Vaults, recently discovered in 1986, are multi-subunit organelles with a molecular mass of ,--,13 MDa. The specific function of vaults is unknown, although they are believed to be involved in internal transport. These ribonucleoproteins are composed of the major vault protein, which comprises ' 70% of the vault's mass, two minor proteins, TEP1 and vPARP, and untranslated RNA(s). It is believed that the protein components of the vault are structural while the RNAs are the functional components. Implications of the vault's involvement in multi-drug resistance in cancer have been made. In some resistant cancer cells, the major vault protein and vRNA(s) are up-regulated up to 15 times when cells are exposed to a cytotoxic drug. Cytotoxic drugs such as doxorubicin are administered as a cancer treatment, but may be ineffective because the drug is actively pumped out of the cell. Multi-drug resistance is the most common failure of chemotherapeutic cancer treatment. In order to prevent the development of multi-drug resistance this research employed the use of small interfering RNA technology to down-regulate the expression of one of the vault RNAs, vRNA3, in cultured GLC4 cells, a small-cell lung cancer cell line. If the vRNA(s) are the functional portion of the vault and a cloned siRNA prevents their up-regulation after drug exposure, the cells should lose their multi-drug resistance, stimulating apoptosis. If successful, this approach may provide an alternative approach to cancer treatment in cells which respond to chemotherapy by increasing the number of vault particles.Initially, the transfection of a plasmid into GLC4 cells was optimized. The best transfection efficiency (N20%) was obtained by using GeneTherapySystems' GenePORTER2 transfection reagent in serum free conditions. To determine if the vault RNAs are the functional portion of the vault complex that confers multi-drug resistance to a cell, a small interfering RNA fragment was designed to specifically knock-down the expression of human vault RNA 3. The siRNA sequence homologous to a portion of vault RNA3 was cloned into an expression vector, and using optimized transfection protocols was transfected into GLC4/REV cells. A Western analysis using caspase-8 antibodies showed no difference in caspase-8 expression in doxorubicin treated and untreated cells. Preliminary results yielded by reverse transcriptase polymerase chain reaction amplification of isolated RNA indicated that the vRNAs were not down-regulated by the siRNAs. / Department of Biology

全球人類健康的頭號殺手--腫瘤目前仍是難以攻克的醫學難題。腫瘤的發生是一個復雜的過程，主要由促癌基因的異常增多或激活及抑癌基因（TSG）的缺失或功能喪失的累積效果導致。近年來基於非基因序列改變所致基因表達水平變化的表觀遺傳學的研究進展表明，啟動子區CpG島甲基化所致的表觀遺傳沉默是抑癌基因轉錄失活的重要機制。Ras和Wnt信號轉導通路在癌病的發生和發展過程中均起到重要的作用，因此針對該兩種信號通路的拮抗因子的表觀遺傳調控及功能學研究將為我們提供有研究及應用前景的候選抑癌基因。 / 作為一種重要的原癌基因，Ras家族基因具有致癌活性的點突變及其導致的過度激活的Ras信號通路被發現廣泛存在於大約30%的人類腫瘤中。然而在一些缺乏Ras基因突變的腫瘤類型中，持續激活的Ras信號通路仍然普遍存在並具有重要作用，昭示著除了Ras基因點突變以外的信號轉導異常激活的機制。與GTP的結合可激活Ras，而RasGAP家族蛋白可通過水解GTP達到使Ras失活的作用。通過采用微陣列比較基因組雜交(aCGH)的實驗手段我們發現6p21.3染色體區具有半接合子缺失， 並於此區域發現了候選抑癌基因RASA5。在以往的研究報道中，RASA5被命名為SynGAP且其功能研究僅限於神經系統。我們的研究發現不同於RasGAP家族的其它基因RASA2-4，RASA5廣泛表達於人類正常器官組織中，並特異性地在腫瘤細胞，特別是鼻咽癌(NPC)，食管鱗狀上皮細胞癌(ESCC)和乳腺癌這些具有野生型Ras基因但Ras信號通路仍被過度激活的細胞中被表觀遺傳沉默。RASA5的異位表達可有效促進腫瘤細胞的雕亡，抑制腫瘤細胞的生長、遷移及“幹性（stemness）“。同時，使用siRNA敲除內源性RASA5可以激發細胞的克隆形成及上皮-間質(EMT)轉化。RASA5的抑癌功能是通過調低Ras-GTP水平並進而抑制其下遊信號通路的活性實現的。過量表達具有致癌活性的點突變的Ras或RasGAP結構域缺失均可部分逆轉這種抑癌作用。此項研究首次證明了RASA5的抑癌功能。 / Wnt/Dvl/β-catenin信號轉導通路在人類腫瘤中存在廣泛的異常激活。我們發現DACT (Dpr/Frodo)家族成員TUSC-T2的表觀遺傳沉默是一種普遍存在於人類腫瘤中的現象。TUSC-T2編碼一種胞質蛋白，外源性表達TUSC-T2可促進腫瘤細胞雕亡並導致腫瘤細胞的克隆形成能力下降。TUSC-T2可與Dvl蛋白結合並下調其活化水平，從而保護GSK-3β蛋白不被Dvl蛋白抑制。GSK-3β可與Axin及APC蛋白形成蛋白質復合物，該復合物可捕捉並降解細胞內信號分子β-catenin。TUSC-T2的過量表達可以抑制β-catenin的激活及其向細胞核內的富集，並進一步阻止β-catenin在細胞核內與Lef/Tcf轉錄因子家族的作用及下遊特定原癌基因，例如c-Myc, CCND1及Fibronectin的表達。因此TUSC-T2具有抑制腫瘤細胞增殖、遷移及上皮-間質(EMT)轉化的作用。 / 綜上所述，我們的研究結果表明RASA5及TUSC-T2是具有抑癌功能的Ras或Wnt/Dvl/β-catenin信號轉導通路抑制因子，其表觀遺傳沉默導致的轉錄失活對於腫瘤的發生發展具有重要意義。同時，針對這兩種抑癌基因的進一步研究將為我們提供富有應用前景的腫瘤標記物。值得註意的是，RASA5課題的研究開創性地闡明了Ras信號通路的拮抗因子的表觀遺傳沉默是一種Ras信號轉導通路於腫瘤細胞中異常激活的新機制。 / Cancer is the top killer of the world, as well as the medical problem difficult to overcome. The conversion of a normal cell to a cancer cell is usually caused by upregulation of oncogenes and downregulation of tumor suppressor genes (TSGs). Epigenetic silencing has been proved to be important in TSGs inactivation, often through methylation of CpG-rich promoter regions. Ras and Wnt signaling pathways are both important for the tumorigenesis, epigenetic and functional studies of antagonists to Ras and Wnt signaling would provide us with candidate TSGs. / Ras is a well-known oncogene. Aberrant mutations of Ras genes occur in approximately 30% of human tumors, causing constitutively activated Ras signaling. However, in certain types of tumors with wild type Ras genes, abnormally activated Ras signaling is still a common and critical event, suggesting alternative mechanisms for Ras signaling hyperactivation. Ras is active when it is bound to GTP, while the hydrolysis of bound GTP and inactivation of Ras is catalyzed by Ras GTPase activating proteins (RasGAPs). Using 1-Mb array CGH (aCGH), we refined a small hemizygous deletion at the 6p21.3 chromosome region that contains a RasGAP family member gene RASA5, which used to be named as SynGAP and studied only in the neuron systems. We demonstrated that RASA5, rather than other RasGAP family members RASA2-4, is broadly expressed in human normal tissues while frequently epigenetically silenced in