胰臟癌 (pancreatic cancer)是一種極度惡性的消化道癌症，目前並沒有尋找到治療胰臟癌的有效方法同時也缺乏早期檢測的手段。其中胰腺癌是胰臟癌最為常見的類型，胰腺癌的平均存活時間只有短六個月，且五年內存活率小於百分之八。本篇研究是針對人類胰腺癌 (pancreatic ductal adenocarcinomas, PDAC) 做研究，胰臟癌的發生也是經由是多階段性的癌化過程，且通常都與致癌基因的活化及抑癌基因的抑制或下調相關。在後期腫瘤微環境中，細胞處在低糖低氧等惡劣環境時會產生未摺疊蛋白反應 (unfolded protein response, UPR) 並導致內質網壓力 (ER stress) 的發生，此時伴護子蛋白GRP78會從內質網中解離並活化，在胰臟癌細胞中也可以看到GRP78的高度表現，但GRP78對於胰臟癌的關係並未完全釐清，本篇會著重於GRP78的探討，證明GRP78是一個具有意義的標靶目標。結果中可以看出不管是人類還是老鼠胰臟癌細胞中，在低葡萄糖環境下都可以誘導出GRP78的上調，此結果在蛋白質及RNA層面都可得到相同結果。並發現在胰臟癌癌化過程中，惡性程度與GRP78表現量成正比。在使用轉染技術降低胰腺癌細胞的GRP78表現量後，可以有效的降低其惡性程度，並且可以降低UPR路徑及葡萄糖代謝速率的表現。最後找出了對於治療胰臟癌有潛力標靶藥物AR-12。

Pancreatic cancer is an extremely malignant and lethal cancer disease, and there is still currently lack of effective treatment for pancreatic cancer treatment. Now we can’t provide any effective way for early detection of pancreatic cancer, neither for effective treatment drug to increase the survival rate for pancreatic cancer patients. Pancreatic ductal adenocarcinomas (PDAC) is the most common type of pancreatic cancer. It encourages us to study the tumor biology of PDAC since PDAC is one of the deadliest human malignancy worldwide. The carcinogenesis of pancreatic cancer is a multi-stage, associated with the activation of oncogenes and inhibition or down-regulation of tumor suppressor genes. In the tumor microenvironment, cancer cell will produce the unfolded protein reactions (UPR) and lead to ER stress when exposed to extreme environments such as hypoglycemia and hypoxia. At this time, the chaperone protein GRP78 will be dissociated and activated from the endoplasmic reticulum to regulate tumor cell proliferation and malignancy. In addition, GRP78 is also highly expressed in different types of cancer cells, but the detail mechanisms of GRP78 involved in pancreatic cancer progression is not fully understood. This study focuses on the study of the functional roles of GRP78 in PDAC development and suggested that GRP78 is a potential therapeutic target for PDAC treatment. Our results showed that pancreatic carcinogenesis could activate UPR pathway by upregulating GRP78 under low glucose tumor environment, and PDAC progression is positively correlated with the amount of GRP78 protein. Knockdown of GRP78 expression could effectively reduce the malignancy of PDAC cells, and modulated UPR pathway and glucose metabolism rate of PDAC cells. Finally, we founded AR-12 is a potential drug for PDAC treatment.

