Dissertations / Theses on the topic 'Pancreatic cancer Sp proteins'

Vascular endothelial growth factor receptor-2 (VEGFR2) is a key angiogenic factor, and angiogenesis is an important physiological process associated with neovascularization, growth, and metastasis of many different tumors. The mechanism of VEGFR2 gene expression was investigated in MiaPaCa-2, Panc-1, and AsPC-1 pancreatic cancer cells transfected with a series of VEGFR2 promoter deletion/mutated constructs, and the results indicated that the GC-rich –60 to –37 region of the promoter was essential for VEGFR2 expression in these cell lines. EMSA and ChIP assays showed that Sp proteins are expressed and bind to the proximal GC-rich region of the VEGFR2 promoter. RNA interference studies on Sp proteins demonstrated that Sp1, Sp3, and Sp4 all contributed to VEGFR2 gene/protein expression in pancreatic cancer cells. VEGFR2 gene expression was also investigated in ZR-75 and MCF-7 breast cancer cells. ZR-75 cells treated with 10 nM 17b-estradiol (E2) increased VEGFR2 mRNA levels/protein expression. The VEGFR2 promoter was induced by E2 in ZR-75 cells, and analysis of the VEGFR2 promoter identified the GC rich -60 to -37 region that was required for E2-mediated transactivation. EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed in ZR-75 cells and bind the proximal GC-rich region of the VEGFR2 promoter. RNA interference was used to determine the relative contributions of Sp proteins on hormonal regulation of VEGFR2 through ER/Sp complexes, and interestingly, in ZR-75 cells, hormone-induced activation of VEGFR2 involves ERa/Sp3 and ERa/Sp4 but not ERa/Sp1. In MCF-7 cells treated with 10 nM E2, VEGFR2 mRNA levels were decreased. Analysis of the VEGFR2 promoter revealed that the same GC-rich region important for E2-mediated upregulation in ZR-75 cells was responsible for E2-dependent downregulation of VEGFR2 gene expression in MCF-7 cells. EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed in MCF-7 cells and bind to the proximal GC-rich region of the VEGFR2 promoter. RNA interference studies showed that Sp1, Sp3, and Sp4 are involved in the E2-mediated downregulation of VEGFR2 in MCF-7 cells, and ERa/Sp protein-promoter interactions are accompanied by recruitment of the corepressor SMRT using the ChIP assay.

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17β-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 → S phase progression. Surprisingly, TCDD alone induced G0/G1 → S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 → S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 → S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.

Cancer is one of the leading causes of death worldwide. Pancreatic ductal adenocarcinoma (PDAC) is characterized by high intrinsic aggressiveness and late diagnosis, causing poor prognosis and resulting in the lowest five-year survival rate among all cancers. Nowadays, there is no biomarker for PDAC diagnosis approved. The survival rate of cancer patients increases when they are diagnosed in the early stages of the pathology; and for this reason, the research of new biomarkers is of great interest. Tumour cells present aberrant glycosylation on their cell surface and also in the secreted glycoconjugates. Hence, a strategy for new tumour biomarker discovery is based on the identification of specific glycoforms. This work has explored the glycosylation of two serum glycoproteins, the alpha-1-acid glycoprotein (AGP) and the ceruloplasmin (CP). The glycosylation of the epithelial mucins MUC1 and MUC5AC in healthy and PDAC tissues has also been analysed.
El càncer és una de les principals causes de mort. L’adenocarcinoma ductal pancreàtic (PDAC) es caracteritza per una alta agressivitat i un diagnòstic tardà, causant un pronòstic desolador i resultant en el càncer amb la taxa relativa de supervivència als cinc anys menor. Actualment no es disposa d’un biomarcador per al diagnòstic del PDAC.La supervivència dels pacients augmenta quan són diagnosticats en els estadis inicials; per aquest motiu, la recerca de nous marcadors és de gran importància. Les cèl·lules tumorals presenten una glicosilació aberrant en la seva superfície cel·lular i també en els glicoconjugats que secreten. Per tant, una estratègia per al descobriment de nous biomarcadors es basa en la identificació de glicoformes específiques. Aquesta tesi ha explorat la glicosilació de dues glicoproteïnes del sèrum, la alfa-1-glicoproteïna àcida i la ceruloplasmina. També s’ha analitzat la glicosilació de les mucines epitelials MUC1 i MUA5AC en teixits sans i de PDAC.

