Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancerrelated deaths with a five-year survival rate of 11%. PDAC tumors are characterized by a dense desmoplastic stromal microenvironment, mediated in part through local cytokine production. PDAC tumors also elicit a systemic inflammatory response in the host; this, combined with a loss of body weight due to muscle and fat wasting, is characteristic of cachexia. Understanding the molecular mechanisms that drive malignant inflammation is critical to improve PDAC therapy and increase patient survival. Oncostatin M (OSM) belongs to the IL-6/GP130 family of cytokines, members of which have been shown to promote PDAC tumor development, inflammation, and cachexia. Much less is known of OSM. My central hypothesis was that OSM promotes pancreatic cancer and cachexia by inducing local and systemic inflammation, fibrosis, and wasting via OSM signaling through the receptor, OSM receptor (OSMR). We investigated effects of exogenous OSM administration in wildtype and IL-6 null mice without cancer. OSM induced systemic fibrosis, bone loss, local muscle wasting, and cardiac dysfunction in presence and absence of IL-6. We further defined the roles of OSM/OSMR in the pancreatic cancer microenvironment and macroenvironment. OSM activated genes involved in inflammation, fibrosis, and tumor progression in both tumor cells and fibroblasts and altered the tumor microenvironment, promoting a dense compaction of tumor cells and cancer associated fibroblasts. Loss of systemic OSM signaling altered tumor metabolism and reduced the stromal compartment without affecting tumor size. Loss of OSMR signaling in tumor cells reduced tumor size and promoted survival. However, systemic loss of OSM or OSMR in host cells did not halt effects of cachexia including muscle dysfunction, atrophy, or inflammation/anemia. Overall, OSM/OSMR signaling in the microenvironment is necessary in modulating tumor phenotype and promoting survival in PDAC but may not be necessary for pancreatic cancer cachexia. / 2024-08-02

Excessive tissue fibrosis in various lung diseases contributes to decline in lung function and subsequent morbidity and mortality. Mechanisms involve complex networks of molecules such as cytokines that are not clearly worked out in conditions such as Idiopathic pulmonary fibrosis (IPF). Furthermore, pulmonary virus infection has been linked to exacerbations of IPF. Previous studies have demonstrated that transient pulmonary over-expression of Oncostatin M (OSM) leads to increased extracellular matrix (ECM) accumulation, Th2-skewed cytokines and Arg1+ M2-like macrophage accumulation in mouse models. OSM can also robustly induce interleukin-33 (IL-33), an IL1 family cytokine or alarmin, both in vivo and in vitro mouse lung systems. Since others have shown that soluble IL-33 exacerbates bleomycin-induced lung fibrosis in mouse models and is associated with Th2-type lung diseases, IL-33 may mediate OSM effects on ECM and Arg1+ macrophage-like cell accumulation. The main hypothesis in this thesis is that OSM can induce IL-33 expression and Arg1+ cells, that OSM can potentiate IL-33 release from virally-infected epithelial cells, and that OSM can prime lungs to subsequent influenza infection and exacerbate pathology. Results demonstrated that OSM induced robust up-regulation of pulmonary IL-33 and Arg1 mRNA and protein expression in vivo, in comparison to another gp130 cytokine, IL-6. However, IL-6 was required for OSM-induced arginase-1 expression in vivo, but not IL-33 expression in vivo. OSM-induced Arg1 expression was also dependent upon IL-33 presence as demonstrated in IL-33-/- animals. This finding implicates a role for both IL-33 and IL-6 in mediating OSM-induced Arg1+ macrophage-like cell accumulation within the lung. Additionally, results showed that a respiratory Influenza A virus infection in vivo alone induced a time-dependent increase in OSM and IL-33 (Day 4), however reduced IL-33 by 7-days post-infection. Influenza infection in AdOSM-primed mice and led to decreased IL-33 expression and eosinophilic infiltration within the lung 5-days post-influenza infection. Collectively, these results demonstrate that OSM can drive Th2-associated pathology correlated to increased IL-33 and Arg1 expression. Contrary to expectations, influenza A virus infection led to a reduction in OSM-induced Th2-phenotype in vivo. Further exploration into the OSM-IL-33 pathway will provide insight into innate immune mechanisms of lung inflammation, virus infection and control of ECM accumulation. / Thesis / Master of Science (MSc)