multiple tumors, especially in certain tumor types such as nasopharyngeal (NPC), esophageal (ESCC) and breast carcinomas (BrCa) with wild-type Ras while Ras cascade is still constitutively active. Ectopic expression of RASA5 led to apoptosis, growth and migration inhibition, as well as ‘stemness’ repression of tumor cells. Meanwhile, knockdown of RASA5 by siRNA promoted the tumor cell colony formation as well as epithelial-mesenchymal transition (EMT). The tumor-suppressive function of RASA5 was exerted through downregulating Ras-GTP level and further inactivating Ras signaling. Such an inhibitory effect could be partially abrogated in the presence of mutated, activated Ras or by deletion of the RasGAP domain. For the first time, our study refined the role of RASA5 as a tumor suppressor. / Wnt/DVL/β-catenin signaling pathway is aberrantly activated in a wide range of human cancers. We identified a DACT (Dpr/Frodo) family member TUSC-T2 as an epigenetically downregulated gene in human tumors. TUSC-T2 encodes a punctate cytoplasmic protein. Ectopic expression of TUSC-T2 dramatically inhibited tumor cell colony formation in silenced tumor cell lines, mainly through inducing apoptosis. TUSC-T2 interacts and downregulates Dishevelled (Dvl) protein, thus protecting glycogen synthase kinase 3β (GSK-3β) from inactivation by Wnt/Dvl and allowing GSK-3β to form a complex with Axin and APC to promote the phosphorylation and proteasomal degradation of β-catenin. Overexpression of TUSC-T2 disrupted β-catenin activation and accumulation in nuclei, thus preventing its binding to transcription factors of the Lef/Tcf family. This caused the downregulation of β-catenin target oncogenes such as c-Myc, CCND1 and Fibronectin as well as the inhibition of tumor cell proliferation and migration. We also observed that TUSC-T2 could inhibit tumor cell EMT. / Taken together, our data demonstrate that RASA5 and TUSC-T2 are functional tumor suppressors epigenetically silenced in multiple tumors through acting as negative regulators of the Ras or Wnt/Dvl/β-catenin cancer pathways, and could be developed as promising biomarkers for human tumors. Of note, our study reveals that epigenetic silencing of the Ras antagonist represents a new mechanism responsible for Ras aberrant activation in cancers with wild-type Ras. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Fan, Yichao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 184-216). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / List of abbreviations --- p.ii-iii / List of tables --- p.iv / List of Figures --- p.v-vii / List of Publications --- p.viii-ix / Abstract in English --- p.x-xii / Abstract in Chinese --- p.xiii-xiv / Table of Contents --- p.xv / Chapter Chapter 1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Cancer epigenetics --- p.4 / Chapter 1.1.1 --- Epigenetic modifications --- p.5 / Chapter 1.1.1.1 --- DNA Methylation --- p.5 / Chapter 1.1.1.2 --- Histone modifications --- p.10 / Chapter 1.1.1.3 --- RNA interference --- p.14 / Chapter 1.1.1.4 --- Nucleosome positioning --- p.15 / Chapter 1.1.2 --- Epigenetic alteration induced Tumor suppressor genes (TSGs) silencing during carcinogenesis --- p.17 / Chapter 1.2 --- Epigenetic alterations in cancer pathways --- p.23 / Chapter 1.2.1 --- Brief introduction of cancer pathways --- p.23 / Chapter 1.2.2 --- Ras pathway --- p.25 / Chapter 1.2.2.1 --- Ras pathway and carcinogenesis --- p.25 / Chapter 1.2.2.2 --- Epigenetic regulation of RasGAP proteins in carcinogenesis --- p.28 / Chapter 1.2.2.3 --- Epigenetic silencing of other negative regulators of Ras signaling --- p.30 / RAS association domain family (RASSF) proteins --- p.30 / PTEN --- p.32 / Sprouty (SPRY) proteins --- p.33 / Chapter 1.2.2.4 --- Hypomethylation induced Ras oncogenes activation --- p.35 / Chapter 1.2.2.5 --- Ras mediates epigenetic regulation through feedback loop --- p.36 / Chapter 1.2.3 --- Wnt pathway --- p.43 / Chapter 1.2.3.1 --- Wnt signaling pathway and carcinogenesis --- p.43 / Chapter 1.2.3.2 --- Epigenetic silencing of negative regulators of Wnt signaling --- p.45 / Chapter 1.2.3.3 --- DACT family proteins and carcinogenesis --- p.48 / Chapter 1.3 --- Application of tumor specific epigenetic alterations as tumor biomarkers and therapeutic targets --- p.49 / Chapter 1.3.1 --- The potential and advantage of tumor specific epigenetic alterations used as tumor biomarkers and therapeutic targets --- p.49 / Chapter 1.3.2 --- Epigenetic-disrupted regulators of Ras signaling as tumor biomarkers and therapeutic targets --- p.50 / Chapter 1.3.3 --- Epigenetic-disrupted regulators of Wnt signaling as tumor biomarkers and therapeutic targets --- p.52 / Chapter Chapter 2 --- Aims of this study --- p.54 / Chapter 2.1 --- To identify epigenetically silenced candidate TSGs as antagonists to Ras or Wnt signaling --- p.55 / Chapter 2.2 --- To elucidate the functional of candidate TSGs --- p.56 / Chapter Chapter 3 --- Materials and Methods --- p.57 / Chapter 3.1 --- Cell lines, tumor samples and routine cell line maintenance --- p.57 / Chapter 3.2 --- Drug and stress treatments --- p.59 / Chapter 3.3 --- DNA and RNA extraction --- p.59 / Chapter 3.4 --- Semi-quantitative RT-PCR and Real time PCR --- p.60 / Chapter 3.5 --- Direct sequencing of PCR products --- p.67 / Chapter 3.6 --- CpG island analysis --- p.67 / Chapter 3.7 --- Bisulfite treatment --- p.67 / Chapter 3.8 --- Methylation-specific PCR (MSP) and bisulfite genomic sequencing --- p.68 / Chapter 3.9 --- Plasmid extraction --- p.69 / Chapter 3.9.1 --- Bacteria culture --- p.69 / Chapter 3.9.2 --- Mini-scale preparation of plasmid DNA --- p.70 / Chapter 3.9.3 --- Large-scale endotoxin-free plasmids extraction --- p.71 / Chapter 3.10 --- Construction of expression plasmids --- p.71 / Chapter 3.10.1 --- Gene cloning and plasmids construction of RASA5 --- p.71 / Chapter 3.10.2 --- Gene cloning and plasmids construction of TUSC-T2 --- p.74 / Chapter 3.11 --- Immunofluorescence Staining --- p.74 / Chapter 3.12 --- Colony formation assay --- p.76 / Chapter 3.13 --- Apoptosis assay --- p.77 / Chapter 3.14 --- Luciferase reporter assay --- p.78 / Chapter 3.15 --- Protein preparation and Western blot --- p.79 / Chapter 3.16 --- Ras Activity Assay --- p.80 / Chapter 3.17 --- Wound healing assay --- p.81 / Chapter 3.18 --- Matrigel invasion assay --- p.81 / Chapter 3.19 --- RNA Interference --- p.81 / Chapter 3.20 --- Statistical analysis --- p.82 / Chapter Chapter 4: --- Epigenetic disruption of Ras signaling through silencing of a Ras GTPase-activating protein RASA5 in human cancers --- p.83 / Chapter 4.1 --- Identification of RASA5 as a downregulated gene residing in the 6p21.3 deletion region --- p.86 / Chapter 4.2 --- RASA5 is widely expressed in human normal tissues but downregulated in tumor cell lines --- p.91 / Chapter 4.3 --- The tumor-specific downregulation pattern of RASA5 is unique in the RASA family genes --- p.95 / Chapter 4.4 --- RASA5 promoter CpG methylation resulted in its transcription inactivation --- p.96 / Chapter 4.5 --- Frequent methylation of RASA5 promoter in multiple primary tumors --- p.101 / Chapter 4.6 --- Cloning and characterization of human RASA5 --- p.104 / Chapter 4.7 --- RASA5 inhibits tumor cell clonogenicity through inducing apoptosis --- p.108 / Chapter 4.8 --- RasGAP domain is required for the tumor suppressive function of RASA5 --- p.111 / Chapter 4.9 --- Certain cancer types harbor wild type Ras but active Ras signaling, with RASA5 epigenetically silenced --- p.114 / Chapter 4.10 --- RASA5 antagonizes Ras signaling pathway --- p.117 / Chapter 4.10.1 --- RASA5 represses Ras signaling through downregulating Ras-GTP level --- p.117 / Chapter 4.10.2 --- Oncogenic mutant form of Ras abrogated colony formation inhibitory effect of RASA5 on tumor cells --- p.120 / Chapter 4.10.3 --- Knockdown of RASA5 promoted the tumor cell colony formation and Ras signaling activation --- p.122 / Chapter 4.10.4 --- RASA5 inhibits ERK1/2 nuclei translocation and activation --- p.123 / Chapter 4.10.5 --- RASA5 negatively regulates Ras target gene expression --- p.125 / Chapter 4.11 --- RASA5 inhibits tumor cell migration and invasion through the Ras/Rac/cofilin signaling --- p.127 / Chapter 4.12 --- RASA5 suppresses tumor cell epithelial-mesenchymal transition (EMT) and stemness --- p.133 / Chapter 4.13 --- RASA5 appears in the cellcell interaction region nanotubes --- p.139 / Chapter 4.14 --- Discussion --- p.141 / Chapter Chapter 5: --- The Wnt/Dvl signaling antagonist TUSC-T2 is a pro-apoptotic tumor suppressor epigenetically silenced in tumors and inhibits tumor cell proliferation and migration --- p.150 / Chapter 5.1 --- Expression of TUSC-T2 is downregulated in human tumors --- p.150 / Chapter 5.2 --- TUSC-T2 promoter methylation results in its transcriptional inactivation --- p.151 / Chapter 5.3 --- Cloning and characterization of TUSC-T2 --- p.155 / Chapter 5.4 --- TUSC-T2 inhibits tumor cell clonogenicity through inducing apoptosis --- p.157 / Chapter 5.5 --- TUSC-T2 inhibits Wnt/Dvl/β-catenin pathway --- p.161 / Chapter 5.6 --- TUSC-T2 suppresses cell migration and EMT through upregulating E-cadherin --- p.165 / Chapter 5.7 --- Discussion --- p.171 / Chapter Chapter 6: --- Conclusions --- p.176 / Chapter 6.1. --- RasGAP family member RASA5 is epigenetically silenced in human cancers, acting as a tumor suppressor through negatively regulating Ras signaling --- p.177 / Chapter 6.2. --- DACT family member TUSC-T2 functions as a candidate TSG silenced by promoter methylation and inhibits Wnt/Dvl/β-catenin pathway --- p.178 / Chapter Chapter 7: --- Future Studies --- p.181 / Chapter 7.1. --- Further functional study of RASA5 and TUSC-T2 --- p.181 / Chapter 7.2. --- Clinical application of epigenetic silenced candidate TSGs --- p.182 / Chapter 7.3. --- Further screening of candidate TSGs as antagonists to cancer pathways --- p.183 / Reference list --- p.184

The gene, cox-1, is over-expressed in cultured GLC4 small cell lung cancer cells concurrent with the development of multi-drug resistance (MDR) as a result of the use of the chemotherapeutic agent used to combat the cancer, doxorubicin. Prevention of MDR has been a tremendous challenge in cancer research and this research is concerned with abolishment of MDR as a cancer survival strategy. RNA-mediated interference technology (RNAi) was employed using siRNA to decrease cox-1 expression and temporarily restore the susceptibility of the cells to doxorubicin. GLC4 cells are of three types: S (sensitive cells never exposed to doxorubicin); ADR (MDR cells cultured in doxorubicin), and; REV (revertant cells previously cultured in presence of doxorubicin but no longer). REV and ADR cells were transfected with cox-1 siRNA. After 24 h, 1x106cells were used for RNA isolation and 1 μg of RNA was used for RT-PCR to assess down-regulation of cox-1 RNA. RT-PCR results indicated that cox-1 RNA was down-regulated