Since the phenomenon was first identified, apoptosis has been proposed to serve as a barrier to the development of cancer in metazoans. Over the years, the inability to carry out apoptosis has been implicated in the initiation, formation and progression of tumors. Moreover, acquired resistance to apoptosis is now believed to represent a major obstacle to the successful application of oncotherapies. Caspases play a central role in the induction and execution of apoptosis. As such, their activity must be tightly regulated. To date, inhibitor of apoptosis proteins (IAPs) are the only known intrinsic regulators of caspase function. The present study focused on the identification of molecular targets differentially expressed in normal and cancer cells that could serve to facilitate the rational design of anti-cancer therapies. We hypothesized that variations in the levels of key apoptotic regulators such as caspases, IAPs and their antagonists could conceivably contribute to the acknowledged resistance of pancreatic cancer cells to cytotoxic therapies. Our first specific aim was to derive an expression profile of apoptotic modulator/effector genes in pancreatic cancer cell lines. Our analysis uncovered a tendency towards up-regulation of IAP expression (namely cIAP-2) and down-regulation of pro-apoptotic factors such as caspases and the Xiap antagonist Xaf-1 in these cell lines. In particular, Xaf1 protein expression appeared to be completely repressed in neoplastic cell lines. Moreover, over-expression of one or more IAPs was observed in several solid malignancies. Lastly, while Xiap expression and subcellular localization were not altered in evolving intraductal lesions and pancreatic tumors, immunohistological surveys uncovered over-expression and nuclear redistribution of cIAP-1, cIAP-2 and survivin in pancreatic adenocarcinomas. Our second objective was to determine if differential expression of IAPs influenced the sensitivity of three human pancreatic cancer cell lines to drug-induced apoptosis. In vitro studies uncovered a good correlation between transcriptional up-regulation of IAPs, caspase-dependent cleavage of Xiap, activation of downstream effector caspase-3 and rapidity of onset of etoposide-induced apoptosis in these cell lines. In particular, endogenous levels of cIAP-2 mRNA appeared to be good predictors of etoposide-responsiveness. However, attempts at sensitizing pancreatic cancer cells to etoposide by down-modulating Xiap expression via over-expression of Xaf-1 or siRNA-mediated degradation of Xiap were unsuccessful. (Abstract shortened by UMI.)

