臺灣博碩士論文加值系統

癌症幹細胞是指具有幹細胞特性的癌細胞，其擁有自我更新以及分化成腫瘤細胞的能力。雖在腫瘤中通常只占一小部分，但被認為是造成癌症惡化或抗藥性的主要原因。為了進一步探討癌症幹細胞之主要控制基因在攝護腺癌的惡化過程所扮演的角色，我們建構出一套可模擬攝護腺癌惡化轉移的細胞模式進行研究。人類攝護腺癌細胞CWR22Rv1經由原位注射打入老鼠攝護腺內，14周後，將轉移到肺組織中的攝護腺癌細胞進行分離培養，並命名為14w CWR22Rv1。我們發現，在癌症轉移惡化過程中，第二型肝細胞生長因子活化抑制者(HAI-2) 的表現有隨之下降的趨勢，同時與癌症幹細胞相關的蛋白質如Nanog和Oct-4，以及其他與癌症惡化相關的蛋白質，如間質蛋白酶 (matriptase)的表現和c-Met等訊息傳遞分子的活化也隨之升高。為了進一步研究HAI-2 在攝護腺癌惡化過程中所扮演的角色，我們利用HAI-2的shRNA減弱HAI-2的基因表現，結果顯示，降低HAI-2的表現量使癌症幹細胞相關分子Sox2的表現增加，並伴隨著細胞侵襲、移動力以及自我更新能力的提升。同時也致使matriptase和c-Met、EGFR以及ERK訊息分子的活化。當將HAI-2過量表現於較為惡化的14w CWR22Rv1細胞時，Sox2、matriptase的表現量以及c-Met分子的活化現象相對受到抑制，並抑制了細胞自我更新的能力。我們進一步的研究顯示間質蛋白酶的大量表達和HAI-2基因表達的減弱，對上述分子在攝護腺癌細胞所造成的影響非常類似，並且有促進細胞自我更新的作用。此外，我們觀察到降低HAI-2的表現量所誘使Sox2表現上升的情況也隨著將c-Met的激酶活性的抑制而減弱。綜合上述結果，這些證據顯示HAI-2在攝護腺癌細胞中可經由間質蛋白酶和c-Met訊息的傳遞路徑來調控癌症幹細胞相關分子，例如Sox2基因的表達。

Cancer stem cells (CSCs)/cancer initiating cells represent a minor population of tumor cells with the properties of self-renewal or cancer progenitor cells, contributing to tumor progression. To further delineate the molecular mechanisms of prostate cancer progression linked to the expression changes of stem cell-related genes, a prostate cancer progression model was established by orthotopic injection of prostate cancer CWR22Rv1 cells into nude mice. Fourteen weeks after the injection, the tumor cells metastazing into the lung were isolated as 14w CWR22Rv1 cells. Interestingly, the expression level of hepatocyte growth factor activator inhibitor type 2 (HAI-2) in 14w CWR22Rv1 cells was down-regulated, while the expression levels of the stem cell-related transcription factors (Oct-4 and Nanog), the activated level of matriptase and the tyrosine phosphorylation level of c-Met were increased in these cells. To further clarify if HAI-2 played a role in modulating prostate cancer malignancy, we used HAI-2 shRNAs to silence HAI-2 expression, and found that the knockdown of HAI-2 enhanced CWR22Rv1 cell invasion, migration, sphere formation, and the expression levels of Sox2. Moreover, HAI-2 knockdown activated matriptase and the phosphorylation of c-Met, EGFR and ERK. In addition, overexpression of HAI-2 or HAI-1 in 14w CWR22Rv1 cells down-regulated the expression of Sox2 and significantly activated matriptase and the phosphorylation of c-Met. Our data further showed that an increase of matriptase activation by overexpression had similar effects to HAI-2-knockdown cells on Sox2 expression, the phosphorylation of c-Met as well as sphere-forming potentials. Furthermore, inhibition of the c-Met kinase activity in HAI-2-knockdown cells reduced the expression of Sox2. Therefore, our data suggest that HAI-2 may play an inhibitory role in regulating the stem cell-related properties of prostate cancer cells through modulating matriptase and c-Met pathways.

致謝 II
摘要 III
Abstract IV
Chapter 1: Introduction 1
1.1 Prostate cancer 2
1.2 Cancer stem cells 2
1.3 Cancer stem cell markers 3
1.4 Hepatocyte growth factor activator inhibitor type 2 (HAI-2) 4
1.5 Matriptase and HAI-1 5
1.6 c-Met signaling 6
1.7 Research Motivation 9
Chapter 2: Materials and methods 10
2.1 Materials 11
2.2 Methods 14
Chapter 3: Results 22
3.1 Establishment and characterization of a lung-metastatic prostate cancer model 23
3.2 Roles of HAI-2 in the expression of stem cell-related transcription factors 25
3.3 Inhibitory roles of HAI-2 in prostate cancer cell invasion, migration and self-renewal capacities 26
3.4 Matriptase and signaling pathways in human prostate cancer cell progression model 27
3.5 Roles of HAI-2 knockdown in matriptase, c-Met, EGFR and ERK in prostate cancer cells 28
3.6 Roles of HAI-1 and HAI-2 on the expression levels of stem cell-related transcription factors in 14w CWR22Rv1 cells 29
3.7 Effects of HAI-1 and HAI-2 overexpression on matriptase, c-Met, EGFR and ERK in 14w CWR22Rv1 cells 30
3.8 Inhibitory roles of HAI-1 and HAI-2 in the sphere-forming capacities of 14w CWR22Rv1 cells 30
3.9 Role of matriptase in the expression of stem cell-related transcription factors and in the phosphorylation levels of c-Met and ERK 31
3.10 Inhibition of c-Met kinase down-regulated the expression of Sox2. 32
Chapter 4: Discussion 34
Chapter 5: Figure 40
Chapter 6: References 61

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