to basal levels seen before exposure to doxorubicin. Ct values for GLC4/ADR and cox-1 down-regulated GLC4/ADR cells were 23 and 34, respectively. The result indicated abundant levels and moderate levels of cox-1 mRNA in the ADR cells and the transfected ADR cells respectively. The relative expression level of cox-1 mRNA was 33% higher in the non-transfected GLCR/ADR cells as compared to the transfected GLCR/ADR cells as shown by the curve. Two hundred thousand cells were used for hemacytometer cell counts in the presence of trypan blue to assess cell viability. cox-1 down-regulation in ADR cells resulted in a significantly higher percentage of non-viable cells (25.4%) as compared to its non-transfected control (20.5%) using a Student’s t-test (*P <0.05). Similarly, fluorescence microscopy confirmed that apoptosis was significantly increased in the ADR cells treated with doxorubicin and cox-1 siRNA simultaneously (69.4%) as compared to its non-transfected control (56.7%) (*= P <0.01). A Western blot analysis performed by Fernando Cuadrado indicated that siRNA transfection decreased the expression of COX-1 by 66% in GLC4/ ADR cells as compared to the non-transfected control using densitometry. However, no conclusive results were obtained using flow cytometry as the flow cytometer was incapable of analyzing the mixed cell population (adherent and suspension) which is a characteristic of this cell line, GLC4. Thus, we have clearly demonstrated that MDR cancer cells can be altered temporarily to become susceptible to doxorubicin, a potentially important finding for the treatment of cancer patients. / Department of Biology

To achieve successful gene therapy, safe and efficient gene delivery vectors are needed. As an alternative to viral vectors, non-viral vectors, incorporating compounds such as cationic polymers and lipids have been widely studied. Much effort has been made to enhance transgene delivery efficiency, such as development of more effective cationic lipids or polymers, optimization of transfection formulations, and investigation on structural-activity of delivery vectors. Gemini surfactant, consisting of two surfactant monomers linked by a spacer group, is a thrust research area for gene therapy as non-viral vectors due to their high stability, longer storage on shelves, easiness to produce. A series of phytanyl substituted asymmetric gemini surfactants, phy-3-m (m = 12, 16, and 18) and phy-7NH-m (m = 12, 16, and 18), were rationally designed and synthesized. Due to the bulky nature and increased hydrophobicity of phytanyl branch, phy-3-m surfactants showed much lower values of critical micelle concentration (CMC) compared to their corresponding symmetric m-3-m. Particle size and transmission electron microscopy (TEM) imaging indicate that this type of gemini surfactants tends to form stacked bilayers rather than spherical or rod-like micelles which are typically observed in gemini surfactants with shorter spacers. Phy-3-m surfactants have higher degree of micelle ionization, indicating that the counter ions of the gemini surfactants can be easily replaced by other anionic ions, such as DNA, which is an advantage of phy-3-m used as transgene vectors. To evaluate transfection ability, transfection assays were carried out in OVCAR-3 cells. Transfection complexes formed by a plasmid pVGtelRL, coding enhanced green fluorescence protein (EGFP) gene, phy-3-m, and a neutral lipid, 1,2-Dioleyl-sn-glycerophosphatidylethanolamine (DOPE), at the charge ratios (+/-) of 2:1, 5:1, 10:1, and 20:1, were incubated with OVCAR-3 cells. Treated cells at all charge ratios except 20:1 showed EGFP signals under fluorescence microscopy. Meanwhile, EGFP expression and cell toxicity was quantified using fluorescence-activated cell sorting (FACS). For each gemini surfactant complex, the transfection efficiency and cytotoxicity go through a maximum, occurring at different values of the charge ratio. Considering both transfection efficiency and cytotoxicity, the optimal charge ratio to formulate the complexes containing phy-3-m was found to be 5:1 for in vitro transfection. Compared to a positive control, 16-3-16, phy-3-m showed higher transfection ability and lower cytotoxicity to OVCAR-3 cells. Initial characterization of transfection complexes was investigated by measuring particle size and zeta potential. At all charge ratios, transfection complexes were positively charged, and greater than +30 mV at 5:1 and 10:1, indicating that the complexes would be stable in solution at the ratio above 2:1. Transfection complexes were larger at lower charge ratio, but particle size dropped with increasing charge ratio (+/-). Comparing particle size and zeta potential with transfection efficiency, no correlation between size/zeta potential and transfection ability was observed. The larger particles may enter cells through caveolin-mediated pathway or phagocytosis, and smaller ones through a clathrin-mediated endocytosis. In addition, phase structures of the complexes were investigated using small angle X-ray scattering (SAXS). The complexes containing phy-3-m gemini surfactants were found to be able to adopt multiple phase structures, such as L, HII, and other highly ordered unidentified phase structures. By contrast, L structure was dominant in the transfection complexes formed by 16-3-16. The ability of phy-3-m system to adopt multiple phases appears correlated with their higher transfection efficiency in OVCAR-3 cells.

