Development of a cargo delivery system and inhibition studies focused on clostridium difficile toxin a

Virulence factors of pathogenic bacteria are to be blamed for life-threatening infections such as diphtheria, anthrax, botulism, and tentanus. In the case of enzymatic exotoxins, disease arises from cytotoxic proteins, and cytotoxicity is acheived only after cell entry. This intrinsic mechanism for cell entry is intriguing from research and medical views. Along with a review on existing cargo delivery systems utilizing protein toxins and the usefulness of such a system, here is described the first reported Clostridium difficile toxin A fusion protein, luciferase-TcdA, and evidence of the successful transport of an active enzyme, luciferase, into the cytosol of vero cells. A feature that makes our system so attractive, is the auto-proteolytic event that releases the cargo protein after internalization. In addition to protein delivery, the exciting success of a peptide-based inhibitor is described. Almost complete inhibition of toxin A was observed in cellulo by a peptide, identified to reversibly bind to the active site, HQSPWHHGGGC, that was functionalized with an epoxy group, HQSPGepoxyHHGGGC. Placement of the functional group is crucial for toxin inhibition. Through studying protein toxins we not only gain insight necessary for defending against deadly diseases, but we can also learn to harness their distinctive properties for the development of novel biotechnology tools, such as a protein translocation system

Dissecting the effect of EGF starvation on the signaling and transcriptomic landscapes of the mouse intestinal epithelium

Die EGFR-Signalübertragung steuert viele verschiedene zelluläre Prozesse in allen Arten von Epithelzellen, einschließlich des Darmepithels. Diese Prozesse reichen von Proliferation und Wachstum über Differenzierung bis hin zu Autophagie und Apoptose. Die vorliegende Studie zielt darauf ab, die Signalveränderungen zu charakterisieren, die im Darmepithel als Reaktion auf EGF-induzierten Hungerstress stattfinden. Kontraintuitiv führte eine 24-stündige EGF-Starre zu einer deutlichen Phosphorylierung von EGFR, MEK1/2 und ERK1/2, was auf eine Aktivierung dieser Signalachse in Darmzellen hindeutet. Diese Veränderungen waren am signifikantesten in den undifferenzierten CD44-reichen Krypta-Basiszellen. Interessanterweise war die EGF-Starvation-induzierte ERK1/2-Phosphorylierung mit der Hochregulierung einer Untergruppe von ERK-Zielgenen verbunden, bei denen es sich zumeist um primäre Zielgene handelt. Die Überexpression des EGFR-Liganden HBEGF und des FGFR-Liganden FGF1 in ausgehungerten Zellen könnte für die hungerbedingte Zunahme der MAPK-Aktivität verantwortlich sein, obwohl eine erhöhte Sekretion dieser Liganden durch ausgehungerte Organoide nicht bestätigt werden konnte. Dennoch wird die kompensatorische Ligandensekretion durch die Beobachtung gestützt, dass die erneute Zugabe von EGF zu ausgehungerten Organoiden die pERK1/2-Spiegel auf den Ausgangswert zurücksetzt, was bedeutet, dass EGF mit einem anderen von ausgehungerten Zellen sezernierten Liganden um den EGFR konkurriert. Zusätzlich zu HBEGF wurde festgestellt, dass andere Gene, die für den Schutz, das Überleben und die Regeneration des Darmepithels bekannt sind, in ausgehungerten Organoiden überexprimiert werden, wie z. B. Reg3b. Insgesamt können die in dieser Studie berichteten EGF-induzierten Veränderungen der MAPK-Signalübertragung und der globalen Genexpression als ein überlebensförderndes Programm interpretiert werden, das bevorzugt in Darmstammzellen und frühen Vorläuferzellen aktiviert wird.EGFR signaling drives many different cellular processes in all kinds of epithelial cells including the intestinal epithelium. Such processes range from proliferation and growth to differentiation to autophagy and apoptosis. The present study aims to characterize signaling changes that take place in the intestinal epithelium in response to EGF starvation-induced stress using epithelial organoids derived from the mouse duodenum and human colorectal tumor tissue. Counterintuitively, 24 h EGF starvation induced a prominent phosphorylation of EGFR, MEK1/2 and ERK1/2 indicating an activation of this signaling axis in intestinal cells. These changes were most significant in the undifferentiated CD44-high crypt base cells. Interestingly, EGF starvation-induced ERK1/2 phosphorylation was associated with upregulation of a subset of ERK target genes that were mostly primary-response targets. Overexpression of the EGFR ligand HBEGF and the FGFR ligand FGF1 in starved cells may account for starvation-driven increase in MAPK activity, although an increased secretion of these ligands by starved organoids was not confirmed. Nevertheless, compensatory ligand secretion is still supported by the observation that EGF re-addition to starved organoids restores pERK1/2 levels to baseline which implies that EGF competes for EGFR with some other ligand secreted by starved cells. In addition to HBEGF, other genes known to promote protection, survival and regeneration of the intestinal epithelium were found to be overexpressed in starved organoids such as Reg3b. Collectively, EGF starvation-induced changes in MAPK signaling and global gene expression reported in this study can be interpreted as a pro-survival program that gets activated preferentially in intestinal stem cells and early progenitors

