臺灣博碩士論文加值系統

目前已知大腸直腸癌之癌化過程為一連串的抑癌基因缺失或致癌基因活化所導致，其中已被證實的包括了APC、KRAS、SMAD4以及p53等。APC的缺失，被認為是腸道上皮細胞增生形成瘜肉的第一步驟，進而累積突變形成惡性腫瘤。近年研究指出，大腸直腸腫瘤檢體中可發現抑癌基因Phosphatase and tensin homolog deleted on chromosome ten (PTEN)的突變或表現量下降，且與臨床上之預後情形與藥物治療有關，但詳細機制尚未明確證實。於是本篇論文利用小鼠模式，探討PTEN在大腸直腸癌化過程中所扮演的角色。實驗使用誘導式Cre/LoxP系統建立K8-CreERT; Apcfx/+ (簡稱為Apcfx/+)與K8-CreERT; Apcfx/+;Ptenfx/+ (簡稱為Apcfx/+;Ptenfx/+)基因轉殖小鼠，利用Keratin 8之驅動子驅使Cre recombinase表現於單層柱狀上皮中，其中包括了腸道上皮細胞，並於小鼠六周成熟時腹腔注射tamoxifen，引起Cre recombinase進入細胞核，剔除Apc與Pten的單等位基因模擬大腸直腸癌化過程，進而探討Pten是否於大腸直腸癌化過程中扮演角色及其調控機制。
結果發現，Apcfx/+;Ptenfx/+小鼠出現直腸腫瘤情形明顯較Apcfx/+小鼠為早且數目較多，顯示Pten之單等位基因缺失會加速Apcfx/+小鼠之直腸腫瘤形成與增加腫瘤數目。進一步利用組織切片分析腫瘤之細胞型態，並利用免疫組織化學染色觀察Apc缺失所活化之下游β-catenin與CyclinD1，發現Apcfx/+與Apcfx/+;Ptenfx/+小鼠所生成之異常增生腺窩病灶(aberrant crypt foci, ACF)與腺瘤(adenoma)皆有β-catenin於細胞質堆積及進核現象，而於腺瘤當中則有CyclinD1高表現細胞核中。利用CyclinD1與BrdU的共同免疫螢光染色卻發現，腫瘤當中表現CyclinD1與BrdU的細胞恰好分開，表示CyclinD1表現不直接反映正進行複製之癌細胞。統計後發現，Apcfx/+;Ptenfx/+小鼠中有62.5%的腫瘤屬於CyclinD1High/BrdULow，而Apcfx/+小鼠中則僅有20%，顯示Pten的單等位基因缺失可能影響了腫瘤細胞表現CyclinD1的情形。
此外為了觀察Pten單等位基因缺失是否影響了大腸直腸癌化過程中重要的基因突變率，首先利用免疫螢光染色觀察腫瘤當中Pten的表現情形，結果發現分別於Apcfx/+和Apcfx/+;Ptenfx/+小鼠中各發現一個腫瘤的Pten的表現量下降。利用腫瘤組織抽取基因組核醣核酸(genomic DNA)，並放大K-ras基因第12個密碼子(codon12)，進行基因定序後並未發現有點突變發生。利用免疫組織化學染色觀察腫瘤中p53的表現量，結果發現Apcfx/+小鼠中有一個腫瘤出現p53過度表現情形，伴隨著Pten的表現量下降及侵襲至黏膜下層(submucosa layer)形成惡性腺瘤(adenocarcinoma)。結果顯示，Pten之單等位基因缺失並非經由增加腫瘤累積已知的抑癌基因缺失或致癌基因活化，導致直腸腫瘤的加速形成或數目增加。
為了尋找Pten之單等位基因缺失是否更容易造成腫瘤中產生未知的基因片段缺失，利用晶片式比較基因體雜交技術(array-based comparative genomic hybridization; array-CGH)偵測異常核醣核酸套數(copy number aberrartion; CNA)。分析後發現Cdkn1a (p21), Ccdc11和Nup85等CNAs於Apcfx/+及Apcfx/+;Ptenfx/+小鼠中的部分腫瘤皆有偵測出。Lrrtm4和Sumo2等CNAs則只有於部分Apcfx/+小鼠之腫瘤中發現，然而Pisd, Pisd-ps1/3, Sfi1和Acta2/Wtap則只有於Apcfx/+;Ptenfx/+小鼠中的部分腫瘤被偵測出。結果顯示，Pten單等位基因缺失可能會造成腫瘤累積不同小片段基因缺失，導致腫瘤加速形成。

