背景和目的：胰腺導管腺癌（簡稱胰腺癌）是世界範圍內惡性程度最高的癌癥之一，目前它的5 年生存率不到5%。大部分的病人在診斷初期就已經發展到了局部浸潤或遠處轉移的階段，因此失去了根治性手術切除的机会。輔助性化療對於胰腺癌病人來說是一個首選的治療方案，但是目前只有一小部分病人對化療藥物有良好的反應，而臨床化療失敗常與腫瘤細胞對化療藥物產生耐藥有關。吉西他濱是目前臨床上常用的一線抗癌藥物，但是它的耐藥現象在胰腺癌病人中廣泛存在，也是阻礙其臨床應用的主要原因之一。盡管已經有很多研究致力於揭示吉西他濱在胰腺癌細胞中的耐藥機理，目前臨床上仍然沒有有效的方法應對吉西他濱耐藥。我們的研究主要是為了探討一些以前沒有报道過的參與吉西他濱耐藥機理的基因，借此揭示胰腺癌細胞的吉西他濱耐藥的深層機制，為臨床上的治療提供理論依據。 / 實驗方法：我們實驗室之前在胰腺癌細胞株Capan2 中用全基因組RNAi篩選的方法確定LLGL1 作為抑癌基因能增強吉西他濱在胰腺癌細胞中的細胞毒性。我們隨後用體外細胞毒性分析實驗和皮下腫瘤動物模型來驗證LLGL1 是否能增強吉西他濱的細胞毒性，用蘇木素-伊紅染色和原味末端轉移酶標記技術分析抑制LLGL1 的表達是否會影響吉西他濱誘導的細胞雕亡反應。我們還應用微陣列分析技術進一步探尋LLGL1 的下遊靶蛋白，用實時定量PCR(qRT-PCR) 、蛋白印跡法（western blotting）、熒光素酶檢測等技術來進一步證實LLGL1 與下遊靶蛋白的關系，用免疫組織化學方法探究LLGL1 下遊靶蛋白在胰腺癌組織中的表達情況，以及該蛋白與LLGL1 的表達相關性，還應用染色體免疫共沈澱的方法探討轉錄因子Sp1(pThr453) 和RNA 聚合酶 II 在LLGL1 下遊靶蛋白的啟動子上的富集情況。 / 實驗結果：LLGL1 能增強吉西他濱在胰腺癌中的細胞毒性，抑制該基因的表達能誘導胰腺癌細胞對吉西他濱的耐藥，而上調該基因的表達則會增強胰腺癌細胞對吉西他濱的細胞毒性反應。OSMR 是LLGL1 的下遊靶蛋白, 其在胰腺癌組織中的表達與LLGL1 呈負性相關，抑制OSMR 的表達可以逆轉由LLGL1表達下調引起的吉西他濱耐藥現象。OSMR 表達上調可以增強腫瘤幹細胞標記物CD44 和CD24 的表達。另外，在胰腺癌細胞中，抑制LLGL1 的表達能激活ERK2/Sp1 信號通路，導致磷酸化Sp1(pThr453)的表達升高。OSMR 啟動子既沒有TATA 元件也沒有INR 元件，但是有Sp1 结合元件可供Sp1 結合。磷酸化Sp1(pThr453)可以結合到OSMR 啟動子的Sp1 结合元件上，從而促使RNA 轉錄酶II 結合到該啟動子上，啟動OSMR 基因的轉錄。 / 結論：我們的研究發現：1，LLGL1 能增強吉西他濱在胰腺癌中的細胞毒性，抑制該基因在胰腺癌細胞中的表達能上調OSMR 的表達，並誘導吉西他濱耐藥；2，OSMR 的表達在胰腺癌組織中與LLGL1 呈負性相關；3，下調LLGL1的表達能激活ERK2/Sp1 信號通路，進一步導致磷酸化Sp1(pThr453)和RNA 轉錄酶II 在OSMR 啟動子上的聚集，最終促使OSMR 的高表達，而下調LLGL1的表達能抑制該調節通路，從而抑制OSMR 的轉錄。 / Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers worldwide. Its 5-year survival rate is less than 5%, because most patients have already developed to the advanced stage of local invasion or distant metastasis once diagnosed, and missed the chances of curable surgical resection. Adjuvant chemotherapy is an alternative therapeutic strategy against PDAC. Yet, only very small proportion of patients could benefit from chemotherapy due to the innate and easily-acquired chemo-resistance in PDAC cells, especially to the first-line chemotherapeutic drug, gemcitabine. Many studies have been conducted to exploring the mechanisms underlying gemcitabine resistance in PDAC cells, but gemcitabine resistance is still the major obstacle impeding PDAC patients benefits from chemotherapy. Our studies aimed to investigate novel genes involved in gemcitabine response and to explore the undefined mechanisms generating gemcitabine resistance in PDAC cells. / Methods: Our colleagues previously performed genome-wide RNAi screening in gemcitabine-sensitive Capan2 cells. Lethal giant larvae homolog 1 (LLGL1) was identified as a potential gemcitabine-sensitizing gene which was then validated by our subsequent in-vitro drug cytotoxicity