Investigation of the role of CUZD1-STAT5 signaling in mammary gland development and breast cancer

In the mammary gland, genetic circuits controlled by the hormones, estrogen, progesterone and prolactin (PRL), act in concert with pathways regulated by the epidermal growth factor (EGF) family to control the growth and morphogenesis of this tissue during puberty, pregnancy and lactation. However, the precise molecular mechanisms that integrate these signaling pathways are unclear. In this study, we identifed CUZD1 (CUB and zona pellucida-like domain containing protein- 1) as a novel mediator of PRL signaling in steroid hormone-primed mouse mammary gland and undertook an examination of its role in growth and differentiation of this tissue during pregnancy. CUZD1 expression is markedly induced in steroid-primed mammary epithelial cells in response to PRL treatment. Cuzd1-null mice exhibited a striking impairment in ductal branching and alveolar development during pregnancy, resulting in a subsequent defect in lactation. Interestingly, phosphorylation and activation of STAT5, a transcription factor that mediates PRL signaling, was absent in Cuzd1-null mammary tissue during pregnancy and lactation. We also noted that the expression of epiregulin (EREG), an EGF family growth factor regulated directly by STAT5, is suppressed in Cuzd1-null mammary gland. Protein interaction studies, using flag-tagged CUZD1 expressed in HC11 mouse mammary epithelial cells, revealed that CUZD1 associates with a multi-protein complex containing JAK1/JAK2 and STAT5. Elevated expression of CUZD1 in HC11 cells stimulated phosphorylation and nuclear translocation of STAT5. Chromatin immunoprecipitation experiments indicated that STAT5 and CUZD1 co-occupy the same regulatory region of the Ereg gene. Over-expression of CUZD1 in mammary epithelial HC11 cells induced tumorigenic characteristics, such as substrate independent growth and migration. Furthermore, HC11-Cuzd1 cells formed mammary tumors in vivo following orthotopic injection into nude and Balb/C mice. Mammary tumor cells derived from these animals showed elevated levels of phosphorylation and nuclear localization of STAT5 and consequent activation of the EGF signaling pathway. Blockade of JAK/STAT5 signaling through the use of a STAT5 inhibitor markedly reduced the production of the EGF family growth factors and inhibited PRL-induced tumor cell proliferation in vitro. It also impaired the progression of CUZD1-driven mammary tumorigenesis in vivo. Collectively, our findings suggest that CUZD1 plays an important role in mammary epithelial cell proliferation during mammary gland development and in tumorigenesis by facilitating JAK-STAT5 signaling and subsequent production of growth factors, such as EREG. CUZD1, therefore, emerges as a critical mediator of PRL action that controls mammary alveolar development during pregnancy and lactation and cell proliferation during tumorigenesis

Characterization of TMEFF2; its role in tumour progression and development of targeting strategies for anti-cancer therapy