L'adénocarcinome pancréatique (PDAC) est aujourd’hui un problème important de santé publique. Les traitements conventionnels contre le cancer ont peu d'impact sur le cours de cette maladie. Les changements génétiques impliqués dans la progression du PDAC concernent souvent des gènes qui codent pour des protéines liées à différentes voies de transduction de signal. Ce fait indique l'importance d’identifier le rôle et les relations entre les multiples voies de signalisation impliquées dans le PDAC. Dans ce travail nous utilisons une approche double-hybride chez la levure pour identifier des interactions impliquant des protéines exprimées dans des cellules cancéreuses pancréatiques. Dans la première partie de cette thèse, un groupe particulier de protéines, les protéines d’échafaudage, contenant de multiples domaines (SH3), a été étudié. Nous avons identifié plusieurs molécules interagissant avec ces protéines, modulant la prolifération cellulaire, la survie (CIZ1, BIRC6, RBBP6), la signalisation (LTBP4, Notch2, TOM1L1, STK24) et la dynamique des membranes (PLSCR1, DDEF2, VCP). Certaines protéines interagissant avec (Vav2, ITSN1, Stac) sont connues pour leurs différents rôles dans le vésicules de transport, l'endocytose ainsi que dans d’autres processus de régulation. Nous avons aussi dentifié des interactions protéiques assurées par d’autres protéines exprimées dans le pancréas, et ce pour identifier les événements qui sont spécifiques aux cellules cancéreuses pancréatiques. PTFIA est un activateur de transcription qui joue un rôle déterminant dans l’organogenèse pancréatique, il est aussi impliqué dans la maintenance du pancréas exocrine. Cette protéine contient un domaine HLH qui facilite la conversion des monomères inactifs en dimères s’activant pendant les étapes appropriées du développement. Nous avons identifié plusieurs molécules interagissant avec cette protéine, modulant la prolifération cellulaire (GCIP); différentiation de macrophage (PRKX) et la tumorigenesis d'intestinal. KLF6 est un facteur de transcription qui peut activer ou inhiber des gènes impliqués dans la régulation du cycle cellulaire. Quelques études présentent KLF6 comme étant un facteur de transcription impliqué dans la prolifération cellulaire. Il a été récemment démontré qu’il était déréglé dans des multiples cancers (prostate et ovaires), dérèglement traduit par une perte d’hétérozygotie. Nous avons identifié plusieurs molécules interagissant avec cette protéine, modulant la Ribosylation (PARP10) et l’adhésion (MSN). ArgBP2 a été récemment identifié comme étant un nouveau marqueur pour le cancer pancréatique et potentiellement une nouvelle cible thérapeutique. ArgBP2 est un substrat et interacteur des tyrosines kinases Arg et Abl, il a aussi été trouvé concentré sur les fibres d'actine. ArgBP2 appartiennent à une famille de protéines d'échafaudage qui incluent la vinexine et le CAP/ponsin; ils participent à la régulation de l’adhésion cellulaire, l’organisation du cytosquelette d'actine et la signalisation, en aval, des récepteurs de facteurs de croissance. La protéine ArgBP2 est caractérisée par un domaine (SoHo) dans la région N-terminal et trois domaines SH3 dans la région de C-terminal. Nous avons identifié la protéine CIP4 (Cdc42 interacting protein 4) comme étant un nouvel interacteur d’ArgBP2. Nous avons montré que, par cette interaction, ArgBP2 pouvait augmenter la capacité de c-Abl à phosphiryler CIP4 et que CIP4, après interaction, pouvait inhiber la phosphorylation d'ArgBP2. L’utilisation de petites molécules capables de perturber les interactions et l'analyse de l’effet de ces pertes d’interactions sur la croissance des cellules cancéreuses pancréatiques, in vivo et in vitro, pourraient permettre d’identifier des agents thérapeutiques potentiels pour le traitement du cancer pancréatique, une des maladies les plus meurtrières connues.

Els pacients amb càncer presenten una taxa de supervivència superior si es diagnostiquen a estadis inicials, per la qual cosa és indispensable disposar de marcadors tumorals adequats. Glicoformes de proteïnes específiques es podrian utilizar com marcadors tumorals. S’han investigat les subformes i glicosilació de l’Antígen Prostàtic Específic (PSA) per millorar la seva capacitat de diagnosis de pacients amb càncer de pròstata vs aquells amb hiperplàsia benigna prostàtica. També s’han avaluat glicoproteïnes sèriques amb alteracions glucídiques en pacients de càncer de pàncrees, comparat amb pacients amb pancreatitis crònica i controls. S’ha observat una disminució de la fucosilació core i sialilació del PSA en càncer de pròstata i un augment de la fucosilació core i Sialyl-Lewis X en algunes Proteïnes de fase Aguda en càncer de pàncrees. Aquest canvis s’haurien d’avaluar en un cohort de pacients més gran per determinar el seu paper en el cribratge, diagnòstic o monitorització dels cancers estudiats.
The survival rate of cancer patients is increased when they are diagnosed at localized stage, for which the availability of adequate tumour markers is crucial. The determination of specific tumour‐associated glycoforms may either improve the specificity of known cancer biomarkers such Prostate-Specific Antigen (PSA) or allow the discovery of new tumour markers. This work has investigated PSA subforms and their glycosylation with the aim to improve the differentiation between prostate cancer and benign prostatic hyperplasia. In addition, serum glycoproteins with altered glycosylation have been evaluated in pancreatic cancer patients, compared to chronic pancreatitis patients and healthy controls. A decrease of PSA core fucosylation and sialylation in Prostate cancer and an increase in some Acute-Phase Proteins core fucosylation and Sialyl-Lewis X in pancreatic cancer were observed. These changes should be evaluated in a larger cohort of patients to determine their role as prostate and pancreatic cancer biomarkers, respectively.

