Measles virus infection – Interplay between virus and host cells

Measles, caused by measles virus (MV), is a highly contagious viral disease causing severe respiratory infection and a typical rash. Despite the availability of a protective vaccine, measles is still the leading vaccine-preventable cause of childhood mortality worldwide. The high mortality associated with the disease is mainly due to an increased susceptibility to secondary infections during the period of immunosuppression that continues for several weeks after recovery. The present study was undertaken to elucidate the role of cytoskeletal components in the regulation of MV infection. The most interesting finding was that MV replication was activated in unstimulated peripheral blood mononuclear cells (PBMC) when globular actin was converted into the filamentous form with jasplakinolide. This provides a new aspect in our understanding of MV infection in PBMC. In the second part of the thesis we investigated MV-induced structural changes of cellular nuclear matrix, which is a proteinaceous framework of the nucleus similar to the cytoskeleton in the cytoplasm. We showed that cleavage of nuclear markers was virusspecific and a general caspase inhibitor rescued MV-infected cells from cell death. Furthermore, we studied MV-induced innate immune mechanisms in lung epithelial and endothelial cells. Our results showed that MV infection resulted in activation of the double stranded RNA (dsRNA) binding molecules melanoma differentiation-associated gene 5 (mda-5), retinoic acid inducible gene I (RIG-I), and toll-like receptor 3 (TLR3) gene expression, followed by high expression of antiviral cytokine mRNA.

Novel Players in the Integrin Signaling Orchestra: TCPTP and MDGI

Metastases are the major cause of cancer deaths. Tumor cell dissemination from the primary tumor utilizes dysregulated cellular adhesion and upregulated proteolytic degradation of the extracellular matrix for progeny formation in distant organs. Integrins are transmembrane adhesive receptors mediating cellcell and cellmatrix interactions that are crucial for regulating cell migration, invasion, proliferation, and survival. Consequently, increased integrin activity is associated with augmented migration and invasion capacity in several cancer types. Heterodimeric integrins consist of an alpha - and beta-subunit that are held together in a bent conformation when the receptor is inactive, but extension and separation of subdomains is observed during receptor activation. Either inside-out or outside-in activation of receptors is possible through the intracellular molecule binding to an integrin cytoplasmic domain or extracellular ligand association with an integrin ectodomain, respectively. Several regulatory binding partners have been characterized for integrin cytoplasmic beta-domains, but the regulators interacting with the cytoplasmic alpha-domains have remained elusive. In this study, we performed yeast two-hybrid screens to identify novel binding partners for the cytoplasmic integrin alpha-domains. Further examination of two plausible candidates revealed a significant coregulatory role of an integrin alpha-subunit for cellular signaling processes. T-cell protein tyrosine phosphatase (TCPTP) showed a specific interaction with the cytoplasmic tail of integrin alpha1. This association stimulated TCPTP phosphatase activity, leading to negative regulation of epidermal growth factor receptor (EGFR) signaling and diminished anchorage-independent growth. Another candidate, mammary-derived growth inhibitor (MDGI), exhibited binding to several different integrin cytoplasmic alpha-tails through a conserved GFFKR sequence. MDGI overexpression in breast cancer cells altered EGFR trafficking and caused a remarkable accumulation of EGFR in the cytoplasm. We further demonstrated in vivo that MDGI expression induced a novel form of anti-EGFR therapy resistance. Moreover, MDGI binding to α-tails retained integrin in an inactive conformation attenuating integrin-mediated adhesion, migration, and invasion. In agreement with these results, sustained MDGI expression in breast cancer patients correlated with an increased 10-year distant disease-free survival. Taken together, the integrin signaling network is far from a complete view and future work will doubtless broaden our understanding further.