TMEFF2 (transmembrane protein with EGF-like and two follistatin motifs 2) is a transmembrane protein expressed in brain and prostate and over-expressed in prostate cancer (Uchida et al. 1999; Horie et al. 2000). The role of TMEFF2 in prostate cancer is controversial. Several data indicate that TMEFF2 has cancer-promoting activity (Glynne- Jones et al. 2001; Ali and Knäuper 2007), while others suggest that TMEFF2 inhibits progression of cancer (Gery et al. 2002; Gery and Koeffler 2003). TMEFF2 is cleaved by membrane-anchored proteases, including disintegrin and metalloproteases (ADAMs) and -secretase (Ali and Knäuper 2007), but the biological meaning of TMEFF2 shedding is not known. It was hypothesized that the opposing findings describing the role of TMEFF2 in prostate cancer result from proteolytic processing of TMEFF2 by different proteases which are co-expressed with TMEFF2 in prostate cancer cells, such as the type II transmembrane serine proteases (TTSPs), prostasin and ADAMs. To support this hypothesis co-expression of TMEFF2 and serine proteases was analyzed in prostate cancer cell lines and clinical samples. The shedding of TMEFF2 by ADAMs and serine proteases was investigated using HEK293 cells expressing AP/V5 TMEFF2 or shedding resistant AP/V5 303-320TMEFF2 mutant (Ali and Knäuper 2007). The data obtained from AP activity assay and Western blot analysis of cell lysates showed that TMEFF2 is cleaved by serine proteases (matriptase and hepsin) and ADAMs (ADAM9, ADAM12). Moreover, serine proteases and ADAMs cleave TMEFF2 in different positions, generating several soluble TMEFF2 fragments. To establish the biological role of TMEFF2 processing, N-terminal TMEFF2 fragments predicted to be generated by TTSPs and ADAMs were expressed in E. coli and mammalian cells. Preliminary experiments using HEK293 and PNT2-C2 cells indicated that soluble TMEFF2 does not signal through ErbB receptors and suggested several signaling pathways that might be regulated by TMEFF2. The fate of TMEFF2 C-terminus following ectodomain shedding was examined by confocal microscopy and Western blotting, indicating that TMEFF2 cytoplasmic domain is likely degraded following the release of TMEFF2-EC

The role of Hedgehog signaling in Cholangiocarcinogenesis: An in vitro and in vivo (mouse model) study

Cholangiocarcinoma (CC) is the second most frequently occurring primary liver tumor worldwide and is receiving medical importance because of its rising number of incidences, poor diagnosis and substandard response to the therapy. It is associated not only with genetic alterations, but other essential modifications of tumour microenvironment, which lead to the activation of various signaling pathways involved in tumour induction and progression. Thus, further exploration of the molecular mechanisms involved in the regulation of CC phenotype is needed for a better understanding of tumor progression and designing of more effective therapies. Hedgehog pathway is one of them, and its augmentation has been observed in various human malignant neoplasms, but its involvement in cholangiocarcinogenesis is still unclear. In this pursuit, this study was conducted in Alb- Cre/LSL-KRASG12D/p53L/L mice and human CC cell lines (TFK-1 and HUCCT-1) to gain a better understanding about the underlying mechanisms by which Hedgehog pathway activation triggers and maintains the process of cholangiocarcinogenesis. The study revealed CC development by 19-20 weeks of age in our mouse model, which was followed by lung metastasis via upregulation of mesenchymal markers. The expression of Hedgehog components like SHH and its downstream elements, SMO and GLI1, was found to be significantly elevated in liver tissue during CC development. Consequently, the expression of CSC transcription factors SOX-2, OCT-4 and NANOG, and CD133 were remarkably enhanced, resulting in stem- like properties and tumor progression in the liver tissue. Blockade of Hedgehog pathway using Cylopamine inhibited the growth of endogenous Hedgehog pathway- dependent tumors by suppressing SMO protein expression and GLI1 nuclear translocation. Moreover, systemic Cyclopamine treatment also inhibited metastasis by inhibiting the expression of invasive markers, thereby prolonging the survival of these mice. Additionally, Hedgehog pathway inhibition by Cyclopamine abrogated NANOG, Oct4, SOX2 and CD133 augmentation. Further, to validate our findings from mouse model studies, we investigated the expression of Hh pathway elements in human TMAs. Our data showed that in the study cohort of 49 patients, more than 80 % of CC samples were SHH and GLI1 positive, thereby implicating the role of Hedgehog pathway in cholangiocarcinogenesis. Based on previously obtained results, we analysed the fraction of CD133+ cells in HUCCT-1 cell line and assessed their tumor forming capacity by implanting 100 or 1000 CD133+ cells in NOD/SCID mice. We observed that CD133+ cells had a remarkably high tumorigenic potential, since as few as 100 cells could result in 100% tumor incidence as compared to unsorted cells. To further validate our hypothesis, we evaluated the expression of Hedgehog pathway elements, SHH and GLI1, in xenografted tumors derived from implantation of CSC marker- based sorted cells. We found that the expression of SHH and GLI1 was markedly upregulated in CD133+ derived tumors as compared with the tumors developed from unsorted cells. Intra-tumoral hypoxia is known to contribute towards therapeutic resistance through modulatory effects on various pathways. In this study, we investigated the relationship between hypoxia and SHH pathway activation and the effect of this interplay on cancer stemness and EMT during cholangiocarcinogenesis. Hypoxia promoted SHH pathway activation, evidenced by upregulated SHH and SMO levels, and enhanced GLI1 nuclear translocation; whereas silencing of HIF-1α impaired SHH upregulation. Hypoxia also enhanced the expression of CSC transcription factors (NANOG, Oct4, SOX2), CD133 and EMT markers (N-cadherin, Vimentin), thereby supporting invasion. Cyclopamine treatment suppressed hypoxia induced SHH pathway activation, consequently reducing invasiveness by downregulating the expression of CSC transcription factors, CD133 and EMT. Cyclopamine also induced apoptosis in CC cells under hypoxia, suggesting that hypoxia induced activation of SHH pathway has modulatory effects on CC progression. Taken together, the results presented here indicate that aberrant activation of Hedgehog signaling has an important early role in tumor induction, metastasis and maintenance of CC cancer stemness. Therefore, it suggests that Hedgehog signaling may hold promise for new diagnostic and therapeutic approaches for CC treatment