APC, Adenomatous Polyposis Coli, has been suggested as a critical gate-keeper involved in the initiation step of colorectal tumor formation. PTEN is a well studied tumor suppressor gene (TSG) in various types of malignancies. However, the causal mechanisms of Pten loss in colorectal tumor progression remain controversial. To investigate the role of Pten in colorectal tumor progression, I generated the K8-CreERT; Apcfx/+ (referred to Apcfx/+) mice as a tumor model, in which one allele of Apc was ablated in simple columnar epithelial cells after tamoxifen injection at 6 weeks old. In addition, I crossed the K8-CreERT; Apcfx/+ mice with the Ptenfx/fx allele to obtain K8-CreERT; Apcfx/+;Ptenfx/+ (referred to Apcfx/+;Ptenfx/+) mice in order to generate a Pten heterozygosity in the Apc heterozygous rectal tumor model.
Compared with Apcfx/+ mice, we found that Apcfx/+;Ptenfx/+ mice developed more rectal tumors with shorter latency. This result suggests that monoallelic ablation of Pten increases tumor numbers and accelerates tumor progression in Apc heterozygous background. To investigate how Pten loss promotes tumor progression, I examined the expression pattern of β-catenin and CyclinD1 which involved in Wnt/β-catenin canonical pathway in the rectal tumors. I had found that the number of rectal tumors with higher percentage of cyclinD1+ cells in the Apcfx/+;Ptenfx/+mice, was more than the Apcfx/+mice. This result suggests an epistatic interaction between the Pten and Apc/β-catenin downstream signaling circuits.
In order to investigate whether the Pten monoallelic lesion would cause the mice developing more tumors which loss of Pten expression, I examined the Pten expression pattern in the rectal tumors using IF staining. This result reveals that only one tumor loss of Pten expression in Apcfx/+ and Apcfx/+;Ptenfx/+ mice. Furthermore, I examined the status of K-ras gene and the expression of p53, there was no K-ras codon12 point mutation detected in tumors. The tumor sections were stained with p53 using IHC staining and revealed that only one tumor over-expressed p53 in Apcfx/+ mice after tamoxifen injection 47 weeks. These results reveals that the Pten monoallelic lesion would not altered the point mutation rate of K-ras and the percentage of p53 over-expression in rectal tumors.
Furthermore, in order to identify other genetic alterations, I used array-CGH to detect the copy number aberrations (CNAs) of the colorectal tumors in the Apcfx/+ and the Apcfx/+;Ptenfx/+ mice. I found that the CNAs of Cdkn1a (p21), Ccdc11, Chst9 and Nup85 were commonly detected in both types of colorectal tumors. The CNAs of Lrrtm4, Sumo2 were specifically detected in rectal tumors of Apcfx/+ mice, whereas Pisd, Pisd-ps1/3, Sfi1 and Wtap were detected only in rectal tumors of Apcfx/+;Ptenfx/+ mice. These findings suggest that some genetic lesions (or hits) may be commonly created during colorectal tumor progression; some lesions may be specifically created in the tumors initiated from Apc and Pten double heterozygous mutations. Taken together, monoallelic Pten loss accelerates tumor progression at least in part by elevating the expression of cyclinD1 and by generating additional genetic lesions in colorectal tumors initiated from epithelial cells with Apc mutations.

Contents i
Abstract in Chinese vi
Abstract in English viii

I. Introduction 1
I-1 Colorectal cancer 1
I-1.1 Epidemiology and physiology of CRC 1
I-1.2 The structure of colon and rectum 2
I-1.3 The progression of CRC 2
I-1.4 The mouse model of CRC 3
I-2 The genetic alterations and colorectal tumor progression 5
I-2.1 The sequential model and the alternative path model 5
I-2.2 APC and Wnt/??catenin canonical pathway 6
I-2.3 The models of Apc deficient mice 7
I-3 Phosphatase and tensin homolog deleted on chromosome
10 (Pten) 9
I-3.1 The structure of PTEN 9
I-3.2 PTEN and the PI3K/AKT pathway 9
I-3.3 The tumor suppressor role of Pten 10
I-3.4 The clinical studies of PTEN in CRC 11
I-4 Specific aims 13
I-4.1 Working hypothesis 13
I-4.2 Aims 13
I-4.3 Experimental approaches 13