Optimisation of the conventional tripartite of pancreatic cancer (PC) treatment have led to significant improvements in mortality, however further knowledge of the underlying molecular processes is still required. Transcript profiling of mRNA expression of over 44K genes with microarray technology demonstrated upregulation of secreted frizzled related protein 4 (sFRP4) and ??-catenin in PC compared to normal pancreata. Their pathway ??? Wnt signalling is integral to transcriptional regulation and aberrations in these molecules are critical in the development of many human malignancies. Immunohistochemistry protocols were evaluated by two independent blinded examiners for antigen expression differences associated with survival patterns in 140 patients with biopsy verified PC and a subset of 23 normal pancreata with substantial observer agreement (kappa value 0.6-0.8). A retrospective cohort was identified from 6 Sydney hospitals between 1972-2003 and archival formalin fixed tissue was collected together with clinicopathological data. Three manual stepwise regression models were fitted for overall, disease-specific and relapse-free survival to determine the value of significant prognostic variables in risk stratification. The models were fitted in a logical order using a careful strategy with step by step interpretation of the results. Immunohistochemistry demonstrated increased sFRP4 membranous expression (> 10%) in 49/95 PC specimens and this correlated with improved overall survival (HR:0.99;95%CI:0.97-6.40;LRchi2=134.75; 1df; ??< 0.001). Increased sFRP4 cytoplasmic staining (> 2/3) in 46/85 patients increased the disease-specific survival (HR:0.52;95%CI:0.31-0.89;LR test statistic =248.40;1df;??< 0.001). Increasing ??-catenin membranous expression (< _60%) in 26/116 patients was associated with an increased risk of overall death (HR:3.18;95%CI:1.14-8.89;LR test statistic =4.61;1df,??< 0.05). Increasing cytoplasmic expression in 65/114 patients was protective and was associated with prolonged survival on univariate, but not multivariate analysis (Disease specific survival HR:0.75;95%CI:0.56-1.00;logrank chi2=3.91;1df; ??=0.05). Increased nuclear ??-catenin expression in 65/114 patients was associated with prolonged survival (disease-specific HR:0.92;95%CI:0.83-1.02; LR test statistic= 49.72;1df;??< 0.001). At the conclusion, 12 patients (8.6%) remained alive, 122 died of their disease (68 males versus 54 females). They were followed for a median of 8.7 months (range 1.0-131.3) months. The median age was 66.5 years (range 34.4-96.0, standard deviation 10.9) years. Pancreatic resection was achieved in 79 patients with 46.8% achieving RO resection. The 30 day post-operative mortality was 2.1%. The overall 1 year survival rate was (33.7% ; 95%CI: 25.78-33.79) with a 5 year survival of (2.87%, 95%CI: 2.83-6.01) and a median survival of (8.90 months; 95%CI: 7.5-10.2). The median disease-specific survival was (9.40; 95%CI: 7.9-10.5 months) and the median time to relapse was 1.2 months (95%CI 1.0-1.2 months). A central tenet of contemporary cancer research is that an understanding of the genetic and molecular abnormalities that accompany the development and progression of cancer is critical to further advances in diagnosis, treatment and eventual prevention. High throughput tissue microarrays were used to study expression of two novel tumour markers in a cohort of pancreatic cancer patients and identified sFRP4 and ??-catenin as potential novel prognostic markers.