論文審定書 i
論文公開授權書 ii
摘要 iii
Abstract iv
第一章 序論 1
第二章 材料與方法 4
2.1細胞株培養 4
2.2基因工程小鼠 (genetic engineering mouse, GEM)小鼠基因型鑑定 4
2.3 細胞增生(proliferation)實驗 5
2.4病毒的製造及利用短髮夾RNA使基因表現的降低 6
2.5體外腫瘤細胞侵襲實驗
7
2.6西方墨點法 8
2.7 定量Quantitative RT-PCR analysis 9
2.8 體外細胞遷移(migration)實驗 10
2.9 細胞群落分析 11
2.10 流式細胞分析法 11
2.11 統計分析 12
第三章 結果 13
3.1確認在胰腺癌細胞中GRP78會受到低葡萄糖環境誘導而提升 13
3.2 在RNA層次中看出胰腺癌細胞中GRP78及其下游UPR pathway會受到低葡萄糖環境誘導而提升 14
3.3 胰臟癌的惡化程度與GRP78表現量成正比 15
3.4 降低GRP78在胰腺癌表現量後確實會降低癌細胞的惡性表徵 15
3.5 葡萄糖代謝能力及UPR路徑會因為降低GRP78在胰腺癌表現量而降低 16
第四章 討論 21
結果圖 24
圖一.胰臟癌細胞在不同環境中誘導GRP78的蛋白表現量 25
圖二. 胰臟癌細胞在低葡萄糖環境中誘導GRP78的表現量，並觀察細胞表徵 25
圖三. 在低葡萄糖環境中觀察小鼠胰臟癌細胞GRP78的蛋白表現量 27
圖四.在低葡萄糖環境下檢測胰臟癌細胞GRP78及下游訊號的表現量 28
圖五.在低葡萄糖環境下檢測胰臟癌細胞GRP78的RNA表現量 29
圖六.在低葡萄糖環境下檢測胰臟癌細胞VEGFA、GLUT1的RNA表現量 30
圖七.在低葡萄糖環境下檢測胰臟癌細胞GRP94、ATF4、XBP1的RNA表現量 31
圖八.比較正常胰臟及胰臟癌腫瘤GRP78的mRNA表現量 32
圖九. 利用胰臟癌基因鼠腫瘤切片進行組織染色比較不同時期的胰臟癌GRP78的表現量 33
圖十.觀察轉染細胞對於GRP78及EMT相關蛋白的表現量差異 34
圖十一.在癒合實驗中比較轉染細胞對於癒合能力的影響 35
圖十二.在聚落形成實驗中比較轉染細胞對於聚落形成能力的影響 36
圖十三.在聚落實驗中觀察轉染細胞對於聚落形成能力的影響 37
圖十四.在細胞增生實驗中觀察轉染細胞對於細胞增生能力的影響 38
圖十五.觀察轉染細胞對於GRP78及代謝相關蛋白的表現量差異 39
圖十六.在低葡萄糖環境下並合併觀察轉染後，對於GRP78的RNA表現量 40
圖十七.在低葡萄糖環境下並合併觀察轉染後，對於GRP78下游訊號的RNA表現量 41
圖十八.在低葡萄糖環境下並合併觀察轉染後，對於GRP94 RNA表現量 42
圖十九.在低葡萄糖環境下並合併觀察轉染後，對於GRP78及下游訊號的蛋白質表現量 44
圖二十.在低葡萄糖環境下並合併觀察轉染後，目標細胞的細胞週期變化 46
圖二十一.在不同環境且搭配轉染細胞中觀察GRP78的表現量，並觀察細胞表徵 47
圖二十二. 搭配GRP78抑制劑AR-12後，在癒合實驗中比較轉染細胞對於癒合能力的影響 49
圖二十三在細胞增生實驗中觀察轉染細胞合併給予AR-12對於細胞增生能力的影響 51
圖二十四.在低葡萄糖環境下並合併給予AR-12後，對於GRP78及下游訊號的蛋白質表現量影響 52
圖二十五.在給予AR-12後，觀察細胞的細胞週期變化 53
參考資料 54

1.Grzesiak, J.J., et al., The integrin-extracellular matrix axis in pancreatic cancer. Pancreas, 2007. 35(4): p. 293-301.
2.Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2018. CA: a cancer journal for clinicians, 2018. 68(1): p. 7-30.
3.Xu, Y.-F., et al., Abnormal distribution of peripheral lymphocyte subsets induced by PDAC modulates overall survival. Pancreatology, 2014. 14(4): p. 295-301.
4.Curley, S.A., et al., Noninvasive radiofrequency treatment effect on mitochondria in pancreatic cancer cells. Cancer, 2014. 120(21): p. 3418-3425.
5.Pereira, E.R., et al., Transcriptional and post-transcriptional regulation of proangiogenic factors by the unfolded protein response. PloS one, 2010. 5(9): p. e12521.
6.Ghosh, R., et al., Transcriptional regulation of VEGF-A by the unfolded protein response pathway. PloS one, 2010. 5(3): p. e9575.
7.Mahadevan, N.R., et al., Transmission of endoplasmic reticulum stress and pro-inflammation from tumor cells to myeloid cells. Proceedings of the National Academy of Sciences, 2011. 108(16): p. 6561-6566.
8.Ni, M. and A.S. Lee, ER chaperones in mammalian development and human diseases. FEBS letters, 2007. 581(19): p. 3641-3651.
9.Wang, M., et al., Role of the unfolded protein response regulator GRP78/BiP in development, cancer, and neurological disorders. Antioxidants & redox signaling, 2009. 11(9): p. 2307-2316.
