STK11/LKB1, a serine/threonine protein kinase, is a key upstream kinase of adenine monophosphate-activated protein kinase (AMPK), a necessary kinase in the control of metabolism for maintaining energy homeostasis. Although it has become clear that LKB1 is mutated in a significant number of Peutz¡VJeghers syndrome (PJS) and sporadic cancers, most frequently in adenocarcinoma of the lung, little is known about how the LKB1 signaling regulates the metabolic process and energy production underlying hypoxia and increased radiosensitivity of lung tumor. Here, we employed lung cancer cells as a model system to dissect the functional roles of LKB1 signaling in human lung adenocarcinoma. We found that LKB1 inhibits lung cancer cell migration, transformation and chemo-resistance in vitro after we restored LKB1 expression in LKB1 null A549 and H460 lung cancer cells. We also found that LKB1 prevents UV-induced DNA damage in human lung cancer cell lines by comet assay and activated UV-induced apopotsis by MTT assays. Furthermore, we designed a systems biology approach to provide a comprehensive protein-protein interaction analysis in order to elucidate the LKB1 tumor suppressor network in vivo. We employed Immunoprecipitation-HPLC- Mass Spectrometry (IP-LC-MS) to identify the novel proteins interacting with LKB1 under different cellular stress conditions. We have identified that LKB1 is involved in CFTR synthesis pathway underlying normoxia condition and participates in the glycolysis and gluconeogenesis pathways underlying hypoxia condition. Together, our findings indicated that LKB1 is involved in the regulation of cell migration, energy metabolism and DNA repair in lung cancer cells, and should provides insights to further exploit the concept of deranged cancer bioenergetics and aberrant growth signals to achieve more effective and selective strategies for lung cancer patients.

No description available.

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse. This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd). Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd. The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse. This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd). Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd. The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.

Leukocyte cell-derived chemotaxin 2 (LECT2) is first isolated as a 16-kDa secreted protein from cultured fluid of phytohemagglutinin-activated human T-cell leukemia SKW-3 cells. Recently LECT2 has shown to be synthesized by human hepatocytes and stimulates the growth of chondrocytes. LECT2 is involved in chemotactic factor to neutrophils and may be associated with rheumatoid arthritis. Besides, LECT2 is evolutionarily conserved and acts as a repressor in the Wnt/£]-catenin signaling pathway. Wnt/£]-catenin signaling is implicated in liver carcinogenesis. However, the exact roles of LECT2 in liver carcinogenesis are not yet well characterized. This study is to investigate the extra roles of LECT2 in Wnt signaling. Our results showed that adenoviral administration of LECT2 over-expression suppress oncogenic processes such as migration, invasion, proliferation and colony formation, as well as alteration in cell cycle distributions. In animal model significantly suppress liver malignancies in orthotopic Novikoff hepatoma. In conclusion, we show that ad-LECT2 gene delivery attenuated cell carcinogenesis process via downregulated Wnt/£]-catenin signaling in vitro and suppressed tumor growth in vivo. Besides LECT2 over-expression represents a novel therapeutically factor for hepatocelluar carcinoma.

No description available.

Yes / Skin development is governed by complex programs of gene activation and silencing, including microRNA-dependent modulation of gene expression. Here, we show that miR-214 regulates skin morphogenesis and hair follicle (HF) cycling by targeting β-catenin, a key component of the Wnt signaling pathway. miR-214 exhibits differential expression patterns in the skin epithelium, and its inducible overexpression in keratinocytes inhibited proliferation, which resulted in formation of fewer HFs with decreased hair bulb size and thinner hair production. The inhibitory effects of miR-214 on HF development and cycling were associated with altered activities of multiple signaling pathways, including decreased expression of key Wnt signaling mediators β-catenin and Lef-1, and were rescued by treatment with pharmacological Wnt activators. Finally, we identify β-catenin as one of the conserved miR-214 targets in keratinocytes. These data provide an important foundation for further analyses of miR-214 as a key regulator of Wnt pathway activity and stem cell functions during normal tissue homeostasis, regeneration, and aging.