The Cellular Oxygen Sensor PHD2 in Cancer Growth

Adequate supply of oxygen is essential for the survival of multicellular organisms. However, in several conditions the supply of oxygen can be disturbed and the tissue oxygenation is compromised. This condition is termed hypoxia. Oxygen homeostasis is maintained by the regulation of both the use and delivery of oxygen through complex, sensitive and cell-type specific transcriptional responses to hypoxia. This is mainly achieved by one master regulator, a transcription factor called hypoxiainducible factor 1 (HIF-1). The amount of HIF-1 is under tight oxygen-dependent control by a family of oxygen-dependent prolyl hydroxylase domain proteins (PHDs) that function as the cellular oxygen sensors. Three family members (PHD1-3) are known to regulate HIF of which the PHD2 isoform is thought to be the main regulator of HIF-1. The supply of oxygen can be disturbed in pathophysiological conditions, such as ischemic disorders and cancer. Cancer cells in the hypoxic parts of the tumors exploit the ability of HIF-1 to turn on the mechanisms for their survival, resistance to treatment, and escape from the oxygen- and nutrient-deprived environment. In this study, the expression and regulation of PHD2 were studied in normal and cancerous tissues, and its significance in tumor growth. The results show that the expression of PHD2 is induced in hypoxic cells. It is overexpressed in head and neck squamous cell carcinomas and colon adenocarcinomas. Although PHD2 normally resides in the cytoplasm, nuclear translocation of PHD2 was also seen in a subset of tumor cells. Together with the overexpression, the nuclear localization correlated with the aggressiveness of the tumors. The nuclear localization of PHD2 caused an increase in the anchorage-independent growth of cancer cells. This study provides information on the role of PHD2, the main regulator of HIF expression, in cancer progression. This knowledge may prove to be valuable in targeting the HIF pathway in cancer treatment.

Integrins on the move

Cancer is a leading cause of death worldwide accounting for 13% of all deaths in 2005. The spread of cancer and formation of metastases is the major cause of mortality among cancer patients. The spread of cancer is based on the cancer cell’s ability to break away from the surrounding tissue and to migrate into new areas in the body. The ability of cells to bind its surroundings and to move is controlled by the mechanical cell surface adhesion receptors called the integrins. Integrins have a critical role in cell adhesion, cell motility and tissue homeostasis. By communicating with ECM, integrins transmit signals from the surrounding environment inside the cell and modulate the function of many important signalling pathways involved in cell survival, development, gene expression, proliferation, motility and cytoskeletal organization. During cell migration integrin-matrix adhesions are formed in front of the cell while rear-adhesions are released during migration. Integrins are endocytosed from the plasma-membrane into the cytoplasm and partly recycled back to new adhesion sites in a process called integrin trafficking. Also, the cell cytoskeleton and protrusions are important in cell migration. Finger-like actin protrusions called filopodia display an interesting cancer relevant cooperation with integrins that is required for cell migration. The expression and function of integrins changes markedly as cells acquire carcinogenic properties. Changed integrin function is partly responsible for detachment of tumor cells from neighbouring cells and for providing enhanced invasive capabilities for tumor cells to disseminate. Similarly, the formation of filopodia is increased in cancer. High myosin-10 expression is related to poor outcome in breast cancer and increased cell migration. The proper function of myosin-10 induced filopodia needs association with β1 integrins. The importance of integrin trafficking and filopodia formation is becoming increasingly more recognized in cancer. This thesis focusses on the role of integrins, integrin trafficking and myosin-10 induced filopodia cancer cell migration.

The interplay of JNK and SCG10 during cortical development and in excitotoxic responses in brain

Initially identified as stress activated protein kinases (SAPKs), the c-Jun Nterminal kinases (JNKs) are currently accepted as potent regulators of various physiologically important cellular events. Named after their competence to phosphorylate transcription factor c-Jun in response to UVtreatment, JNKs play a key role in cell proliferation, cell death or cell migration. Interestingly, these functions are crucial for proper brain formation. The family consists of three JNK isoforms, JNK1, JNK2 and JNK3. Unlike brain specific JNK3 isoform, JNK1 and JNK2 are ubiquitously expressed. It is estimated that ten splice variants exist. However, the detailed cellular functions of these remain undetermined. In addition, physiological conditions keep the activities of JNK2 and JNK3 low in comparison with JNK1, whereas cellular stress raises the activity of these isoforms dramatically. Importantly, JNK1 activity is constitutively high in neurons, yet it does not stimulate cell death. This suggests a valuable role for JNK1 in brain development, but also as an important mediator of cell wellbeing. The aim of this thesis was to characterize the functional relationship between JNK1 and SCG10. We found that SCG10 is a bona fide target for JNK. By employing differential centrifugation we showed that SCG10 co-localized with active JNK, MKK7 and JIP1 in a fraction containing endosomes and Golgi vesicles. Investigation of JNK knockout tissues using phosphospecific antibodies recognizing JNK-specific phosphorylation sites on SCG10 (Ser 62/Ser 73) showed that phosphorylation of endogenous SCG10 was dramatically decreased in Jnk1-/- brains. Moreover, we found that JNK and SCG10 co-express during early embryonic days in brain regions that undergo extensive neuronal migration. Our study revealed that selective inhibition of JNK in the cytoplasm significantly increased both the frequency of exit from the multipolar stage and radial migration rate. However, as a consequence, it led to ill-defined cellular organization. Furthermore, we found that multipolar exit and radial migration in Jnk1 deficient mice can be connected to changes in phosphorylation state of SCG10. Also, the expression of a pseudo-phosphorylated mutant form of SCG10, mimicking the JNK1- phopshorylated form, brings migration rate back to normal in Jnk1 knockout mouse embryos. Furthermore, we investigated the role of SCG10 and JNK in regulation of Golgi apparatus (GA) biogenesis and whether pathological JNK action could be discernible by its deregulation. We found that SCG10 maintains GA integrity as with the absence of SCG10 neurons present more compact fragmented GA structure, as shown by the knockdown approach. Interestingly, neurons isolated from Jnk1-/- mice show similar characteristics. Block of ER to GA is believed to be involved in development of Parkinson's disease. Hence, by using a pharmacological approach (Brefeldin A treatment), we showed that GA recovery is delayed upon removal of the drug in Jnk1-/- neurons to an extent similar to the shRNA SCG10-treated cells. Finally, we investigated the role of the JNK1-SCG10 duo in the maintenance of GA biogenesis following excitotoxic insult. Although the GA underwent fragmentation in response to NMDA treatment, we observed a substantial delay in GA disintegration in neurons lacking either JNK1 or SCG10.

