Browsing by discipline "Translationaalinen syöpäbiologia"
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(2018)TAUSTA: Verisuonten uudismuodostuksen, läpäisevyyden ja rappeutumisen säätelyssä avainasemassa on Angiopoietiini-Tie-järjestelmä, joka koostuu angiopoietiineista 1-4 (Ang1-4) sekä niiden reseptoreista Tie1 ja Tie2. Ang1:n aikaansaama Tie-reseptorien aktivaatio johtaa verisuonten endoteelisolujen välisten sidosten tiukentumiseen ja homeostasian ylläpitoon. Ang2 puolestaan pystyy salpaamaan Tie2-reseptorin, jolloin verisuonten läpäisevyys lisääntyy ja endoteeli alkaa rappeutua. Paradoksaalisesti Ang2 pystyy myös aktivoimaan Tie1:n tietyissä olosuhteissa, jolloin verisuonten läpäisevyys vähenee. TAVOITTEET: Tämän tutkimuksen tarkoituksena oli selvittää Ang2:n yli-ilmentymisen ja Tie1-poistogeenisyyden vaikutusta verisuonten läpäisevyyteen hiirten keuhkokudoksessa. Lisäksi tutkittiin anti-Tie1 (DX2240) ja anti-Ang2 (MEDI3617) vasta-aineiden vaikutusta verisuonten läpäisevyyteen ja tulehdusreaktion voimakkuuteen LPS-indusoidussa verenmyrkytyksessä. MENETELMÄT: Määritettiin valkosolujen, punasolujen ja fibrinogeenin määrä immunohistokemiallisten värjäysten avulla Ang2:ta yli-ilmentävien hiirten, Tie1-poistogeenisten hiirten ja näiden yhdistelmähiirten keuhkoissa. Lisäksi määritettiin keuhkolaskimoiden seinämien paksuus sekä nanopartikkelien vuoto anti-Tie1 ja anti-Ang2 vasta-aineita saaneiden hiirten keuhkoissa tulehduksen ja verisuonten läpäisevyyden arvioimiseksi. TULOKSET: Ang2:n yli-ilmentymisen ja Tie1-poistogeenisyyden ei havaittu aiheuttavan merkittävää eroa verisuonten läpäisevyydessä. Anti-Tie1 ja anti-Ang2-vasta-aineet eivät myöskään vähentäneet merkittävästi verisuonten läpäisevyyttä verenmyrkytyksessä.
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(2018)Kaposi’s sarcoma herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), was discovered following the AIDS-epidemic as the causative agent of Kaposi’s sarcoma (KS), an angiogenic endothelial tumor of the skin, and of two rare lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Infection by KSHV displays two life cycle phases; latent and lytic replication. In latency, the virus stays dormant within the host, expressing only a few genes and no viral particles are produced. Latency is the default mode of infection, however, upon appropriate induction the virus reactivates to express all of its genes and replicate viral DNA during the productive lytic replication, culminating with the release of infectious progeny particles and lysis of the host cells. Virus reactivation from latency to the lytic replication is an essential step in the KS pathogenesis. Upon KSHV infection, endothelial cells (EC) undergo reprogramming towards spindle cell, the principal proliferating cell in advanced KS lesions. The transcription factor prospero related homeobox gene Prox1 has an important role in mediating the effects of KSHV on EC reprogramming, contributing to the KS development. Prox1 is the master regulator of lymphatic endothelial cell fate, and its expression is manipulated during the KSHV infection. However, the role of Prox1 in the KSHV life cycle and lytic reactivation has not been studied. To elucidate the role of Prox1 in KSHV reactivation from latency, the effect of ectopic expression of Prox1 on the lytic gene and protein expression in both latent and reactivated KSHV-infected cells was studied. This led to a significant increase in KSHV lytic gene and protein expression, suggesting Prox1 as a positive regulator of KSHV lytic replication. Moreover, Prox1 wild-type, but not its DNA-binding deficient mutant, could significantly increase the release of infectious virions. To investigate the expression levels of Prox1 during KSHV infection, infection kinetics assay was performed, which showed an increase in the Prox1 levels during acute infection. Intriguingly, this was followed by a progressive decrease in the Prox1 levels as latency was established. In conclusion, the focus of this thesis is to investigate the role of Prox1 in KSHV reactivation, and to provide a deeper insight into the virus reactivation mechanisms that can be utilized for future therapeutic strategies against KSHV-mediated tumorigenesis of KS. Keywords: KSHV, Kaposi’s sarcoma, Prox1, virus reactivation, lytic replication
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(2016)Glioblastoma Multiforme (GBM) is the most common and aggressive types of glioma in adults. Autophagy allows degradation and recycling of cellular components such as damaged proteins and dysfunctional organelles to sustain the metabolism and homeostasis of rapidly growing cells. Recent reports suggest that autophagy may promote tumor cell survival under stress conditions and can be an emerging target for cancer therapy. Autophagy inhibitor combined with TMZ can induce glioblastoma cell death and improve the radiotherapy efficacy. The Neuropilin-1 (NRP1) is 120 kDa-130 kDa type I integral transmembrane protein. It was originally identified as a co-receptor for the class 3 semaphorins (SEMA3) involved in axon guidance and found to interact with VEGF/VEGFR2 to promote angio-genesis. Recent studies have revealed its much broader roles on tumor progression. In various types of human cancers, NRP1 is often up-regulated and associated with aggressive clinical tumor behaviour. NRP1 overexpression is