Role of mitochondria in liver diseases

252 p.La disfunción mitocondrial desempeña un papel clave en el inicio y desarrollo de las enfermedades hepáticas crónicas. La proteína J controlada por metilación (MCJ) es un inhibidor endógeno de laactividad mitocondrial, y previamente hemos podido demostrar niveles significativamente aumentados deMCJ en pacientes con hígado graso, daño hepático inducido por paracetamol y lesión hepática debido a la colestasis, lo que sugiere una posible asociación entre MCJ y la disfunción mitocondrial. La enfermedad hepática alcohólica causa más de 2 millones de fallecimientos en el mundo y es la segunda causa detrasplante hepático. Sin embargo, carece de un tratamiento específico. Por otro lado, las tasas actuales de trasplante hepático cubren menos del 10% de las necesidades globales, y entre las estrategias paraaumentar el grupo de donantes, se ha propuesto el uso de hígado con criterio expandido, aquellos queprovienen de hígados añosos o esteatóticos. Sin embargo, el uso de estos órganos aumenta el fallo hepático post trasplante, ya que su capacidad regenerativa está limitada y sufren una alta susceptibilidad hacia el daño por isquemia. En ambos modelos la disfunción mitocondrial es un indicador temprano deldaño hepático y hemos podido comprobar la sobreexpresión de MCJ en estadios avanzados. De hecho, elsilenciamiento hepático de MCJ (1) recupera la actividad mitocondrial, alivia la esteatosis y evita la inflamación y el estrés oxidativo en modelos preclínicos de enfermedad hepática alcohólica, y (2) acelerala regeneración hepática y reduce la lesión isquémica en ratones jóvenes, pero significativamente,también en ratones añosos y esteatóticos, promoviendo su uso para el trasplante hepático, reduciendo así la escasez existente de donantes. En resumen, este proyecto muestra la contribución de la disfunción mitocondrial, en especial de la proteína MCJ, en el desarrollo de la enfermedad hepática alcohólica y la regeneración limitada junto con mayor susceptibilidad isquémica que se observa en hígados con un metabolismo comprometido, con el objetivo de establecer dicha proteína como futura diana terapéutica para el tratamiento de enfermedades hepáticas crónica