assay in LLGL1-inhibited Capan2 and SW1990 cells and in vivo subcutaneous xenograft mouse model. Hematoxylin & Eosin staining and terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling were applied for the assessment of apoptotic effects induced by gemcitabine in subcutaneous xenografts. We did gene expression microarray analysis to explore the potential downstream targets of LLGL1. Western blotting, qRT-PCR, and luciferase assay were applied to validate the downstream target of LLGL1 that were figured out by microarray analysis. We also did immunohistochemical staining to investigate the expression levels and correlationship of LLGL1 and its downstream target in PDAC specimens. Chromatin immunoprecipitation was performed to explore the enrichment of the transcriptional factor Sp1(pThr453) and RNA polymerase II (Pol II) at the promoter of the downstream targets of LLGL1. / Results: LLGL1 was identified as a gemcitabine-sensitizing gene, whose inhibition remarkably reduced gemcitabine response in gemcitabine-sensitive Capan2 and SW1990 cells, and ectopic expression induced gemcitabine response in gemcitabine-resistant PANC1 cells. Oncostatin M receptor (OSMR) was identified as a downstream target of LLGL1, whose expression was negatively correlated with LLGL1, and knockdown of OSMR significantly reversed gemcitabine resistance induced by LLGL1 inhibition in Capan2 and SW1990 cells. Additionally, activation of OSMR signaling was associated with the elevated expression of cancer stem cell markers, CD44 and CD24, both of which had already been identified to contribute to gemcitabine resistance in PDAC cells. Moreover, OSMR up-regulation induced by LLGL1 inhibition in SW1990 cells depended on the activation of ERK2/Sp1 signaling and subsequent accumulation of Sp1(pThr453) and Pol II at the TATA-less, INR-less but Sp1-binding-site-rich promoter of OSMR, while ectopic expression of LLGL1 in PANC1 cells inactivated ERK2/Sp1 signaling and subsequently reduced the enrichment of Sp1(pThr453) and Pol II at OSMR promoter. / CONCLUSIONS: Our studies revealed the novel tumor suppressive role of LLGL1 as a gemcitabine-sensitizing gene in PDAC cells. Loss of LLGL1 resulted in the activation of ERK2/Sp1 signaling and up-regulation of OSMR expression, and ultimately desensitized gemcitabine response in PDAC cells. More importantly, ectopic expression of LLGL1 disrupted such regulatory axis and improved gemcitabine response. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhu, Yinxin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 154-183). / Abstracts also in Chinese.

