時，KLF10喪失使KRASG12D誘導的腫瘤發生快速進展。雖然單獨KRASG12D的突變會引起癌前期胰腺上皮內瘤變（PanIN）緩慢演進至高程度的PanINs，但KRASG12D和KLF10缺失的組合會導致人類胰腺（PDAC）腫瘤的快速發展。 然而KLF10的丟失也加速了KRASG12D; P53L / L搭配的小鼠動物模型的胰腺癌快速發展，並通過參與SDF-1 / CXCR4的路徑的活化了轉移前。在我的研究的第二部分中，我們研究了另一種TGFβ1/ SMAD下游阻遏物TGFβ誘導因子同源框1（TGIF1），以研究TGIF1在PDAC發展中的潛在作用。我們選擇性刪除TGIF1，使用Cre-Loxp系統與Pdx-1Cre KrasG12D和Pdx-1Cre; KRASG12D; P53L / L模型搭配，以研究TGIF1缺失對PDAC惡性程度的影響。我們觀察到Pdx-1Cre; KRASG12D; TGIF1L / L; P53L / L PDAC模型顯示胰腺癌形成伴隨著高頻率的肺和肝轉移。我們進一步發現TGIF1缺失透過活化HAS2-CD44信息傳遞和上調PD-L1促進KRASG12D誘導的胰腺癌的發展。最後我們的研究結果皆顯示這些KLF10-TGIF1所調控的訊息傳遞網絡可能可以成為治療胰腺癌轉移的新治療目標。

The overall five-year survival rate for pancreatic cancer reported as low as 5% a decade ago, even now still less than 10%. The main issue in pancreatic cancer is patients in the early stages of pancreatic cancer have no obvious symptoms which leads to be diagnosed almost delay to advanced stages when patients harbored abdominal pain, jaundice or ascites. To explore what makes pancreatic cancer becoming highly invasive and metastatic may prolong survival for pancreatic cancer patients. Several studies have demonstrated that the increased TGF-β secretion in the tumor microenvironment may associate with the survival and poor prognosis of pancreatic cancer patients. Importantly, Smad4, a member of the SMAD family of signaling transduction, which acts as a pivotal mediator of TGF-β, was identified to be inactivated in more than 50% of pancreatic cancer. Alternatively, inactivation of the downstream factors in the TGF-β/SMAD signaling pathway may also affect the development of pancreatic. My thesis work is divided into two parts, the first part of the thesis is to investigate the effect of Krueppel-like factor 10 (KLF10), also named TGFβ-inducible early growth response protein 1(TIEG-1) on pancreas development and pancreatic cancer progression. We demonstrated that conditional KLF10 deletion in the pancreatic epithelium had no discernable impact on pancreatic development or physiology. However, when combined with the activated KRASG12D allele, KLF10 loss enabled rapid progression of KRASG12D –induced tumorigenesis. While activation of KRASG12D alone elicited premalignant pancreatic intraepithelial neoplasia (PanIN) that progressed slowly to high grades of PanINs, the combination of KRASG12D and KLF10 deficiency resulted in the rapid development of tumors resembling pancreatic ductal adenocarcinoma (PDAC) in humans. KLF10 loss also accelerated PDAC development of KRASG12DP53L/L compound mice and altered the prometastatic phenotype by involving the activation of SDF-1/CXCR4 stemness pathway. In the second part of my study, we investigate another TGFβ1/SMAD downstream repressor TGFβ-induced factor homeobox 1 (TGIF1) to study the potential role of TGIF1 in PDAC development. We selective deleted TGIF1, by using Cre-Loxp system to cross with Pdx-1Cre KrasG12D and Pdx-1CreKRASG12D P53L/L models in order to clarify the impact of TGIF1 deletion on malignant progression of PDAC. We observed that Pdx-1CreKRASG12DTGIF1L/LP53L/L PDAC model displayed the high frequency of lung and liver metastasis during PDAC formation in mutant mice. We further revealed that TGIF1 loss contributed to the progression of KRASG12D-induced PDAC through activation of HAS2-CD44 signaling pathway and upregulation of PDL1. Lastly, our results suggested that these KLF10-TGIF1 involving signaling networks might potentially become new therapeutic nodes for the treatment of pancreatic cancer metastasis.