independently correlated with poor prognosis in human glioma and contributes to balance the glioblastoma cell proliferation and survival. In cancer cells, it interacts with diverse growth factor receptors including TGFR, c-Met, FGFR, EGFR as well as PDGFR to promote their signal-ling pathways in tumor cell survival, proliferation, migration and invasion. Although these functions of NRP1 mainly rely on its ectodomain, the cytoplasmic domain of NRP1 has been recently found to be essential for the internalization of NRP1-binding complex. In addition, the C-terminal SEA sequence on its cytoplasmic domain have potential to bind intracellular PDZ domain-containing molecules. Tyrosine phosphorylation of p130Cas has been identified to regulate the downstream pathways of NRP1, which is dependent on NRP1 intracellular domain. However, the downstream trafficking of NRP1 is poorly understood and its tumor-promoting function relevance remains ambiguous. The p62/Sequestosome 1, encoded by SQSTM1 gene, is an intracellular protein commonly found in inclusion bodies. It is asso-iated with protein aggregation diseases in liver and brain. Owing to its ability to interact with multiple important cellular intermediates, it works as a 'hub' adaptor linked to nuclear factor-kappaB (NF-κB) activation, protein aggregates formation, selective autophagy, adipogenesis and tumorigenesis. The p62 has a critical role on autophagy via regulating the collection and delivery of ubiquitinylated cargos to the autophagosome via its PB1, UBA and LIR domains. On the other hand, recent study has revealed a new role of p62 as a negative regulator in the autophagy regulation. High level of p62 is able to suppress the autophagy by pro-moting mTORC1 activation. This route forms a feed-forward loop for increasing level of p62 due to the reduced autophagy. Thus, p62 plays a critical role in regulation of autophagy. Here we observed that suppression of NRP1 in glioblastoma cell clearly exhibits a defected autophagy accompanied by marked accumulation of p62, the autophagic adaptor. Overexpression of NRP1 by glioblastoma cells shows enhanced autophagy flux. These data suggests the role of NRP1 in autophagy promotion. In addition, we mapped out that p62 binds to the cytoplasmic domain of NRP1 mediating its pro-autophagy effects. PB1 domain of p62 overexpression enhances the p62-positive aggregates and NRP1/p62 interaction. Taken together, our results define a novel role of NRP1 in the regulation of autophagy through its association with p62. In summary, our present results provide novel insights into the molecular basis of the emerging interplay between NRP1 and autophagy, the identification of a new cytoplasmic protein that binding to intracellular domain of NRP1 and the implications of the p62-mediated signalling loop for NRP1-promoted autophagy in GBMs. Since efforts to inhibit autophagy to improve GBM therapy have thereby attracted great interest, our findings may provide valuable clues for future cancer therapeutic strategies.
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(2016)Tumor cells exhibit uncontrolled proliferation, which is supported and accelerated by a constant supply of nutrients carried by blood vessels. Tumor angiogenesis, the formation of new blood vessels, besides its contribution to tumor growth, also allows the dissemination of tumor cells into distant organs. In addition to the hematogenous routes, the tumor cells metastasize through lymphatic vasculature as well. Tumor-associated lymphangiogenesis, the formation of new lymphatic vessels, is a key process in this regard. Multiple growth factor pathways regulate angiogenesis and lymphangiogenesis. Among the most important vascular growth factors implicated in this regulation are vascular endothelial growth factors (VEGFs) and angiopoietin growth factors (Angs). It has been shown that targeting VEGF/VEGFR-2 pathway could inhibit tumor growth. Many studies during the last decade have demonstrated that attenuating VEGFR-3 function inhibits primary tumor growth and also metastasis. Selective antibodies against Ang2 were shown to inhibit tumor growth and metastasis in different tumor models in mice. However, the question regarding whether combining different therapeutic methods, namely anti-VEGFR-2, anti-VEGFR-3 and anti-Ang2, will have additive benefits in comparison to single-agent therapies still remains. We aimed to test the inhibitory effects of simultaneous targeting of all VEGF pathways and Ang2 on primary tumor growth in human lung carcinoma xenografts in immunodeficient mice. To achieve this, we used soluble VEGF – trap (Aflibercept), VEGF-C/D – trap and antibodies against Ang2. Our results show that triple therapy significantly improves the inhibition of primary tumor growth in comparison to monotherapies and dual therapies. Combination of all 3 treatments also improved the reduction of intra-tumor blood vessels and lymphatic vessels. The effects of triple targeting on controlling metastasis, however, was not significant in orthotopic breast cancer model, mainly due to great variation in tumor growth in this model. However, a clear trend of reduced metastasis in several organs (liver, kidneys) was observed. Overall, this study suggests that attenuating all VEGF pathways and Ang2 could improve the inhibitory effects of anti-angiogenic treatments.
Now showing items 1-4 of 4