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive human malignancies, is thought to be initiated by KRAS activation. Here, we find that transcriptional activation mediated by the GLI family of transcription factors, although dispensable for pancreatic development, is required for KRAS induced pancreatic transformation. Inhibition of GLI using a dominant-negative repressor (Gli3T) inhibits formation of precursor Pancreatic Intraepithelial Neoplasia (PanIN) lesions in mice, and significantly extends survival in a mouse model of PDAC. Further, ectopic activation of the GLI1/2 transcription factors in mouse pancreas accelerates KRAS driven tumor formation and reduces survival, underscoring the importance of GLI transcription factors in pancreatic tumorigenesis. Interestingly, we find that although canonical GLI activity is regulated by the Hedgehog ligands, in the context of PDAC, GLI transcription factors initiate a unique ligand-independent transcriptional program downstream of KRAS, that involves regulation of the RAS, PI3K/AKT, and NF-кB pathways. We identify I-kappa-B kinase epsilon (IKBKE) as a PDAC specific target of GLI, that can also regulate GLI transcriptional activity via positive feedback mechanism involving regulation of GLI subcellular localization. Using human PDAC cells, and an in vivo model of pancreatic neoplasia, we establish IKBKE as a novel regulator pf pancreatic tumorigenesis that acts as an effector of KRAS/GLI, and mediates pancreatic transformation. We show that genetic knockout of Ikbke leads to a dramatic inhibition of initiation and progression of pancreatic intraepithelial viii neoplasia (PanIN) lesions in mice carrying pancreas specific activation of oncogenic Kras. Furthermore, we find that although IKBKE is a known NF-кB activator, it only modestly regulates NF-кB activity in PDAC. Instead, we find that IKBKE strongly promotes AKT phosphorylation in PDAC in vitro and in vivo, and that IKBKE mediates reactivation of AKT post-inhibition of mTOR. We also show that while mTOR inhibition alone does not significantly affect pancreatic tumorigenesis, combined inhibition of IKBKE and mTOR has a synergistic effect leading to significant decrease tumorigenicity of PDAC cells. Together, our findings identify GLI/IKBKE signaling as an important oncogenic effector pathway of KRAS in PDAC that regulates tumorigenicity, cell proliferation, and apoptosis via regulation of AKT and NF-кB signaling. We provide proof of concept for therapeutic targeting of GLI/IKBKE in PDAC, and support the evaluation of IKBKE as a therapeutic target in treatment of pancreatic cancer, and IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts. Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR. In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells. These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies in the United States, with an average five-year survival rate of just 6.7%. One unifying aspect of PDAC is mutational activation of the KRAS oncogene, which occurs in over 90% of PDAC. Therefore, inhibiting KRAS function is likely an effective therapeutic strategy for this disease, and current research in our lab and others is focused on identifying downstream effectors of KRAS signaling that may be therapeutic targets. miRNAs are powerful regulators of gene expression that can behave as oncogenes or tumor suppressors. Dysregulation of miRNA expression is commonly observed in human tumors, including PDAC. The mir-17~92 cluster of miRNAs is an established oncogene in a variety of tumor contexts, and members of the mir-17~92 cluster are upregulated in PDAC, but their role has not been explored in vivo. This dissertation encompasses two studies exploring the role of miRNAs in pancreatic tumorigenesis. In Chapter II, I demonstrate that deletion of the mir-17~92 cluster impairs PDAC precursor lesion formation and maintenance, and correlates with reduced ERK signaling in these lesions. mir-17~92 deficient tumors and cell lines are also less invasive, which I attribute to the loss of the miR-19 family of miRNAs. In Chapter III, I find that Dicer heterozygosity inhibits PDAC metastasis, and that this phenotype is attributable to an increased sensitivity to anoikis. Ongoing experiments will determine whether shifts in particular miRNA signatures between cell lines can be attributed to this phenotype. Together these findings illustrate the importance of miRNA biogenesis, and the mir-17~92 cluster in particular, in supporting PDAC development and progression.