Pancreatic cancer is the 4th leading cause of cancer related death in the United States with a median survival time of less than 6 months. Pancreatic ductal adenocarcinoma (PDAC) accounts for greater than 85% of all pancreatic cancers, and is marked by early and frequent mutation of the KRAS oncogene, with activating KRAS mutations present in over 90% of PDAC. To date, though, targeting activated KRAS for cancer treatment has been very difficult, and targeted therapies are currently being sought for the downstream effectors of activated KRAS. Activation of KRAS stimulates multiple signaling pathways, including the MEK-ERK and PI3K-AKT signaling cascades, but the role of downstream effectors in pancreatic tumor initiation and progression remains unclear. I therefore used primary pancreatic ductal epithelial cells (PDECs), the putative cell of origin for PDAC, to determine the role of specific downstream signaling pathways in KRAS activated pancreatic tumor initiation. As one third of KRAS wild type PDACs harbor activating mutations in BRAF , and KRAS and BRAF mutations appear to be mutually exclusive, I also sought to determine the effect of activated BRAF (BRAF V600E ) expression on PDECs and the signaling requirements downstream of BRAF. I found that both KRAS G12D and BRAF V600E expressing PDECs displayed increased proliferation relative to GFP expressing controls, as well as increased PDEC survival after challenge with apoptotic stimuli. This survival was found to depend on both the MEK-ERK and PI3K-AKT signaling cascades. Surprisingly, I found that this survival is also dependent on the IGF1R, and that activation of PI3K/AKT signaling occurs downstream of MEK/ERK activation, and is dependent on signaling through the IGF1R. Consistent with this, I find increased IGF2 expression in KRAS G12D and BRAF V600E expressing PDECs, and show that ectopic expression of IGF2 rescues survival in PDECs with inhibited MEK, but not PI3K. Finally, I showed that the expression of KRAS G12D or BRAF V600E in PDECs lacking both the Ink4a/Arf and Trp53 tumor suppressors is sufficient for tumor formation following orthotopic transplant of PDECs, and that IGF1R knockdown impairs KRAS and BRAF-induced tumor formation in this model. In addition to these findings within PDECs, I demonstrate that KRAS G12D or BRAF V600E expressing tumor cell lines differ in MEK-ERK and PI3K-AKT signaling from PDECs. In contrast to KRAS G12D or BRAF V600E expressing PDECs, activation of AKT at serine 473 in the KRAS G12D or BRAF V600E expressing tumor cell lines does not lie downstream of MEK, and only the inhibition of PI3K alone or both MEK and the IGF1R simultaneously results in loss of tumor cell line survival. However, inhibition of MEK, PI3K, or the IGF1R in KRAS G12D or BRAF V600E expressing tumor cell lines also resulted in decreased proliferation relative to DMSO treated cells, demonstrating that all three signaling cascades remain important for tumor cell growth and are therefore viable options for pancreatic cancer therapeutics.

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.

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.

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), 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.

El càncer de pàncrees és altament agressiu i representa la cinquena causa de mort al mon occidental. Anteriorment, en el nostre laboratori, vam identificar que el receptor tissular del plasminogen (tPA) hi està sobre-expressat i juga un paper important el la progressió tumoral. En la present tesi hem profunditzat en l'estudi del mecanisme molecular de tPA i seus receptors en aquest càncer.
En primer lloc hem caracteritzat en detall la interacció de tPA amb Annexina A2 (principal receptor de tPA en endoteli i altament expressada en pàncrees) demostrant que les dades publicades sobre la seqüència responsable de la interacció no eren correctes. A més a més hem caracteritzat les proteïnes de lisats cel·lulars pancreàtics que interaccionen amb tPA mitjançant un assaig pull down i posterior anàlisi proteòmic. de tot identificant un conjunt de possibles lligands de tPA. D'entre aquests hem seleccionat galectina 1, una lectina que mai s'ha descrit que interaccioni amb tPA, per realitzar la caracterització bioquímica i funcional del seu paper com a nou lligand de tPA en càncer de pàncrees.
Pancreatic cancer is a highly aggressive disease and represents the fifth cause of death in occidental world. Our laboratory has previously reported tissue type plasminogen activator (tPA) over expression in this cancer and its role in tumoral progression. During the present thesis we have studied tPA and its molecular mechanism through its receptors in this tumor.
We have first characterized tPA interaction with annexin A2 (its main receptor in endothelium and highly expressed in pancreas). Our results showed that published data about the sequence responsible of this interaction was not correct. We have also identified a set of new putative tPA receptors in pancreatic cell lisates using a pull down assay and proteomic analysis. One of the proteins identified was galectin 1, a lectin with not know relation with tPA. We performed a biochemical and functional characterization of the interaction between these two proteins in pancreatic cancer.