Resistin-like molecule alpha (RELMα) is a secreted protein implicated in murine models of allergen-induced asthma, bleomycin-induced pulmonary fibrosis, and helminth infection. Transient pulmonary overexpression of Oncostatin M by Adenovirus vector (AdOSM) induces lung inflammation biased toward Th2 cytokines, eosinophil and alternatively activated (AA/M2) macrophage accumulation. In AdOSM-treated C57Bl/6 and BALB/c mice, we observed RELMα mRNA and protein markedly induced. RELMα is recognized as a marker of AA/M2 macrophages, and we observed by chromogenic in situ hybridization that RELMα mRNA co-expresses with the macrophage marker CD68, and RELMα mRNA was also highly induced in columnar airway epithelial cells upon AdOSM treatment. Assessing IL-6 as a comparator gp130 cytokine, AdIL-6 induced RELMα at significantly lower levels, however maximal induction of RELMα by AdOSM in C57Bl/6 mice required IL-6, assessed in IL-6–/– mice. Maximal induction of RELMα by AdOSM also required IL-33 in C57Bl/6 mice but not in BALB/c mice, assessed in IL-33–/– mice. We investigated functions of RELMα in response to OSM, in RELMα–/– mice. Inflammatory cell infiltration and Th2-associated cytokine responses were not altered in RELMα–/– in comparison to wildtype mice. However, RELMα-deficiency resulted in less accumulation of CD206+ AA/M2 macrophages, IFNγ+ Th1 cells in the lung, reduced induction of extracellular matrix gene mRNAs for COL1A1, COL3A1, MMP13, TIMP1, and reduced parenchymal alpha smooth muscle actin. RELMα–/– mice also showed less airway epithelial hyperplasia, increased epithelial cell damage/death (assessed morphologically) and increased LDH and soluble CK18 in response to AdOSM. Our findings suggest that RELMα does not modulate Th2 cytokines, but does participate in matrix deposition, airway remodelling mechanisms, and protection from inflammation-induced damage due to OSM-overexpression in lungs of C57Bl/6 mice. / Dissertation / Master of Science (MSc)

Les adénomes hépatocellulaires (AHC) sont des tumeurs bénignes rares qui se développent le plus souvent chez la femme jeune suite à la prise de contraceptifs oraux. Les complications principales sont l’hémorragie et plus rarement, la transformation maligne en carcinome hépatocellulaire (CHC). Des travaux récents ont permis d’identifier 3 groupes moléculaires principales d’AHC qui se définissent par (1) l’inactivation du facteur de transcription HNF1A (H-HCA), (2) l'activation de la voie Wnt/ß-caténine (bHCA) ou (3) la présence d’infiltrats inflammatoires (IHCA).Afin d’identifier les voies de tumorigenèse associées au développement d’AHC inflammatoires (IHCA), une analyse transcriptomique comparant des IHCA à des foies non tumoraux a été réalisée au laboratoire, ce qui a permis d’identifier dans ce groupe tumoral une activation de la voie IL-6/JAK/STAT3. Nous avons recherché de nouvelles altérations géniques et nous avons caractérisé le mécanisme d'activation de la voie IL-6/JAK/STAT dans les IHCA. Les conséquences fonctionnelles sur la voie STAT3 des différents mutants ont été analysées par une modélisation de leur expression dans des lignées hépatocellulaires. Par ailleurs, nous avons réalisé des études génomiques intégrées (analyse CGH-SNP, méthylome et séquençage exome) sur une large série de 250 AHC avec pour objectif d’affiner la classification moléculaire des AHC, d’identifier de nouveaux gènes altérés dans ces tumeurs et d’élucider les mécanismes de transformation maligne des AHC en CHC.Dans le groupe des IHCA, ces analyses nous ont permis d’identifier de nombreux oncogènes activés par mutation somatique ; de plus, trois de ces gènes n’avaient jamais été décrits comme étant mutés dans des tumeurs humaines. Nous avons identifié des mutations activatrices du récepteur à l’IL-6, gp130 dans 60% des IHCA. Nous avons aussi retrouvé des mutations de FRK, une src-like kinase, dans 10% des IHCA, du facteur de transcription STAT3 dans 5% des IHCA, du gène GNAS dans 5% des cas, et de la tyrosine kinase JAK1 dans 1% des cas. Toutes les mutations identifiées étaient somatiques, monoalléliques et mutuellement exclusives. Nous avons pu montrer, dans des systèmes de lignées cellulaires hépatocellulaires, que l'expression des formes mutées de ces gènes est capable d’activer la voie IL-6/STAT3 en absence du ligand IL-6, contrairement aux protéines sauvages. Nous avons identifié des inhibiteurs pharmacologiques qui permettent d’inhiber de façon spécifique ces mutants et qui pourraient être utilisés en clinique pour le traitement des IHCA.Grâce à une technique de CGH-SNP, nous avons identifié des événements récurrents de pertes et gains de chromosomes associés aux groupes moléculaires d’AHC. De façon similaire, l’étude de la méthylation dans les AHC a permis de mettre en évidence un pattern spécifique à chaque sous groupe. Nous avons montré