Die physiologische Relevanz von ADAM17 in der Darmkarzinogenere

Die physiologische Bedeutung von ADAM17 besteht in der irreversiblen proteolytischen Regulation von wichtigen Signalwegen wie dem EGFR Signalweg, wobei ADAM17 für die proteolytische Freisetzung von löslichen ErbB-Liganden wie Amphiregulin (AREG) verantwortlich ist. Lösliche EGFR Liganden binden an Rezeptoren der ErbB Rezeptor Tyrosinkinasefamilie, woraufhin sich Homo- oder Heterodimere der ErbB Rezeptoren bilden, welche durch Phosphorylierung aktiviert werden und intrazellulär u.a. Proliferationssignale weiterleiten. Die Expression von ADAM17, AREG und EGFR ist in Tumoren erhöht, was sich physiologisch durch signifikant verstärkte proteolytische Freisetzung von AREG auf intestinalen Epithelzellen äußert. Im Verlauf dieser Arbeit ließ sich dies in vitro bei humanen Kolorektalzelllinien, ex vivo in murinen 3D Organoidzellkulturen und in vivo in Gewebelysaten und Kolonkulturen von ADAM17 defizienten hypomorphen ADAM17ex/ex Mäusen zeigen. Um die pathophysiologische Relevanz von ADAM17 während der Darmkarzinogenese zu bestimmen, wurde im Rahmen dieser Arbeit zum einen ein entzündungsbasiertes Kolitis assoziiertes Darmkrebsmodell und zum anderen ein genetisch prädispositioniertes Modell mit hypomorphen ApcMin/+ ADAM17ex/ex Mäusen etabliert und analysiert. Die Mutation im Adenomatous polyposis coli (Apc) Gen hat eine Dysregulation des Wnt-Signalwegs zur Folge, was zur spontanen Entwicklung einer Vielzahl intestinaler Neoplasien führt. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass die ADAM17 Defizienz im CAC Mausmodell zu einer signifikant erhöhten Ausbildung von Karzinomen, assoziiert mit verstärkten Entzündungsmerkmalen, im Kolon führt. Bemerkenswerterweise zeigen die genetisch prädispositionierten ApcMin/+ Mäuse bei ADAM17 Defizienz eine signifikant reduzierte Tumoranzahl und verminderte Zellproliferation, die einheitlich geringgradigen Dysplasien entsprechen.One of the physiological roles of ADAM17 is the proteolytic regulation of important pathways like the EGFR (epidermal growth factor receptor) pathway. ADAM17 cleaves membrane bound ErbB (erythroblastosis oncogene B) ligands such as Amphiregulin (AREG). The soluble ligands bind to receptors of the ErbB receptor tyrosine kinase family and trigger phosphorylation of both homo- or heterodimers of the ErbB receptors. Phosphorylation of ErbB receptors has been shown to activate intracellular signals such as proliferation. Expression and activation of ADAM17, AREG and EGFR is highly upregulated during tumorigenesis, which leads to increased shedding of AREG on intestinal epithelial cells as presented in this thesis. This effect was shown in vitro using human colorectal cancer cells, ex vivo in an established 3D murine organoid culture system and in vivo using tissue lysates and colon cultures from ADAM17 deficent hypomorphic ADAM17ex/ex mice. To identify the pathophysiological relevance of the previous results, the effect of ADAM17 deficiency on tumor formation was investigated in vivo in murine colitis associated cancer (CAC) as a model for inflammation associated colorectal cancer. Further a genetically predisposed colorectal cancer model was chosen, whereby ApcMin/+ mice crossbred with hypomorphic ADAM17ex/ex mice were generated and evaluated in this work. The mutated Adenomatous polyposis coli (Apc) gene results in dysregulation of the Wnt signaling pathway, which leads to a spontaneous development of multiple intestinal neoplasia (Min) in this mouse model. In summary, tumor formation is altered in ADAM17 deficiency CAC mouse model and hypomorphic ADAM17ex/ex mice developed higher numbers of carcinoma coupled with increased intestinal inflammation. Remarkably, the genetically predisposed ApcMin/+ mice deficient for ADAM17 showed a significantly reduced tumor number, diminished cell proliferation and low grade dysplasi

Role of NF-κB in autophagy-controlled inflammatory responses and in intestinal epithelial cell fate decisions