[Truncated abstract] The Phase II clinical candidate, [(trans-Pt(NH3)2Cl)2{μ-trans-Pt(NH3)2(H2N(CH2)6NH2)2}]4+ (BBR3464 or 1,0,1/t,t,t) shows a unique binding profile when compared to the anticancer agent cis-[Pt(NH3)2Cl2] (cisplatin) and dinuclear platinum complexes of the general formula [(trans-Pt(NH3)2Cl)2(H2N(CH2)nNH2)]2+. There is evidence that the increased efficacy of 1,0,1/t,t,t results from the presence of the charged central linker, which can alter the mode of binding to DNA. This alternate binding mode may be due to an electrostatic and hydrogen bonding association of the central platinum moiety in the minor groove that occurs prior to covalent binding (termed “pre-association”) . . . This research shows that 0,0,0/t,t,t is an adequate model to study the pre-association process of 1,0,1/t,t,t and that it binds in the minor groove of DNA. Therefore it is likely that 1,0,1/t,t,t pre-associates in the minor groove of DNA prior to covalent binding. This work supports the conclusions reached in NMR studies of the binding of 1,0,1/t,t,t with the 1,4-GG sequence (Qu et al. JBIC. 8, 19-28 (2003)), which showed simultaneous binding in the major and minor groove. The findings of the current work may also explain the observed binding mode of 1,0,1/t,t,t, which can bind to DNA in both the 3',3' and 5',5' directions (Kasparkova et al. JBC. 277, 48076-48086 (2002)). These unique binding characteristics are thought to be responsible for the increased efficacy of 1,0,1/t,t,t, and in light of the current results the observed binding mode most likely stems from the electrostatic pre-association of the central platinum moiety.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL. Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL. Further, in the hematopoietic system stem cell aging is characterized by accumulation HSCs with poor self-renewal capacity and myeloid biased differentiation. Despite improved appreciation of the cell intrinsic and cell extrinsic mechanisms driving age-associated HSC functional exhaustion, no interventions have proven effective in delaying HSC aging to date. Here, we show that genetic inactivation of the Phf6 prevents age- associated HSC functional decline. Immunophenotypic and single cell transcriptomics profiling demonstrated markedly decreased accumulation of immunophenotypically-defined HSCs, reduced myeloid bias and decreased upregulation of transcriptional programs associated with stem cell aging in old hematopoietic-specific Phf6 knockout mice. Functionally, Phf6 knockout HSCs from aged mice demonstrated increased hematopoietic reconstitution capacity and preservation of lymphoid differentiation potential. Mechanistically, analysis of long-term HSCs from old Phf6 knockout mice revealed reduced levels of ongoing DNA damage and downregulation of genotoxic stress-induced transcriptional signaturesconducive of HSC aging. These results identify Phf6 as an important epigenetic regulator of HSC aging, whose inactivation counters the functional deterioration of HSC activity induced with age. Microprotein encoding genes are a class of genes which encode poly-peptide gene products comprised by 100 or fewer amino acids. Until recently, many such genes had been considered of low- or no-coding potential given the technical limitations associated with identification of such small proteins. However, recently prominent examples of microprotein encoding genes have been reported with a wide variety of regulatory functions. Therefore, we hypothesized that novel microprotein genes exist within the human genome with oncogenic and tumor suppressive roles. To test this hypothesis, we developed a pipeline for identification of microproteins based on conservation of the open reading frame. Leveraging PLATE-seq to generate a high-dimensional readout in a loss-of-function screen, we then screened for microproteins with potential tumor suppressive or oncogenic function. From this, we identified a brain- specific, 65 amino-acid microprotein encoded in within LINC00617 (TUNAR) which is conserved at the protein level across vertebrates. We experimentally validated the protein-level expression of the TUNAR microprotein. In vitro and in vivo knockout and overexpression experiments demonstrate a role for TUNAR as a tumor suppressor in glioma. Specifically, we show that loss of Tunar in the mouse brain results in lower expression of Fermt1 and genes in the integrin signaling pathway. Consistently, overexpression of TUNAR in human glioblastoma multiforme cell lines significantly impeded cellular migration suggesting a role of Tunar in glioma cell dissemination. Finally, human glioma sequencing and copy number data were mined to determine the prognostic significance of the loss of TUNAR in human gliomas. These analyses demonstrated that copy number loss of TUNAR is associated with poor outcomes in lower grade gliomas and that TUNAR expression and glioma grade are strongly, negatively correlated suggesting that TUNAR likely has tumor suppressive effects in human glioma.