Novel cancer-related regulatory targets for the signalling sphingolipids sphingosine-1-phosphate and sphingosylphosphorylcholine

The signalling sphingolipid sphingosine-1-phosphate (S1P) is necessary for development of the immune system and vasculature and on a cellular level regulates migration, proliferation and survival. Due to these traits S1P has an important role in cancer biology. It is considered a primarily cancer-promoting factor and the enzyme which produces it, sphingosine kinase (SphK), is often over-expressed in tumours. S1P is naturally present in the blood, lymph, tissue fluids and cell cytoplasm and functions through its cell surface receptors (S1P1-5) and as an intracellular second messenger. Sphingosylphosphorylcholine (SPC) is closely related to S1P and has similar regulatory functions but has not been extensively studied. Both S1P and SPC are able to evoke either stimulatory or inhibitory effects on cancer cells depending on the context. The aim of this thesis work was to study novel regulatory targets of S1P and SPC, which mediate the effects of S1P/SPC signalling on cancer cell behaviour. The investigated targets are the transcription factor hypoxia-inducible factor 1 (HIF-1), the intermediate filament protein vimentin and components of the Hippo signalling pathway. HIF-1 has a central role in cancer biology, as it regulates a multitude of cancer-related genes and is potently activated by intratumoural hypoxia through stabilization of the regulatory subunit HIF-1α. Tumours typically harbour high HIF-1α levels and HIF-1, in turn, facilitates tumour angiogenesis and metastasis and regulates cancer cell metabolism. We found S1P to induce follicular thyroid cancer cell migration in normal oxygen conditions by increasing HIF-1α synthesis and stability and subsequently HIF-1 activity. Vimentin is a central regulator of cell motility and is also commonly over-expressed in cancers. Vimentin filaments form a cytoskeletal network in mesenchymal cells as well as epithelial cancer cells which have gone through epithelial-mesenchymal transition (EMT). Vimentin is heavily involved in cancer cell invasion and gives tumours metastatic potential. We saw both S1P and SPC induce phosphorylation of vimentin monomers and reorganization of the vimentin filament network in breast and anaplastic thyroid cancer cells. We also found vimentin to mediate the anti-migratory effect of S1P/SPC on these cells. The Hippo pathway is a novel signalling cascade which controls cancer-related processes such as cellular proliferation and survival in response to various extracellular signals. The core of the pathway consists of the transcriptional regulators YAP and TAZ, which activate predominantly cancer-promoting genes, and the tumour suppressive kinases Lats1 and Lats2 which inhibit YAP/TAZ. Increased YAP expression and activity has been reported for a wide variety of cancers. We found SPC to regulate Hippo signalling in breast cancer cells in a two-fold manner through effects on phosphorylation status, activity and/or expression of YAP and Lats2. In conclusion, this thesis reveals new details of the signalling function of S1P and SPC and regulation of the central oncogenic factors HIF-1 and vimentin as well as the novel cancer-related pathway Hippo.