Es wird vermutet, dass das Zusammenspiel von NF-κB-Signalen und Autophagie die Entzündung in verschiedenen zellulären Kontexten und als Reaktion auf unterschiedliche Stimuli reguliert. Der molekulare Mechanismus, durch den diese beiden Signalwege bei der Regulierung der Entzündungsreaktion zusammenwirken, ist jedoch noch nicht bekannt. Mithilfe biochemischer Analysen und bildgebender Verfahren haben wir zum ersten Mal die Interaktion zwischen dem autophagischen Marker LC3 und der NF-κB/p65-Untereinheit als Reaktion auf verschiedene Stressbedingungen charakterisiert. Wir konnten zeigen, dass die Anhäufung von LC3 im Zellkern nach der NF-κB-Aktivierung mit p65 interagiert, was durch die Ubiquitinierung des p65-Proteins gefördert und durch den Cargo-Rezeptor p62 erkannt wird. Zusammengenommen weisen diese Daten auf eine neue Rolle von p62 beim Transport von im Kern ubiquitiniertem p65 zu Autophagosomen hin, wo es abgebaut wird, um die entzündungsbedingte NF-κB-Hyperaktivierung zu kontrollieren. Diese Erkenntnisse sind wichtig für die Entwicklung neuer therapeutischer Strategien gegen Krankheiten, die mit einer gestörten Autophagie und einer konstitutiven NF-κB-Aktivität einhergehen. Die NF-κB-Signalübertragung spielt nicht nur eine entscheidende Rolle bei Entzündungen und der Tumorbildung, sondern ist auch für Entwicklungsprozesse wichtig. Durch die Etablierung von 3D-Organoid-Kulturen aus dem Dünndarm und unter Verwendung verschiedener Mauslinien weisen wir im zweiten Teil der Arbeit nach, dass NF-κB eine wichtige Funktion bei der Zelldifferenzierung und der Erhaltung von Stammzellen in vivo und ex-vivo spielt. Wir konnten zeigen, dass die Proliferation und das Absterben von Darmepithelzellen (IEC) bei Mäusen mit ubiquitärer Unterdrückung der NF-κB-Aktivität unverändert sind, während die Zahl der Becherzellen auf Kosten der Paneth-Zellen zunimmt. Zusammenfassend lässt sich sagen, dass unsere Ergebnisse eine neue IEC-immanente Rolle von NF-κB bei Entscheidungen über das Zellschicksal und die Differenzierung aufzeigen, die über die Regulierung der Wnt-Signale und der Sox9-Expression stromabwärts von NF-κB erfolgt. Die hier beschriebenen Erkenntnisse verbessern unser Verständnis der NF-κB-Funktionen in der Stammzellbiologie, die, wenn sie dereguliert sind, auch Auswirkungen auf die Entzündung des Darms und die Tumorentstehung haben.The interplay between NF-κB signaling and autophagy has been suggested to regulate inflammation in different cellular contexts and in response to different stimuli. However, the molecular mechanism by which these two pathways interact to regulate the inflammatory response remains elusive. By using biochemical analysis and imaging techniques, we characterized for the first time the interaction of autophagic marker LC3 and NF-κB/p65 subunit in response to different stress conditions. We demonstrated that the accumulation of LC3 within the nucleus interacts with p65 following NF-κB activation, which is promoted by ubiquitination of p65 protein and recognized by the cargo receptor p62. Together, these data identify a novel role for p62 in trafficking nuclear-ubiquitinated p65 to autophagosomes for degradation to control inflammation-driven NF-κB hyperactivation. These findings are important for developing novel therapeutic strategies against diseases involving defective autophagy and constitutive NF-κB activity. In addition to its critical role in inflammation and tumor formation, NF-κB signaling is essential in developmental processes. Establishing 3D organoid culture from the small intestine and using different mouse lines, we prove in the second part of the thesis that NF-kB plays an important function in cell differentiation and stem cell maintenance in vivo and in ex-vivo. We demonstrated that while intestinal epithelial cell (IEC) proliferation and death are unaltered in mice with ubiquitous suppression of NF-κB activity, goblet cell numbers increase at the expense of Paneth cells. In summary, our results revealed a novel IEC-intrinsic role of NF-κB in cell fate decisions and differentiation which occur via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The findings described here improve our understanding of NF-κB functions in stem cell biology which, when deregulated, also have an impact on intestinal inflammation and tumorigenesis