que l’instabilité chromosomique augmente progressivement dans les lésions borderline et dans les CHC développés sur AHC comparés aux AHC classiques. Le séquençage exome de 5 transformations malignes de AHC en CHC a identifié un nombre plus important de mutations dans les AHC qui ont transformé comparé aux AHC classiques ; ce nombre est significativement augmenté dans la partie CHC des tumeurs. La comparaison de la partie bénigne et maligne des tumeurs a mis en évidence l'activation de ß-caténine comme un évènement précoce dans le processus de transformation et a révélé la présence de mutations somatiques fréquentes dans le promoteur de la télomèrase (TERT), identifiées principalement dans la partie maligne des tumeurs.En conclusion, cette étude a permis d’identifier des mécanismes distincts conduisant à l'activation de STAT3 dans les IHCA, renforçant le rôle de la voie JAK-STAT3 dans la tumorigenèse bénigne hépatocellulaire ainsi que le lien entre Src kinases et inflammation. Ces travaux ont permis d’affiner la classification moléculaire des AHC avec des corrélations étroites... / Hepatocellular adenomas (HCA) are rare benign tumors that develop most often in young women after taking oral contraception. The main complications are hemorrhage and rarely, malignant transformation to hepatocellular carcinoma (HCC). Recent work in the laboratory identified three main HCA molecular groups that are defined by (1) inactivation of the transcription factor HNF1A (H-HCA), (2) activation of the Wnt/ß-catenin pathway (bHCA) or (3) the presence of inflammatory infiltrates (IHCA).To identify tumorigenesis pathways associated with the development of inflammatory HCA (IHCA), a transcriptome analysis comparing IHCA to non-tumor liver was performed in the laboratory, leading to the identification of an activation of the IL-6/JAK/STAT3 pathway in these tumors. We sought new gene alterations and we characterized the activation mechanism of the IL-6/JAK/STAT pathway in IHCA. The functional consequences of the different mutants on the STAT3 pathway were analyzed by modeling their expression in hepatocellular cell lines. In addition, we performed integrated genomic studies (CGH-SNP analysis, methylome and exome sequencing) on a wide range of 250 HCA with the aim to refine the molecular classification of HCA, to identify new genes altered in these tumors and to elucidate the mechanisms of malignant transformation of HCA to HCC.In the group of the IHCA, we identified many oncogenes activated by somatic mutation; in addition, three of these genes were never been described as mutated in human tumors. We identified activating mutations in the IL-6 receptor gp130 in 60% of IHCA. We also found mutations in FRK, a src-like kinase, in 10% of IHCA, of the transcription factor STAT3 in 5% of IHCA, of the GNAS gene in 5% of cases, and of the tyrosine kinase JAK1 in 1% of the cases. All identified mutations were somatic and monoallelic and were mutually exclusive. We have shown in hepatocellular cell lines that the expression of mutated forms of these genes is able to activate the IL-6/STAT3 pathway in the absence of the IL-6 ligand, in contrast to wild-type proteins. We have identified pharmacological inhibitors that specifically inhibit the mutants and that could be used for the clinical treatment of IHCA.Using a CGH-SNP technique, we identified recurrent chromosomes gains and losses associated with the HCA molecular groups. Similarly, the study of methylation in HCA highlighted a specific pattern in each subgroup. We showed that chromosomal instability increases gradually in borderline lesions and in HCC developed on HCA compared to classical HCA. Exome sequencing of 5 malignant transformation of HCA to HCC identified a large number of mutations in the transformed HCA compared to classical HCA; and this number is significantly increased in HCC tumors counterpart. Comparison of benign and malignant tumors highlighted the activation of ß-catenin as an early event in the transformation process and revealed frequent somatic mutations in the promoter of the telomerase gene (TERT), identified mainly in the malignant part of tumors.In conclusion, this study has led to the identification of distinct mechanisms leading to the activation of STAT3 in IHCA, strengthening the role of the JAK-STAT3 pathway in benign hepatocellular tumorigenesis and the relationship between Src kinases and inflammation. This work helped to refine the molecular classification of HCA with tight correlations between genotype and phenotype, and led to advances in the identification of major genetic determinants involved in the process of malignant transformation.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 229-253.

