Introduction: The outcomes following radical treatment for bladder cancer (BC) remain poor, with 5 year overall survival (OS) rates of approximately 50% and over 5000 deaths per year in the U.K. There has been paucity of significant therapeutic developments since the introduction of cisplatinum based chemotherapy in the 1970’s. The aim of this study was to identify putative drug targets for the treatment of this aggressive form of cancer. Methods: A tissue microarray (TMA) was constructed from the cystectomy specimens of 497 BC patients and 70 controls, linked to a clinical database with extended follow up. The online database Oncomine® was interrogated to identify putative treatment targets which were subsequently evaluated using in-vitro models of high grade invasive bladder cancer (using the J82 and T24 cell lines). In-vitro modelling was conducted using siRNA target knockdown during proliferation, chemo-sensitivity, migration and Matrigel™ invasion assays. Expression of the putative targets was then correlated with tumour characteristics and patient outcomes, by IHC and automated image analysis of the TMA. Results: The proteins CYR61 and CTGF were selected from Oncomine® and studied in conjunction with the HGF/MET axis, on the basis of known interactions in other cancer types. siRNA knockdown of both proteins abrogated HGF induced Matrigel™ invasion in both cell lines. CYR61 knockdown significantly reduced HGF induced cell migration and foetal calf serum (FCS) induced Matrigel™ invasion in both cell lines. Knockdown of both proteins also significantly increased the sensitivity of both cell lines to cisplatinum. CYR61 expression was significantly increased in BC samples compared to normal controls and an independent predictor of OS at 6 years (HR 1.493, p=0.030). In contrast, loss of CTGF expression was significantly associated with increasing tumour stage and worse OS. MET expression was reduced in BC compared to controls and not predictive of survival following cystectomy. Conclusions: The in-vitro findings for CTGF as a treatment target were encouraging, although these findings were not supported by the TMA data. CYR61 promotes an aggressive bladder cancer phenotype and knockdown reverses features of EMT and increases chemo-sensitivity. Clinical cohort correlation confirms CYR61 to be a promising treatment target in bladder cancer.

Yes / Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates β-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis. / Cancer Research UK (CRUK Beatson Institute C596/A17196, CRUK Glasgow Centre C596/A18076 and S.Z. C596/A12935)

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Made available in DSpace on 2014-06-11T19:23:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-25Bitstream added on 2014-06-13T19:09:53Z : No. of bitstreams: 1 voorwald_fa_me_jabo.pdf: 10335054 bytes, checksum: ceb747d5abc4cf9e53f8a440a6bd0557 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os órgãos reprodutores das fêmeas caninas sofrem transformações morfológicas e funcionais durante o ciclo estral, sob influência do estrógeno e da progesterona. A proteína CYR61 (proteína rica em cisteína, 61kilodaltons), mediada por citocinas inflamatórias, interleucinas, integrinas e outros, regula processos como proliferação e diferenciação celular, quimiotaxia, angiogênese e tumorgênese. É identificada em endometriose em mulheres, ratas e fêmeas de babuíno, acometidas por endometriose ou hiperplasia endometrial. Objetivou-se com este estudo imunolocalizar e quantificar a expressão da CYR61 em órgãos reprodutores das fêmeas caninas saudáveis, sob diferentes níveis séricos de progesterona e estradiol, e acometidas por piometra. Os tecidos coletados das 100 fêmeas caninas foram submetidos à técnica de imunoistoquímica com anticorpo anti-CYR61, e apresentaram marcação citoplasmática positiva nos componentes epiteliais em maior intensidade quando comparada aos componentes estromais. Foi identificada correlação linear positiva de área com marcação citoplasmática no ovário, corno uterino e corpo do útero, na fase lútea do ciclo estral. As tubas uterinas demonstraram maior expressão e correlação linear positiva com a fase proliferativa do ciclo estral. É possível concluir que a expressão do CYR61 está diretamente relacionada com as alterações morfológicas e funcionais dos órgãos reprodutores femininos, provocadas pelos hormônios ovarianos nas diferentes fases do ciclo estral, de forma particular sobre cada tecido, de acordo com suas funções fisiológicas / The reproductive organs of female dogs suffer morphological and functional changes during the estrous cycle, under the influence of estrogen and progesterone. CYR61 protein (cystein-rich protein, 61kilodaltons) mediated by inflammatory cytokines, interleukins, integrins and others, regulates the processes such as cell proliferation and differentiation, chemotaxis, angiogenesis and tumorgenesis. It was identified in endometriosis in women, rats and female baboon, who suffers from endometriosis or endometrial hyperplasia. The objective of this study was the immunolocalization and quantification of CYR61 expression in reproductive organs of healthy canine females under different levels of progesterone and estradiol, and females affected by pyometra. The tissues collected from 100 females dogs were subjected to immunohistochemistry analyses with anti-CYR61, and showed cytoplasmatic positive reaction in higher intensity on epithelial components, when compared with stromal components. This study identified positive linear correlation of cytoplasmatic area with positive reaction on the ovary and uterus in luteal phase of the estrous cycle. The fallopian tubes showed higher expression and positive linear correlation with estradiol, in proliferative phase of the estrous cycle. It was concluded that the CYR61 expression is directly related to the morphological and functional changes of the female reproductive organs, caused by the ovarian hormones at different stages of the estrous cycle in a particular way on each tissue, according to their physiological functions

<p>Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Gemcitabine is a nucleoside pyrimidine analog that has long been the backbone of chemotherapy for PDAC, both as a single agent, and more recently, in combination with nab-paclitaxel. Since gemcitabine is hydrophilic, it must be transported through the hydrophobic cell membrane by transmembrane nucleoside transporters. Human equilibrative nucleoside transporter-1 (hENT1) and human concentrative nucleoside transporter-3 (hCNT3) both have important roles in the cellular uptake of the nucleoside analog gemcitabine. While low expression of hENT1 and hCNT3 has been linked to gemcitabine resistance clinically, mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. We identified that the matricellular protein Cysteine-Rich Angiogenic Inducer 61 (CYR61) negatively regulates expression of hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 significantly increased expression of hENT1 and hCNT3 and cellular uptake of gemcitabine. CRSIPR-mediated knockout of CYR61 sensitized PDAC cells to gemcitabine-induced apoptosis. Conversely, adenovirus-mediated overexpression of CYR61 decreased hENT1 expression and reduced gemcitabine-induced apoptosis. We demonstrate that CYR61 is expressed primarily by stromal pancreatic stellate cells (PSCs) within the PDAC tumor microenvironment, with Transforming Growth Factor- β (TGF-β) inducing the expression of CYR61 in PSCs through canonical TGF-β-ALK5-Smad signaling. Activation of TGF-β signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in an in vitro co-culture assay with PDAC cells. Our results identify CYR61 as a TGF-β induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.</p> / Dissertation