Roles and Regulation of Sphingosine Kinase 2 in Cancer

The two mammalian sphingosine kinases, SK1 and SK2, produce the bioactive signalling lipid sphingosine-1-phosphate (S1P), which generally promotes cell survival, proliferation and migration. In line with this, SK1 is often found to be upregulated in human cancers, and overexpression of SK1 leads to neoplastic transformation of cells and tumour growth. However, despite generating the same product, most evidence to date suggests that SK2 acts in an opposing manner to promote cell death. In contrast, knockout mouse models indicate that there is at least some functional redundancy between the two SK isoforms, and targeting SK2 via genetic or pharmacological approaches in cancer models results in reduced tumour growth. Clearly, the roles of SK2 are poorly understood, but it is apparent that these unique and complex functions of SK2 are largely dictated by its differential subcellular localisation. SK1 is generally a cytoplasmic protein, but it can also be translocated to the plasma membrane where it mediates cell survival, proliferation and oncogenic signalling through the production of S1P. SK2, however, has been reported to localise to the nucleus, endoplasmic reticulum and mitochondria, and at these locations it appears to possess anti-proliferative and pro-apoptotic functions. SK2 has also been reported to localise to the plasma membrane, but its specific roles here have not been well characterised. Therefore, the main aims of this study were to explore the roles of SK2 in cancer, and to characterise novel mechanisms that regulate SK2 subcellular localisation, such as interacting proteins and post translational modifications, in order to gain a better understanding of this complex enzyme and the potential benefits of targeting SK2 in cancer. To explore the roles of SK2 in cancer, we examined the expression of SK2 in various human tumour samples using publically available datasets, and found that SK2 shows statistically significant upregulation in many cancers, but only to modest levels up to 2.5- fold over normal tissues. As high-level SK2 overexpression has been previously shown to cause cell death, we explored the effects of low, close to physiological levels of SK2 overexpression. By engineering a series of human and mouse cell lines overexpressing graded levels of SK2, we found that low-level SK2 overexpression increased cell survival and proliferation, and activated oncogenic signalling pathways. Notably, low-level SK2 overexpression (5- to 10-fold over endogenous levels) was sufficient to induce neoplastic transformation of mouse fibroblasts, resulting in efficient tumour formation in vivo. These findings coincided with decreased nuclear localisation and increased plasma membrane localisation of SK2, as well as increases in extracellular S1P formation. Hence, we have shown for the first time that SK2 can have a direct role in promoting oncogenesis. Furthermore, the Pitson laboratory previously identified a novel SK2-interacting protein, cytoplasmic dynein 1 intermediate chain 2 (IC-2), through a yeast two-hybrid screen, and characterising this interaction formed another part of these studies. We confirmed that SK2 interacts physiologically with the dynein complex in cells via the IC subunit, and being a retrograde-directed transport motor complex, we found that dynein mediates the translocation of SK2 away from the plasma membrane. Interestingly, although IC-2 was identified in the yeast two-hybrid screen, SK2 interacts more robustly with the highly-related IC-1 isoform, which is abundantly expressed in the brain. Strikingly, we found that IC-1 is downregulated 17-fold in glioblastoma multiforme (GBM) patient samples, which correlated with poorer survival of patients with this form of brain tumour. In line with a role for dynein in transporting SK2, low IC-1 expression in GBM cells coincided with more SK2 localised to the plasma membrane, where we had found it to accumulate in an oncogenic setting. Re-expression of IC-1 in these cells reduced plasma membrane localised-SK2 and extracellular S1P formation, and notably, decreased tumour growth and tumour-associated angiogenesis in vivo. Thus, these findings demonstrate a novel tumour-suppressive function of dynein IC-1, and uncover new mechanistic insights into SK2 regulation. Through previous mass spectrometric analyses performed by the Pitson laboratory, it is evident that SK2 contains multiple uncharacterised phosphorylation sites that are not shared with SK1. We explored the function of one such site, Ser363, and found it to potentially regulate nuclear localisation of SK2. Furthermore, we identified SK2 as a bona fide substrate of glycogen synthase kinase 3 (GSK3) in vitro and in cells, involving residues Ser437 and Ser441, and we found that other phosphorylation events may act to regulate SK2 catalytic activity. Overall, the studies outlined here have revealed a previously unreported role for SK2 in driving oncogenesis, and have described the characterisation of novel mechanisms that regulate the subcellular localisation of SK2. Therefore, these findings support the use of SK2 inhibitors as promising anti-cancer therapeutic agents. Furthermore, as the opposing functions of SK2 are largely dictated by it subcellular localisation, these findings may also assist in the development of new strategies to target oncogenic SK2 in cancer.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201