<p>Lung cancer is the leading cause of cancer related deaths in both the US and Canada and efforts still need to be made towards understanding the disease. The role of inflammation in the promotion of cancer development represents a newer avenue of research. The glycoprotein (gp)-130 cytokine interleukin-6 (IL-6) has a well established role in promoting inflammation and recent evidence suggests roles in development of certain tumors in animal models. Less is known of the related family member oncostatin M (OSM) and the functions of either IL-6 or OSM in lung cancer development is not known. Based on the hypothesis that these cytokines promote lung cancer development, IL-6 and OSM were overexpressed in the lungs of two separate mouse models for lung cancer utilizing adenovirus vectors encoding IL-6 or OSM. The first mouse model utilized a Cre-conditional oncogene KRAS G12D (developed by Tyler Jacks) in which endotracheal administration of adenovirus (Ad)-encoded Cre-recombinase resulted in increases in lung densities in a dose-dependent fashion over a period of 6 weeks that were measurable by CT scanning and histology. Increases in cytokines IL-6 and kertinocyte chemoattractant (KC) were detectable in the bronchoalveolar lavage (BAL) by week 4, as well as marked increases in alveolar macrophage numbers. Macrophages were also shown as a possible target for Cre-mediated recombination and mutant KRAS expression. Administration of either AdIL-6 or AdOSM as well as AdCre resulted in a trend toward increases in tumor burden with AdOSM based on experiments terminated at 4 weeks. The second mouse model involved endotracheal administration of the lewis lung carcinoma (LLC) cell line, which after 7 days resulted in detectable tumor burden. Administration of either AdIL-6 or AdOSM and LLC cells simultaneously was shown to increase tumor burden relative to AdDl70 co-administration. These results suggest a possible role of IL-6 or OSM in promoting lung tumor development in animal models and may ultimately reveal gp130 cytokines IL-6 or OSM as a possible therapeutic target for the treatment of lung cancer.</p> / Master of Science (MSc)

The overall objective of this study was to investigate how NS0 cell proliferation is regulated in protein-free media. The hypothesis was that during the adaptation to growth factor-free media, animal cell lines start to produce their own autocrine growth factors to support proliferation, and after some time in a culture the effects of these factors are lost which results in cessation of proliferation. A chemically defined, protein-free and animal component-free medium was developed for the NS0 cells. This medium was comprised of a basal hybridoma medium to which phosphatidyl¬choline, cholesterol, β-cyclodextrin, ferric citrate and amino acids were added. A fed-batch process was then developed in this medium. The feed profile was optimised in a step-wise manner with a final feed solution containing glucose, glutamine, lipids, amino acids, vitamins, sodium selenite and ethanolamine. Specifically, supplementation with lipids (cholesterol) had a drastic effect on cell growth. Calcium, magnesium and potassium were not depleted during culture and a feed containing also iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield further. More than 8 x 106 viable cells mL-1 and 600 mg antibody L-1 was obtained in the final fed-batch. This corresponded to a 4.3-fold increase in viable cell yield and an 11.4-fold increase in product yield compared to bioreactor batch culture when the dilution of the fed-batch culture was also accounted for. The presence of autocrine growth factors in NS0 cell cultures was initially investigated by studying the effects of conditioned medium (CM). Concentrated CM had a significant positive effect on cell growth and part of this effect could be attributed to factor(s) eluting from a gel-filtration column at 20-25 kDa. In the search for cell-derived factors affecting cell growth the following proteins were identified as released/secreted by the NS0 cells; cyclophilin A, cyclophilin B, cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerise, macrophage migration inhibitory factor (MIF), β2-microglobulin, niemann pick type C2, secretory leukocyte protease inhibitor (SLPI), thioredoxin-1, TNF-α, tumour protein translationally controlled-1 and ubiquitin. Zymogram