Curcumin is the active component of tumeric, and this polyphenolic compound has been extensively investigated as an anticancer drug that modulates multiple pathways and genes. We demonstrated that curcumin inhibited 253JB-V and KU7 bladder cancer cell growth, and this was accompanied by induction of apoptosis and decreased expression of the proapoptotic protein survivin and the angiogenic proteins vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). Since expression of survivin, VEGF and VEGFR1 are dependent on specificity protein (Sp) transcription factors, we also investigated the effects of curcumin on downregulation of Sp protein expression as an underlying mechanism for the apoptotic and antiangiogenic activity of this compound. Curcumin decreases expression of Sp1, Sp3 and Sp4 in blader cancer cells indicating that the cancer chemotherapeutic activity of curcumin is due, in part, to decreased expression of Sp transcription factors and Sp-dependent genes. Betulinic acid (BA) and curcumin are phytochemical anticancer agents, and we hypothesized that both compounds decrease EGFR expression in bladder cancer through downregulation of specificity protein (Sp) transcription factors. BA and curcumin decreased expression of EGFR, Sp1, Sp3, Sp4 and Sp-dependent proteins in 253JB-V and KU7 cells; EGFR was also decreased in cells transfected with a cocktail (iSp) containing small inhibitory RNAs for Sp1, Sp3 and Sp4 showing that EGFR is an Sp-regulated gene. Methyl 2-cyano-3,11-dioxo-18?-olean-1,12- dien-30-oate (CDODA-Me) is a synthetic triterpenoid derived from glycyrrhetinic acid which inhibits proliferation of KU7 and 253JB-V bladder cancer cells. CDODA-Me also decreased expression of specificity protein-1 (Sp1), Sp3 and Sp4 transcription factors. Similar results were observed for a structurally-related triterpenoid, methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me), which is currently in clinical trials for treatment of leukemia. Celastrol, a naturally occurring triterpenoid acid from an ivy-like vine exhibits anticancer activity against bladder cancer cells. Celastrol decreased cell proliferation, induced apoptosis and decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and several Sp-dependent genes like Fibroblast growth factor receptor 3 (FGFR3). In vivo studies using KU7 cells as xenografts showed that celastrol represents novel class of anticancer drugs that acts, in part, through targeting downregulation of Sp transcription factors.

Indiana University-Purdue University Indianapolis (IUPUI)
Sequestration at the aggresome and degradation through autophagy are two approaches by which a cell can counteract the toxic effect of misfolded proteins. Tuberous sclerosis (TS) and cancer cells can become dependent on autophagy for survival due to the high demand for protein synthesis, thus making protein clearance a potential therapeutic target. Because of its histone deacetylase (HDAC) inhibitory activity, we hypothesized that 4-phenylbutyrate (4-PBA) inhibits HDAC6 and aggresome formation to induce TS cell death. We found that 4-PBA treatment increases cell death and reduces bortezomib-induced aggresome formation. To link these results with HDAC inhibition we used two other HDAC inhibitors, trichostatin A (TSA) and tubastatin, and found that they also reduce bortezomib-induced protein aggregation. Because tubulin is a target of HDAC6, we next measured the effect of the HDAC inhibitors and 4-PBA treatment on tubulin acetylation. As expected, tubastatin increased tubulin acetylation but surprisingly TSA and 4-PBA did not. Because 4-PBA did not significantly inhibit HDAC6, we next hypothesized that 4-PBA was alternatively inducing autophagy and increasing aggresome clearance. Surprisingly, autophagy inhibition did not prevent the 4-PBA-induced reduction in protein aggregation. In conclusion, we found 4-PBA to induce cell death and reduce aggresome levels in TS cells, but we found no link between these phenomena. We next hypothesized that loss of the Ral guanine nucleotide exchange factor Rgl2 induces cell death via autophagy inhibition in pancreatic adenocarcinoma (PDAC) cells. KRas is mutationally activated in over 90% of PDACs and directly activates Rgl2. Rgl2 activates RalB, a known regulator of autophagy, and Rgl2 has been shown to promote PDAC cell survival. We first confirmed that loss of Rgl2 does increase cell death in PDAC cells. Initial experiments using doubly tagged fluorescent p62 and LC3 (autophagy markers) suggested that loss of Rgl2 inhibited autophagosome accumulation, but after developing a more sophisticated quantitation method we found loss of Rgl2 to have no effect. We also measured endogenous LC3 levels, and these experiments confirmed loss of Rgl2 to have no effect on autophagy levels. Therefore, loss of Rgl2 increases cell death in PDAC cells, but does not have a significant effect on autophagy.