II. Materials and methods 14
II-1 Mice 14
II-1.1 Tg (K18-EGFP; K8-CreERT) 14
II-1.2 Apcfx/fx 14
II-1.3 Ptenfx/fx 14
II-2 DNA preparation and genotyping 15
II-2.1 Genomic DNA preparation (Alkaline hydrolysis method) 15
II-2.2 Genotyping (polymerase chain reaction, PCR analysis) 15
II-2.2.1 Cre allele 15
II-2.2.2 Apc flox allele 15
II-2.2.3 Pten flox allele 16
II-3 Administration of tamoxifen and BrdU 16
II-3.1 Tamoxifen administration 16
II-3.2 Bromodeoxyuridine (BrdU) administration 16
II-4 Whole mount EGFP visualization 17
II-5 Histology and immunostaining analysis 17
II-5.1 Tissue processing and sectioning 17
II-5.2 Hematoxylin and eosin staining (H&E staining) 17
II-5.3 Immunohistochemical staining (IHC) 18
II-5.4 Immunofluorescent staining (IF) 19
II-5.5 Indirect immunofluorescence with tyramide signal
amplication (TSA) 19
II-6 Tumor-free analysis 20
II-7 Detection of K-ras G12D point mutation 20
II-8 Array-based comparative genomic hybridization (array-CGH) 21
II-8.1 Genomic DNA extraction (phenol/ chloroform method) 21
II-8.2 Array-CGH and data analysis 22
II-8.3 Genomic PCR analysis 22
II-8.3.1 Ccdc11 22
II-8.3.2 AK047074 22
II-8.3.3 Cdkn1a (p21) 22
II-8.3.4 Sfi1 23
II-8.3.5 Sumo2, Nup85 23
II-8.3.6 Lrrtm4 23
III. Results 24
III-1 Generation of the K8-CreERT; Apcfx/+; Ptenfx/+ transgenic mice 24
III-2 Pten monoallelic lesion accelerated the rectal tumor progression and increased tumor number in Apcfx/+ mice 25
III-3 Comparing the histology and the expression pattern of Apc, β-catenin and CyclinD1 in the tumors of Apcfx/+ and Apcfx/+;Ptenfx/+ mice 26
III-4 Screening for the expression pattern of Pten, p53, and the point mutation of Kras gene in colorectal tumor progression 28
III-5 Genome wide DNA alterations in colorectal tumors 30

IV. Discussions 32
IV-1 Using K8-CreERT;Apcfx/+ mice as a model to investigate the role of Pten in colorectal tumor progression 32
IV-2 The biological studies of Pten in CRC 34
IV-3 Signal transduction of CRC in Apcfx/+ and
Apcfx/+;Ptenfx/+ mice 36
IV-4 Pten haplo-insufficiency in colorectal tumor progression 38
IV-5 Genomic alterations in early stage colorectal adenoma 39

References 42
Figures 61
Figure 1 Generation of the CRC transgenic mice model 61
Figure 2 Whole mount EGFP expression of rectal tumors in Apc fx/+ and Apc fx/+;Pten fx/+ mice 62
Figure 3 The tumor free curve of Apcfx/+ and Apc fx/+;Ptenfx/+ mice 63
Figure 4 Comparison of the rectal tumor size at different time point between Apcfx/+ and Apcfx/+;Ptenfx/+ mice 64
Figure 5 Comparison of the rectal tumor number at different time point between Apcfx/+ and Apcfx/+;Ptenfx/+ mice 65
Figure 6 Comparison of the β-catenin/CyclinD1 expression profile and BrdU labeling at different stage of colorectal tumor between Apcfx/+ and Apcfx/+;Ptenfx/+ mice 67
Figure 7 The distribution CyclinD1+ and BrdU+ cells in rectal tumors 68
Figure 8 The percentage of CyclinD1High/BrdULow tumors is higher in Apcfx/+;Ptenfx/+ mice 69
Figure 9 Pten expression was down-regulated in some of
the colorectal tumors 70
Figure 10 Sequences of the Kras oncogene in colorectal tumors from Apcfx/+ and Apcfx/+;Ptenfx/+ mice 71
Figure 11 p53 over expression in colorectal tumors 72
Figure 12 The statistics of K-ras and p53 status in the tumors of Apcfx/+ and Apcfx/+;Ptenfx/+ mice 73
Figure 13 The diagram of Array based Comparative Genome Hybridization (Array-CGH) 74
Figure 14 The copy number deletions detected by array-CGH 75
Figure 15 Genomic PCR validation of the candidate genes detected in array-CGH 76
Figure 16 The proposed model for alternative genomic
aberration path of colorectal tumor progression in Apcfx/+ and Apcfx/+;Ptenfx/+ mice 77
Tables 78
Table 1 Specific-deleted CNAs detected in the tumors derived from Apcfx/+ mice or Apcfx/+;Ptenfx/+ mice 78
Table 2 Common CNAs detected in the tumors derived from Apcfx/+ and Apcfx/+;Ptenfx/+ mice 79

Appendix 80
Appendix 1 The sequential model of chromosomal instability in colorectal tumor progression 80

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