Indiana University-Purdue University Indianapolis (IUPUI) / In this thesis, I investigated the preclinical utility and antitumor efficacy of TRAIL delivered by Ad-IU2, a prostate-specific replication-competent adenovirus (PSRCA), against androgen-independent prostate cancer. Through transcriptional control of adenoviral early genes E1a, E1b and E4, as well as TRAIL by two bidirectional prostate-specific enhancing sequences (PSES), expression of TRAIL as well as adenoviral replication was limited to prostate-specific antigen and prostate-specific membrane antigen (PSA/PSMA)-expressing cells. Ad-IU2 replicated efficiently in and was restricted to PSA/PSMA-positive prostate cancer cells and induced 5-fold greater apoptosis in androgen-independent CWR22rv and C4-2 prostate cancer cells than the PSRCA control not expressing TRAIL. Ad-IU2 exhibited superior killing efficiency in PSA/PSMA-positive prostate cancer cells at doses 5 to 8-fold lower than that required by a non-TRAIL expressing PSRCA to produce a similar effect. This enhanced cytotoxic effect was not observed in non-prostatic cells, however. As an enhancement of its therapeutic efficacy, Ad-IU2 exerted a bystander effect through either direct cell-to-cell contact or soluble factors present in conditioned media from Ad-IU2-infected cells. In vivo, Ad-IU2, as compared to a control PSRCA, markedly suppressed the growth of subcutaneous CWR22rv xenografts at six weeks post-treatment (3.1 vs. 17.1-fold growth of tumor). The treatment of androgen-independent prostate cancer with Ad-IU2 prior to external beam radiation therapy (EBRT) significantly reduced clonogenic survival with dose reduction factors of 4.91 and 2.43 for CWR22rv and C4-2 cells, respectively. Radio-sensitization by Ad-IU2 was restricted to PSA/PSMA-positive cells. Combinatorial radio-gene therapy resulted in accumulation of cells in G1 phase and a perturbation of the radiation-induced G2 phase arrest. This multi-modal approach combining viral lysis, apoptosis-inducing gene therapy, and radiation therapy could have great impact in achieving complete local tumor control while reducing radiation dose and associated treatment morbidities. This would result in improvement of the clinical outcome of patients with high risk prostate cancer.

Simple Summary Gene therapy is a novel approach to treat diseases by introducing corrective genetic information into target cells. Adeno-associated virus vectors are the most frequently applied gene delivery tools for in vivo gene therapy and are also studied as part of innovative anticancer strategies. Here, we report on the natural preference of AAV2 vectors for hepatocellular carcinoma (HCC) compared to nonmalignant liver cells in mice and human tissue. This preference in transduction is due to the improved intracellular processing of AAV2 vectors in HCC, resulting in significantly more vector genomes serving as templates for transcription in the cell nucleus. Based on this natural tropism for HCC, novel therapeutic strategies can be designed or existing therapeutic approaches can be strengthened as they currently result in only a minor improvement of the poor prognosis for most liver cancer patients. Abstract Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current—or develop novel—strategies for treating HCC.

目的： 尽管过去几十年癌症的化疗取得了很大进步，但晚期非小细胞肺癌的预后仍然较差。表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor tyrosine kinase inhibitors，EGFR TKIs）给晚期非小细胞肺癌的患者带来了新的希望。然而，EGFR TKIs的总体效果有限，且不良反应较多，价格也较昂贵。如果能找到EGFR TKIs的疗效预测因子，则该治疗就可以只给予那些最有可能从中获益的人，从而提高成本效果，并使治疗变得更加个体化。 / 已有单组研究在接受EGFR TKIs治疗的患者中对有或没有某个标志物的人的预后进行了比较，发现EGFR基因突变、EGFR基因拷贝数增加、EGFR蛋白表达和KRAS基因突变这4个生物标志物可能能够预测EGFR TKIs的疗效。然而，此类研究的方法学是有缺陷的。要确定以上生物标志物是否有预测作用，应该在评估EGFR TKIs疗效的随机对照试验中作亚组分析，对该治疗在有某个生物标志物及没有某个生物标志物的患者中的疗效进行比较，检测治疗与生物标记物的交互作用。 / 但是，现有的随机对照试验通常样本量较小，统计效能不足，难以从中得到确定的结论。因此，我们做了一个随机对照试验的系统综述，以总结现有的最佳证据，对EGFR TKIs与上述4个生物标志物的交互作用进行评估。 / 方法： 我们检索了PubMed，EMBASE，考科蓝图书馆，中国生物医学文献数据库（中文），万方数据库（中文），美国临床肿瘤学会和欧洲肿瘤学会的会议摘要，以及相关原始研究、系统综述与Meta分析、临床指南、共识及专家意见的参考文献。检索时间截至2012年6月。合格研究为非重复、提供了具体数据且符合下列所有条件的研究：1）研究对象：晚期非小细胞肺癌患者；2）干预措施：EGFR TKIs单药治疗或联合其他药物治疗；3）对照措施：安慰剂对照，空白对照或化疗，或者它们任一种加上干预组的基线治疗；4）结局指标：无进展生存期和/或总生存期；5）研究设计：随机对照试验；6）根据上述任一种或多种生物标志物的状态作了亚组分析。 / 两名研究者平行独立地从合格研究中提取了患者特征、治疗方案、结局、生物标志物分析和方法学质量等方面的资料。对每一个研究，我们都根据生物标志物阳性亚组的风险比（hazard ratio）和阴性亚组的风险比计算了一个风险比之比（ratio of hazard ratios）来测量该标志物对疗效的预测能力或者说治疗与该生物标志物的交互作用。然后，采用随机效应模型对来自不同研究的风险比之比进行Meta分析；采用Cochran Q检验和I²评估研究间的异质性；通过敏感性分析考察原始研究的方法学质量等因素对结果的影响；采用Begg漏斗图和Egger检验来检测发表偏倚存在的可能性。 / 结果： 共有18个合格研究入选。可用于各个生物标志物分析的患者数量从1763到3246不等。原始研究普遍对关于方法学质量的信息报告得不够充分；有的研究可能存在重要偏倚。与安慰剂相比，EGFR TKIs可以有效延长无进展生存期和总生存期，但对总生存期的效果相对较小。除了在EGFR基因突变的患者中EGFR TKIs延长无进展生存期的效果明显好于化疗外，其它情形下，不管是无进展生存期还是总生存期，EGFR TKIs与化疗的效果均相当。 / 以无进展生存期为结局的风险比之比，在EGFR基因突变状态不同的亚组间（野生型亚组为参照）为0.37（95% 置信区间[CI]：0.22-0.60，P < 0.0001），EGFR基因拷贝数状态不同的亚组间（未增加的亚组为参照）为0.72（95% CI：0.52-0.99，P = 0.04），EGFR蛋白表达状态不同的亚组间（无表达的亚组为参照）为0.99（95% CI：0.78-1.26，P = 0.93），KRAS基因突变状态不同的亚组间（野生型亚组为参照）为1.35（95% CI：1.02-1.80，P = 0.04）。这些结果提示EGFR TKIs治疗与EGFR基因突变，EGFR基因拷贝数及KRAS基因突变之间可能存在交互作用。以总生存期为结局的风险比之比，在EGFR基因突变、EGFR基因拷贝数、EGFR蛋白表达及KRAS基因突变状态不同的亚组间分别为0.84（95% CI：0.64-1.11，P = 0.22）、0.92（95% CI：0.69-1.23，P = 0.57）、0.86（95% CI：0.70-1.05，P = 0.14）和1.37（95% CI：0.89-2.10，P = 0.15）。 / 就统计学显著性、异质性和稳定性而言，关于其它3个生物标志物的结果不如EGFR基因突变的相关结果确定，关于总生存期的结果不如无进展生存期的相关结果确定。没有证据表明本研究中存在发表偏倚。 / 结论： EGFR基因突变可用于确定哪些患者更有可能从EGFR TKIs治疗中获益。EGFR基因拷贝数增加和KRAS基因突变可能也有类似用途，但它们与治疗的交互作用是独立存在的还是由于它们与EGFR基因突变的相关性而获得的，目前尚不清楚。在EGFR野生型的患者中，选择化疗似乎比EGFR TKIs更好，因为它的副作用相对较少，且更为便宜。 / 本研究的结果为当前的临床指南提供了全面的证据支持。其它3个标志物在EGFR野生型患者中的预测价值可能还值得进一步的探讨，但我们更建议未来的研究在探讨治疗与生物标志物的交互作用时进行多因素分析。 / Objective: Despite the many new progresses in chemotherapy, the prognosis of advanced non-small cell lung cancer (NSCLC) remains poor. The introduction of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) seems to offer new promises for advanced NSCLC patients. However, EGFR TKIs have a limited overall efficacy, clear adverse events and large costs. It has become particularly appealing to identify, through new biomarkers, patients who are more likely to benefit from the treatment so that the treatment can be more personalized and effective. / EGFR mutations, EGFR gene copy number gain, EGFR protein expression and KRAS mutations were indicated as potential predictive biomarkers for the efficacy of the treatment in single-arm studies that compared survival of treated patients with and without a biomarker. However, such comparisons are flawed and the appropriate study design to evaluate the value of a biomarker in predicting efficacy which is known as interaction in epidemiology is the randomized controlled trial with stratified analysis that compared the efficacy of EGFR TKIs between patients with and without the biomarker. / As trials in this field are usually small in sample size and insufficiently powered for drawing a robust conclusion, we conducted this systematic review to summarize the evidence from all relevant randomized controlled trials that have data for investigating the interaction between EGFR TKIs and the 4 biomarkers. / Methods: PubMed, EMBASE, the Cochrane Library, Chinese Biomedical Literature Database (in Chinese), Wanfang Data (in Chinese), the abstracts of conferences of the American Society of Clinical Oncology and European Society of Medical Oncology, the reference list of relevant original studies, systematic reviews and meta-analyses, guidelines, consensus, and expert opinions were searched up to June 2012. / Eligible studies had to be non-duplicate, extractable studies meeting all the following criteria: 1) Population: patients with advanced NSCLC; 2) Intervention: EGFR TKIs alone or EGFR TKIs plus other treatments; 3) Control: placebo, no treatment, or chemotherapy, with or without the baseline treatments in the intervention arm; 4) Outcome: progression-free survival and/or overall survival; 5) Study design: randomized controlled trial; 6) Subgroup analyses were conducted according to the status of one or more of the 4 biomarkers. / Data on patients’ characteristics, treatment protocols, outcomes, biomarker analysis and methodological quality were extracted by two researchers independently. Within a study, we defined the measure of the value of a biomarker in predicting efficacy or biomarker-treatment interaction as the hazard ratio in patients with the biomarker relative to that in those without the marker. The ratio of hazard ratios from relevant studies was then combined by using the random-effect model. / Heterogeneity among studies was assessed by the Cochran’ Q test and I². Sensitivity analyses were conducted to examine the impact of factors such as methodological quality on the results. Begg’s funnel plots and Egger’s tests were used to examine the possibility of publication bias. / Results: Eighteen studies were included. The number of patients available for analyses on different biomarkers varied from 1,763 to 3,246. Data on the methodological quality of included studies are generally under-reported. Some studies seemed to have important biases. EGFR TKIs are in general effective in increasing progression-free and overall survival as compared with placebo although the effect size is smaller for overall survival than for progression free survival. EGFR TKIs are comparable to chemotherapy in their effect in prolonging both progression-free and overall survival, except in EGFR mutation group in which EGFR TKIs seem much more effective than chemotherapy in prolonging progression-free survival. / Importantly, for progression-free survival, the summary ratio of hazard ratios was 0.37 (95% confidence interval [CI]: 0.22-0.60, P < 0.0001) for EGFR mutations (versus wild-type), 0.72 (95% CI: 0.52-0.99, P = 0.04) for EGFR gene copy number gain (versus no gain), 0.99 (95% CI: 0.78-1.26, P = 0.93) for EGFR protein expression (versus negative), and 1.35 (95% CI: 1.02-1.80, P = 0.04) for KRAS mutations (versus wild-type), indicating interaction may exist between EGFR TKIs and EGFR mutation, EGFR gene copy number and KRAS mutations. For overall survival, the summary ratio of hazard ratios for EGFR mutations, EGFR gene copy number gain, EGFR protein expression and KRAS mutations was 0.84 (95% CI: 0.64-1.11, P = 0.22), 0.92 (95% CI: 0.69-1.23, P = 0.57), 0.86 (95% CI: 0.70-1.05, P = 0.14) and 1.37 (95% CI: 0.89-2.10, P =0.15), respectively. / In general, the results on EGFR gene copy number gain, KRAS mutations and EGFR protein expression were less certain than those on EGFR mutations in terms of statistical significance, consistency and robustness, and the results on overall survival were less certain than those on progression-free survival. Publication bias did not seem present in the study. / Conclusions: EGFR mutations and possibly EGFR-GCN and KRAS mutations can help identify who are more likely to benefit from EGFR TKIs treatment. However, it is not clear whether the interaction with EGFR-GCN and KRAS mutations are independent or obtained through their relation with EGFR mutations. Furthermore, in EGFR wild-type patients, given that chemotherapy is cheaper and of fewer side effects, chemotherapy seems clearly a better choice than EGFR TKIs. / Our findings provided the most comprehensive evidence for the recommendations of current guidelines. Although the predictive value of the other 3 biomarkers in wild-type EGFR patients may be worth further investigation, we suggest that multivariate analyses are explored in future studies of biomarker-treatment interactions. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yang, Zuyao. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 88-104). / Abstracts also in Chinese. / Yang, Zuyao.