Das Pankreaskarzinom ist eine der aggressivsten Krebsarten mit einer extrem schlechten Prognose für die Patienten (Robert Koch-Institut 2012). Der Entwicklung neuer Substanzen für die Therapie wird eine große Bedeutung zur Verbesserung der Behandlungsergebnisse zugesprochen (Li D et al. 2004). Das Ziel der vorliegenden Dissertation war, die Wirkung des für die Behandlung von Bandwurminfektionen eingesetzten Anthelmintikums Niclosamid auf verschiedene Pankreaskarzinom-Zelllinien zu untersuchen. Die humanen Pankreaskarzinom-Zelllinien (MIA PaCa-2, Panc-1 und L3.6pl) wurden mit aufsteigenden Konzentrationen des Medikaments Niclosamid für 24 Stunden be¬handelt (0,1; 1; 3; 10; 30 µM, Inkubation in RPMI-1640-Medium). Nachfolgend wurden die vitalen Zellen unter dem Lichtmikroskop ausgezählt. Mit Hilfe einer modifizierten Boyden-Kammer wurde die Migration der Zellen durch eine poröse Membran (Poren¬größe 8 µm) untersucht. FACS-Analysen nach Färbung mit Annexin und Propidium¬iodid dienten zur Bestimmung der Apoptose- und Nekroserate sowie zum Nachweis der Veränderungen im Zellzyklus. Um Effekte von Niclosamid auf die Signaltrans¬duktion aufzuzeigen, wurden qRT-PCR-Analysen ausgewählter Wnt- und Hedgehog-Zielgene durchgeführt. Bereits eine Konzentration von 3 µM Niclosamid führte zu einer signifikanten Reduktion der vitalen Zellen in allen drei Zelllinien. Bei MIA PaCa-2 und Panc-1 konnte sogar ab 0,1 µM Niclosamid eine signifikante Abnahme der Zellzahl beobachtet werden (MIA PaCa-2 auf 48 % und Panc-1 auf 55 %); p≤0,05. Außerdem konnte bei einer Konzentration von 0,5 µM eine Inhibition der Zellmigration in MIA PaCa-2 auf 28 % und Panc-1 auf 27 % des Ausgangswertes gezeigt werden. In der FACS-Analyse zeigte sich bei Konzentrationen von 1 µM (Panc-1) und 10 µM (MIA PaCa-2 und L3.6pl) Niclosamid eine signifikante Zunahme der apoptotischen und nekrotischen Populationsanteile. Weiterhin konnte eine dosisabhängige Induk-tion des Zellzyklusarrestes bei Konzentrationen zwischen 0,1-1 µM beobachtet wer-den. Signifikante Effekte auf die Expression der Wnt-Zielgene fanden sich bei allen drei Zelllinien. So kam es bei mit Niclosamid behandelten Zellen zu einem Rückgang der Expression der kanonischen Wnt-Komponenten Axin2 und MMP7 (Panc-1, L3.6pl), Cyclin D1 (MIA PaCa-2, Panc-1, L3.6pl) und BCL9 (MIA PaCa-2). Eine ein¬deutige Beeinflussung der Komponenten des nicht-kanonischen Wnt-Signalweges (c-jun, Wnt5a) und des Hedgehog-Signalweges (SHH, SMO, Ptch1, Gli1) konnte nicht ermittelt werden. Niclosamid hatte demnach mindestens drei verschiedene Wirkmechanismen: Es hemmte die Zellproliferation und Migration, förderte die Apoptose und Nekrose und be¬wirkte einen Zellzyklusarrest der Pankreaskarzinom-Zellen. Außerdem konnte mit der PCR der hemmende Einfluss von Niclosamid auf den kanonischen Wnt-Signalweg, einen der wichtigsten Signalwege in der Entwicklung des Pankreaskarzinoms, aufge¬zeigt werden. Zusammenfassend lässt sich sagen, dass in Niclosamid ein neuer In¬hibitor des Wnt-Signalweges in Pankreaskarzinom-Zelllinien identifiziert werden konnte. Die Ergebnisse der vorliegenden Dissertation berechtigen möglicherweise zur Annahme, dass Niclosamid zukünftig eine aussichtsreiche chemotherapeutische Substanz zur Behandlung des Pankreaskarzinoms darstellen könnte.