Methyl 2-cyano-3,11-dioxo-18b-olean-1,12-dien-30-oate (CDODA-Me) is a synthetic triterpenoid that inhibits growth of Panc1 and Panc28 pancreatic cancer cell lines and activates peroxisome proliferator-activated receptor B (PPARB)-dependent transactivation in these cells. CDODA-Me has also induced p21 and p27 protein expression and downregulated cyclin D1; however, these responses were receptor-independent. CDODA-Me induced apoptosis, which was accompanied by receptor-independent induction of the proapoptotic proteins early growth response-1 (Egr-1), nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), and activating transcription factor-3 (ATF3). Induction of NAG-1 in Panc28 cells was p38-mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3-K)-dependent, but Egr-1-independent, whereas induction in Panc1 cells was associated with activation of p38-MAPK, PI3-K and p42-MAPK and was only partially Egr-1-dependent. Specificity protein (Sp) transcription factors Sp1, Sp3 & Sp4 are overexpressed in multiple tumor types and negative prognostic factors for survival. Since Sp proteins regulate genes associated with survival (survivin), angiogenesis [vascular endothelial growth factor and its receptors] and growth [cyclin D1, epidermal growth factor receptor], research in this laboratory has focused on development of anticancer drugs that decrease Sp protein expression. Arsenic trioxide, curcumin, 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid (CDDO), CDDO-Me, and celastrol exhibit antiproliferative, antiangiogenic and proapoptotic activity in many cancer cells and tumors. Treatment of cancer cells derived from urologic and gastrointestinal tumors with arsenic trioxide decreased Sp1, Sp3 and Sp4 transcription factors and cotreatment with the proteosome inhibitor MG132 did not inhibit downregulation of Sp proteins in these cancer cells. Mechanistic studies suggested that compound-dependent downregulation of Sp and Sp-dependent genes was due to decreased mitochondrial membrane potential and induction of reactive oxygen species, and the role of peroxides in mediating these responses was confirmed using hydrogen peroxide, demonstrating that the mitochondriotoxic effects of these compounds are important for their anticancer activities. Moreover, repression of Sp and Sp-dependent genes by CDDO-Me and celastrol was due to downregulation of microRNA-27a and induction of ZBTB10, an Sp repressor, and these responses were also reversed by antioxidants. Thus, the anticancer activity of CDDO-Me and celastrol is due, in part, to activation of ROS which in turn targets the microRNA-27a:ZBTB10?Sp transcription factor axis to decrease growth inhibitory, pro-apoptotic and antiangiogenic genes and responses.

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.

Infiltration of myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis in many types of cancer. The polypeptide chemokine PK2 (Bv8, PROK2) has been shown to regulate myeloid cell mobilization from the bone marrow, leading to activation of the angiogenic process, as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 were shown to display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. In this study we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the context of glioblastoma and pancreatic cancer xenograft tumor models. For the highly vascularized glioblastoma, PKRA7 was associated with decreased blood vessel density and increased necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 appears to be mediated by the blockage of myeloid cell migration/infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of certain pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both types of tumor. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by two distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy.
Dissertation