Chemotherapy in childhood cancer patients is associated with reduced bone density that can result in osteoporotic fracture in survivors. A significant proportion of paediatric patients experience a reduction in plasma 25 - hydroxyvitamin D3 [ 25 ( OH ) D3 ] and 1,25 - dihydroxyvitamin D3 [ 1,25 ( OH ) 2D3 ] levels during treatment, the basis of which is unknown. A balance between the bioactivation and degradation of 1,25 ( OH ) 2D3 is responsible for maintaining homoeostatic levels of 1,25 ( OH ) 2D3 at the correct set - point. Whereas the cytochrome P450 enzyme, CYP27B1 ( 25 - hydroxyvitamin D3 1 α - hydroxylase ), catalyses the hydroxylation of the precursor 25 ( OH ) D3 to generate 1,25 ( OH ) 2D3, catabolic inactivation and cleavage of 1,25 ( OH ) 2D3 is achieved by the mitochondrial cytochrome P450 enzyme, 25 - hydroxyvitamin D3 24 - hydroxylase ( CYP24 ), which is highly expressed in bone and kidney cells. Since many of the signalling pathways which regulate the expression of CYP24 are also activated by chemotherapeutic drugs, we hypothesised that the drugs could cause the degradation of plasma 25 ( OH ) D3 and 1,25 ( OH ) 2D3 by increasing CYP24 expression, the principal means of facilitating the bio - inactivation and degradation of plasma 25 ( OH ) D3 and 1,25 ( OH ) 2D3. Using the kidney cell - lines, COS - 1 and HEK293T cells, we now report that chemotherapeutic drugs, represented by daunorubicin hydrochloride ( an anthracycline antibiotics ), etoposide and vincristine sulphate ( vinca alkaloids and related compounds ) and cisplatin ( an alkylating agent ), were able to enhance CYP24 promoter activity in kidney cell lines transfected with a CYP24 promoter - luciferase construct, either by themselves or in the presencedaunorubicin hydrochloride and etoposide, two of the strongest inducers of CYP24 promoter activation under our experimental conditions, demonstrate that these drugs acted in a concentration - dependent manner. In addition to stimulating promoter activity on their own, the drugs also amplified the induction of the CYP24 promoter by 1,25 ( OH ) 2D3. Synergistic increases were generally observed when the cells were treated simultaneously with 1,25 ( OH ) 2D3 and a drug. The two kidney cell lines generally responded in a similar manner when challenged with the drugs, either in the presence or absence of 1,25 ( OH ) 2D3. Interestingly, the hydroxylated derivative of daunorubicin hydrochloride, doxorubicin hydrochloride which is also a commonly used chemotherapeutic drug, had no effect of promoter activity. Further studies with daunorubicin hydrochloride demonstrated that the effects of the drug per se were not mediated by oxidative stress and the vitamin D receptor was not required for daunorubicin hydrochloride per se to stimulate CYP24 promoter activity. However, daunorubicin hydrochloride caused a modest increase in the expression of the vitamin D receptor and this could contribute to its synergistic activity with 1,25 ( OH ) 2D3. In the presence of etoposide, there was also a tendency for the kidney cells to express higher levels of the vitamin D receptor. A key role for the extracellular signal - regulated protein kinase ( ERK ) 1, ERK2 and ERK5 mitogen - activated protein ( MAP ) kinases was demonstrated for the inductive action of daunorubicin hydrochloride and etoposide, with CYP24 promoter - specific transcription factors located in the first - 298bp being likely targets of the ERK activity. Studies with a dominant negative mutant of MKK4, one of the two immediate upstream activators of the c - jun N - terminal kinase isoforms, demonstrated that this MAP kinase also played a crucial role in inductive actions of the of 1,25 ( OH ) 2D3. Dose - response studies with drugs. Consistent with their use in anti - cancer therapy, all of the above drugs killed the human promyelocytic HL60 leukaemic cells at very low concentrations but had no effect on the viability of kidney or liver cells, either at concentrations used in our experiments or at higher levels. Our data provide novel biochemical evidence that some of the commonly used chemotherapeutic drugs could cause an increase in the transcriptional activation of the promoter, most likely via the MAP kinases activating the transcription factors which bind to the CYP24 promoter. Such an effect could contribute to the reduction in plasma 25 ( OH ) D3 and 1,25 ( OH ) 2D3 in some of the patients undergoing chemotherapy. / Thesis (Ph.D.)--School of Paediatrics and Reproductive Health, 2005.