10.Yuan, X., et al., GRP78 promotes the invasion of pancreatic cancer cells by FAK and JNK. Molecular and cellular biochemistry, 2015. 398(1-2): p. 55-62.
11.Wu, H.-M., et al., Reversed expression of GRIM-1 and GRP78 in human non–small cell lung cancer. Human pathology, 2014. 45(9): p. 1936-1943.
12.Reddy, R.K., et al., Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors role of ATP binding site in suppression of caspase-7 activation. Journal of Biological Chemistry, 2003. 278(23): p. 20915-20924.
13.Fu, Y., J. Li, and A.S. Lee, GRP78/BiP inhibits endoplasmic reticulum BIK and protects human breast cancer cells against estrogen starvation–induced apoptosis. Cancer research, 2007. 67(8): p. 3734-3740.
14.Zhou, H., et al., Novel mechanism of anti-apoptotic function of 78-kDa Glucose-Regulated Protein (GRP78) endocrine resistance factor in breast cancer, through release of b-cell lymphoma 2 (bcl-2) from bcl-2-interacting killer (bik). Journal of Biological Chemistry, 2011. 286(29): p. 25687-25696.
15.Li, J., et al., The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells. Cell death and differentiation, 2008. 15(9): p. 1460.
16.Misra, U.K., S. Payne, and S.V. Pizzo, Ligation of prostate cancer cell surface GRP78 activates a proproliferative and antiapoptotic feedback loop: a role for secreted prostate-specific antigen. Journal of Biological Chemistry, 2011. 286(2): p. 1248-1259.
17.Shani, G., et al., GRP78 and Cripto form a complex at the cell surface and collaborate to inhibit transforming growth factor β signaling and enhance cell growth. Molecular and cellular biology, 2008. 28(2): p. 666-677.
18.Ma, Y. and L.M. Hendershot, The role of the unfolded protein response in tumour development: friend or foe? Nature Reviews Cancer, 2004. 4(12): p. 966.
19.Lee, A.S., GRP78 induction in cancer: therapeutic and prognostic implications. Cancer research, 2007. 67(8): p. 3496-3499.
20.Pyrko, P., et al., The unfolded protein response regulator GRP78/BiP as a novel target for increasing chemosensitivity in malignant gliomas. Cancer research, 2007. 67(20): p. 9809-9816.
21.Pootrakul, L., et al., Expression of stress response protein Grp78 is associated with the development of castration-resistant prostate cancer. Clinical Cancer Research, 2006. 12(20): p. 5987-5993.
22.Zhuang, L., et al., Expression of glucose‐regulated stress protein GRP78 is related to progression of melanoma. Histopathology, 2009. 54(4): p. 462-470.
23.Jamora, C., G. Dennert, and A.S. Lee, Inhibition of tumor progression by suppression of stress protein GRP78/BiP induction in fibrosarcoma B/C10ME. Proceedings of the National Academy of Sciences, 1996. 93(15): p. 7690-7694.
24.Dong, D., et al., Critical role of the stress chaperone GRP78/BiP in tumor proliferation, survival, and tumor angiogenesis in transgene-induced mammary tumor development. Cancer research, 2008. 68(2): p. 498-505.
25.Fu, Y., et al., Pten null prostate tumorigenesis and AKT activation are blocked by targeted knockout of ER chaperone GRP78/BiP in prostate epithelium. Proceedings of the National Academy of Sciences, 2008. 105(49): p. 19444-19449.
26.Booth, L., et al., OSU-03012 suppresses GRP78/BiP expression that causes PERK-dependent increases in tumor cell killing. Cancer biology & therapy, 2012. 13(4): p. 224-236.
27.Lee, H., et al., Ganetespib induces G2/M cell cycle arrest and apoptosis in gastric cancer cells through targeting of receptor tyrosine kinase signaling. International journal of oncology, 2017. 51(3): p. 967-974.
28.Zhang, J., et al., Celecoxib derivative OSU-03012 inhibits the proliferation and activation of hepatic stellate cells by inducing cell senescence. Molecular medicine reports, 2015. 11(4): p. 3021-3026.
29.Zhang, S., et al., OSU-03012, a novel celecoxib derivative, is cytotoxic to myeloma cells and acts through multiple mechanisms. Clinical cancer research, 2007. 13(16): p. 4750-4758.