electrophoresis further identified aspartic acid, papain-like cysteine (including cathepsin L) and serine protease activity in the CM. Pro/cathepsin L, CypB, EGF, IFN-α/β/γ, IGF-I/II, leukaemia inhibitory factor, IL-6, IL-11, IL-25, MIF, oncostatin M, TGF-β and TNF-α were excluded as involved in autocrine regulation of NS0 cell proliferation. The serine protease activity was suggested to affect the cells negatively and since the serine protease inhibitor SLPI is also present in NS0 CM, a balance in serine protease activity may be crucial for optimal cell growth. Further, the receptor gp130, known to be associated with myeloma cell growth, was shown to be essential for NS0 cell proliferation as demonstrated by siRNA gene silencing. The results suggested that autocrine regulation of proliferation in NS0 cell cultures involves the receptor subunit gp130. / QC 20100920

[Truncated abstract] Fibrosis is a feature of many pulmonary conditions, including idiopathic pulmonary fibrosis (IPF), which is characterised by the accumulation of fibroblasts/myofibroblasts and excessive deposition of collagen. IPF is a disease of unknown aetiology that is unresponsive to current therapy and is typically fatal. The inflammatory cytokine interleukin (IL)-6 is elevated in patients with IPF and recent studies have shown that IL-6-induced signalling is altered in lung fibroblasts from patients with IPF. IL-6 belongs to the gp130 cytokine family, which is a group of ten structurally related cytokines, that all require the membrane bound glycoprotein gp130 to activate intracellular signalling pathways. Gp130 activates intracellular signalling through the Shp2-ERK1/2 and STAT1/3 pathways to mediate cellular activities. This thesis tests the hypothesis that gp130-mediated signalling is dysregulated in the development and progression of pulmonary fibrosis. To address this hypothesis, I assessed the role of gp130-mediated signalling in a mouse model of bleomycin-induced lung fibrosis. This thesis utilised two novel gp130 mutant mice strains with directed and enhanced gp130-mediated Shp2-ERK1/2 (gp130¿STAT/¿STAT) or STAT1/3 (gp130757F/757F) signalling. I observed complete protection from fibrosis in gp130¿STAT/¿STAT mice up to 60 days after bleomycin treatment and profound fibrosis in gp130757F/757F mice compared to wt controls. The enhanced fibrosis observed in gp130757F/757F mice was diminished by monoallelic deletion of STAT3 (gp130757F/757F;STAT3+/-), identifying gp130-STAT3 signalling as a novel promoter of lung fibrosis. ... In addition, IL-6/11 activation of gp130-mediated signalling modulated transforming growth factor (TGF)-ß-induced effects on adult fibroblast proliferation and myofibroblast differentiation. Interaction between IL-6/11 and TGF-ß1 on fibroblast proliferation was dependent on both the gp130-ERK1/2 and gp130-STAT1/3 pathways. Loss of either pathway abrogated the effects of IL-6 and IL-11 on TGF-ß1- 4 induced fibroblast proliferation. However, it was clear that gp130-STAT3 signalling inhibited TGF-ß1-induced myofibroblast differentiation of primary lung fibroblasts. The inhibition of myofibroblast differentiation was associated with gp130-STAT3 dependent inhibition of TGF-ß1-induced Smad3 phosphorylation. These results indicate that IL-6 and IL-11 promote myofibroblastic differentiation of lung fibroblasts, while gp130-STAT3 signalling inhibits TGF-ß1-induced Smad3 phosphorylation and myofibroblastic differentiation of lung fibroblasts While the pathogenesis of IPF is unknown, it is believed that excessive collagen deposition, aberrant fibroblast behaviour and an inflammatory response are critical to the progression of this disease. It has been shown here that IL-6 family cytokines mediate the development and progression of bleomycin-induced lung fibrosis by increasing collagen synthesis, fibroblast proliferation, myofibroblast differentiation and inflammation through gp130-STAT3 signalling. This thesis has demonstrated that differential activation of cytoplasmic signalling pathways by a membrane bound receptor can have a profound effect on pulmonary responses to injury. Furthermore, this thesis is the first study to identify the gp130-STAT3 pathway as a therapeutic target in the treatment of IPF.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 86-111).

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