國立中山大學研究生學位論文審定書 i
摘要 ii
Abstract iv
Chapter I 1
Abstract 2
Introduction 4
Material & Methods 7
Genetically modified mice and mouse genotyping 7
Immunohistochemistry 8
Western blot analysis 8
Mouse cytokine array analysis 9
RNA extraction and microarray detection 9
Complementary DNA microarray analysis 9
GeneGo analysis 10
Real-time–quantitative PCR analysis (RT–qPCR) 11
Cell proliferation assay 11
Primary pancreatic cell culture 11
Plerixafor treatment 11
Wound-healing assay 12
Luciferase reporter assay 12
Soft agar colony formation assay 13
Retroviral production and infection of target cells 13
Mice and injections 13
Statistical analysis 14
Results 15
KLF10 is not required for normal pancreas development in mice. 15
KLF10 loss in the pancreas rapidly provokes mutant Kras-induced PanIN to PDAC. 16
Homozygous deletion of KLF10 accelerates development of metastatic PDAC in Pdx-1-Cre; LSL-KrasG12D/+; p53L/L model. 18
Pathological and immunohistological analysis of PDAC derived from Pdx-1-Cre; LSL-KrasG12D/+; p53L/L; KLF10L/L model. 20
Anti-apoptosis in response to TGFβ and induction of EMT to enhance cell migration in KLF10 null PDAC tumor cells. 21
Characterization of the gene expression profile of Pdx1-Cre; LSL-KrasG12D/+; p53L/L; KLF10L/L PDAC tumor cells. 23
KLF10 loss induces SDF1 expression to promote in vitro cell migration of PDAC. 24
SDF1/CXCR4 antagonist, Plerixafor delays PDAC development and metastasis on mouse pancreatic cancer. 26
Discussion 30
Figures 39
Figure 1. Depletion of KLF10 in the mouse pancreas does not perturb pancreatic development and function. 41
Figure 2. Concomitant KLF10 loss and activated KrasG12D to drive rapid malignant PDAC development in mice. 44
Figure 3. KLF10 deletion promotes metastatic PDAC in cooperation with KrasG12D activation and P53 deficiency. 47
Figure 4. IHC characterization of metastatic PDAC in PKKP mice. 51
Figure 5. Loss of KLF10 modulates TGFb1 and WNT pathways to promote EMT and cell motility in PDAC. 55
Figure 6. cDNA microarray analysis of primary PDAC cells from PKKP and PKP mice. 58
Figure 7. KLF10 loss induces SDF-1 up-regulation resulting in increased c-Jun phosphorylation and enhanced cell migration of PDAC in vitro. 62
Figure 8. Inhibition of SDF-1/CXCR4 signaling blocks PDAC formation in PKKP and PKP mice. 65
Tables 66
Table 1. Comparison of incidence of metastatic lesions present in various organs of Pdx-1Cre LSL-KrasG12D p53L/L and Pdx-1CreLS L-KrasG12DKLF10L/L p53L/L mice. 67
Table 2. Top 10 differentially regulated gens between murine Pdx-1Cre LSL-KrasG12D p53L/L and Pdx-1CreLS L-KrasG12DKLF10L/L p53L/L PDAC cells. 68
Table 3 List of the primary antibodies used in this study, and information on working dilutions of antibodies for Western blotting, immunohistochemistry (IHC) and immunofluorescence (IF) analyses. 69
Table 4. List of the primer sequences used in this study 72
Chapter II 73
Abstract 74
Introduction 76
Material and Methods 79
Genetically modified mice and mouse genotyping 79
Immunohistochemistry (IHC) and immunofluorescence (IF) 80
Human tissue array analysis 81
Western blot analysis 81
Mouse cytokine array analysis 81
Real-time–quantitative PCR analysis (RT–qPCR) 82
Cell proliferation assay 82
Wound-healing assay 82
Glucose tolerance test (GTT) 82
Complementary DNA microarray analysis 83
GeneGo analysis 83
Murine primary PDAC cell culture, cytokine and inhibitor treatment 84
Xenograft isogenic mice 84
Genomic DNA isolation and bisulfite treatment 85
Methylation-specific polymerase chain reaction (MSP) 85
Statistical analysis 86
Lentivirus production and shRNA for gene knockdown 86
Retroviral production and infection of target cells 86
RESULTS 87
Pancreas-specific TGIF1-deficiency in mice does not alter pancreatic development or induce tumorigenesis. 87
An activating KrasG12D mutation coupled with TGIF1 loss is sufficient to initiate pancreatic tumorigenesis in mice. 88
TGIF1 loss accelerates PDAC development with reduced survival and increased metastatic behaviors 90
TGIF1 loss alters tumor microenvironment to suppress tumor immune response in PDAC 91
TGIF1-deficient PDAC displays enhanced EMT program and cancer stem cell (CSC)-like phenotype 94
TGIF1 loss stimulates HAS2 expression to activate HY/CD44 signaling in KrasG12D p53 loxp/loxp PDAC model. 96
TGIF1 loss links to epigenetic regulation of tumorigenesis in PDAC. 98
Assessment effect of HAS2 inhibitor, 4-methylumbelliferone (4-MU), on HA/CD44 signaling and growth, migration of PDAC cells in vitro. 99
Discussion 101
Figures 109
Figure 9. TGIF1 expression in PDAC and TGIF1 ablation in the pancreas do not interrupt pancreatic development in mice. 112
Figure 10. Conditional TGIF1 deletion accelerates progression to PDAC in cooperation with KrasG12D. 114
Figure 11. Conditional TGIF1 deletion promotes the development of highly metastatic PDAC in KrasG12D P53L/L models. 116
Figure 12 Modulation of the inflammatory cytokine profile and tumor immune response in the PDAC microenvironment by TGIF1. 120
Figure 13. TGIF1 loss promotes EMT and CSC activity to increase PDAC cell migration and invasiveness. 125
Figure 14. cDNA microarray analysis compared the differential gene expression of murine primary PDAC cells derived from PKP and PKTP mice. 129
Figure 15. Epigenetic modulation of TGIF1 and targeting HAS2 suppresses TGIF1 loss-induced PDAC cell migration in vitro. 133
Tables 134
Table 1 List of the primay antibodies used in this study, and information on working dilutions of antibodies in Western blotting (WB), immunohistochemistry (IHC) and immunofluorescence (IF). 135
Table 2. List of the primers used in this study. 139
Table 3. List of the MSP primers used in this study. 140
Table 4. Comparison of incidence of metastatic lesions present in various organs of PKP and PKTP mouse models. 141
References 142
Publication 154

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