Doctor of Philosophy / Department of Biology / Michael A. Herman / All multicellular organisms consist of a variety of cell types. One of the mechanisms to generate this cellular diversity is the asymmetric cell division, which requires the establishment of cell polarity. In Caenorhabditis elegans hermaphrodites, 807 of 949 somatic cell divisions are asymmetric. The centrosome and the Wnt signaling pathway both have been shown to regulate cell polarity and subsequently asymmetric divisions in many model organisms. However, it is not clear whether the Wnt signaling pathway manipulates the cell polarity through specific cellular organelles, such as the centrosome. To address this question, we examined a centrosomal component, SPD-5, to see whether it cooperates with the Wnt signaling pathway to regulate certain asymmetric cell divisions. We showed that SPD-5, which was originally found to be critical for the embryonic development, also played a role during certain post-embryonic cell divisions in C. elegans. Specifically the asymmetric divisions of seam cells that required SPD-5 function were also known to be regulated by the Wnt signaling pathway. Thus the stem-cell like seam cell divisions could be an intriguing system to study the interaction of centrosomes and the Wnt pathway. We found that SPD-5 was required for a successful cell division, similar to other centrosomal components. This suggests that SPD-5 still functions as a centrosomal component during C. elegans post-embryonic development. It has been shown that establishment of seam cell polarity relies on the asymmetric localization of certain Wnt pathway components. Interestingly, we found that SPD-5 was required for the proper localization of several Wnt components in a way that was independent of a key MTOC (microtubule-organizing center) member γ-tubulin. In addition, SPD-5 genetically interacted with the Wnt pathway components APR-1/APC and POP-1/Tcf to regulate asymmetric divisions of seam cells. These data suggest that SPD-5 interacts with the Wnt signaling pathway in controlling the polarity of seam cells. Overall, our results suggest a novel role of SPD-5 in cooperating with the Wnt signaling pathway to regulate cell polarity and asymmetric cell division, in addition to its function as a centrosomal component.

Oral squamous cell carcinoma (OSCC) accounts for majority of head and neck cancers and ranks as the sixth most common cancer in the world. OSCC belongs to the most understudied cancers and little is known about molecular mechanisms underlying its etiology and progression to metastasis. A hallmark of cancer is the enhanced posttranslational modification of cell surface proteins with complex N-glycans. Our studies have shown that induced protein N-glycosylation via activation of the core N-glycosylation-regulating gene, DPAGT1, is associated with reduced E-cadherin adhesion, as well as deregulation of several oncogenic signaling pathways, including Wnt/β-catenin and Hippo. Modest increases in DPAGT1 expression are associated with dramatic amplification of Wnt/β-catenin activity and increased expression and nuclear localization of the Hippo pathway effectors TAZ /YAP. The goal of this study was to align the expression and localization of DPAGT1, complex N-glycans, β-catenin, and TAZ/YAP with the progression of oral cancer in vivo from dysplasia to OSCC. Human oral tissues from different stages of OSCC pathogenesis were characterized for DPAGT1/β-catenin/α-catenin/YAP/TAZ expression and localization and correlated with cell surface expression of complex N-glycans by PHA lectin staining and with expression of primitive cell surface markers, CD44, CD24 and CD29. Results showed that high DPAGT1 expression and nuclear TAZ became increasingly associated with disorganized E-cadherin junctions as oral epithelium progressed from mild to severe dysplasia to OSCC. This correlated with increasing expression of cell surface complex N-glycans and CD44. These studies suggest that DPAGT1/β-catenin/TAZ and high PHA staining represent novel signatures for OSCC pathogenesis.