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Human tumorigenesis is a process in which a normal cell needs to acquire multiple characteristics to become malignant and metastatic. In short, these so called cancer hallmarks include increased proliferation and cell survival, as well as the ability to invade into the surroundings, induce angiogenesis, and finally metastasize to distant sites. These traits are regulated in a variety of different ways. However, some embryonic signaling pathways, including the Notch pathway, are able to regulate many of these processes. Furthermore, it has been shown that these signaling pathways can be deregulated in cancer, and that their untimely activation can lead to malignancies. In this study, Kaposi's sarcoma herpesvirus (KSHV) associated malignancies, namely Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), as well as melanoma have been used as model cancers. In all these malignancies, the tumor cells show alterations in cell identity and lineage marker expression, i.e. signs of cellular de- or transdifferentiation. In addition, the Notch pathway has been shown to be overly active in all of them. Thus, this thesis has focused on how the pro-tumorigenic traits are affected by cell plasticity and reprogramming in these cancers, and how the signaling pathways leading to these phenotypes, most notably Notch, are in turn regulated. Firstly, the results show that in vivo expression of a KSHV oncogene, viral (v-)cyclin, leads to activation of Notch signaling through Notch3 upregulation as well as fine-tuning of the NF-κB pathway through Cdk6 mediated phosphorylation. These changes in turn lead to defects in T-lymphocyte differentiation and immune functions, as well as to the development of T-cell lymphomas. Secondly, this work demonstrates that KSHV infection in primary lymphatic endothelial cells (LECs) in three dimensional (3D) cell culture model leads to activation of a morphogenic process, endothelial to mesenchymal transition (EndMT), and increased invasiveness through activation of the Notch pathway and matrix metalloproteinase MT1-MMP. Lastly, the data show that the changes in cell plasticity contributing to tumorigenic traits are not confined to virally induced cancers. Melanoma cell interaction with LECs leads to activation of the Notch pathway and increased adhesive, invasive, and metastatic properties of the tumor cells. In conclusion, the results show that regulation of cell plasticity through the Notch pathway takes place in different types of cancers, and it can affect several steps of tumorigenesis. A thorough and comprehensive understanding of the processes discovered herein may help develop better and more efficient treatments for these largely fatal malignancies. Monien normaalin solun ominaisuuksien tulee muuttua, ennen kuin solu muuntuu syöpäsoluksi ja pystyy leviämään elimistössä. Solun täytyy muun muassa pystyä jakautumaan hallitsemattomasti, tunkeutumaan ympäristöönsä, erittämään veri- ja imusuonien kasvuun vaikuttavia tekijöitä sekä lopulta pystyä hyödyntämään veri- ja imusuonistoa levitäkseen ympäriä kehoa. Syöpäsolujen eri ominaisuuksia säätelevät tyypillisesti eri signalointireitit, mutta eräät sikiönkehityksen aikana aktiivisesti toimivat reitit, kuten Notch-signalointi, voivat vaikuttaa moniin syöpäsolun ominaisuuksiin. Tällaisten signalointireittien yliaktiivisuus onkin liitetty syövän syntyyn ja leviämiseen. Tässä tutkimuksessa on käytetty malleina Kaposin sarkoomaan liittyvän herpesviruksen (KSHV) aiheuttamia syöpiä, Kaposin sarkoomaa (KS) ja primaaria efuusiolymfoomaa (PEL), sekä melanoomaa. Näissä kaikissa syövissä on havaittavissa, että syöpäsolujen soluidentiteetti on heterogeeninen, ja että syöpäsolut pystyvät ohjelmoitumaan uudelleen kasvuolosuhteidensa mukaan. Näille syöville on yhteistä myös, että Notch-signalointireitti on aktivoitunut. Tutkimukseni aiheena oli solujen muovautuvuus- ja ohjelmoitumiskyvyn vaikutukset syövän syntyyn ja leviämiseen ja näiden prosessien säätely. Työni ensimmäisessä osassa osoitin, kuinka KSHV:n onkogeenin v-sykliinin ilmentyminen hiiressä johtaa Notch- ja NF-κB- signalointireittien aktivoitumiseen ja sitä kautta T-solujen erilaistumisen ja toiminnan häiriöön sekä T-solulymfooman kehittymiseen. Seuraavaksi näytin, kuinka KSHV-infektio imusuonten seinämän soluissa johtaa Notch-signaloinnin aktivoitumiseen ja solujen uudelleenohjelmoitumiseen mesenkyymisolujen kaltaisiksi, jolloin ne pystyvät tehokkaammin tunkeutumaan ympäristöönsä. Lopuksi osoitin, että edellä kuvatut mekanismit eivät ole aktiivisia ainoastaan syöpävirusten aiheuttamissa kasvaimissa, vaan että myös melanoomasolujen interaktio imusuonten solujen kanssa aktivoi Notch-signalointia ja johtaa syöpäsolujen lisääntyneeseen adheesio-, invaasio- ja metastasoimiskykyyn. Notch-signalointi ja syöpäsolujen muovautumis- ja uudelleenohjelmoitumiskyky säätelevät siis monentyyppisten syöpien kehittymistä ja leviämistä. Näiden prosessien perusteellinen tuntemus mahdollistaa parempien ja tehokkaampien hoitojen kehittämisen näitä huonoennusteisia syöpiä vastaan.

Nuclear factor kappa-B (NF-kappaB) activates multiple genes with overlapping roles in cell proliferation, inflammation and cancer. Using an unbiased approach we identified human CDK6 as a novel kinase phosphorylating NF-kappaB p65 at serine 536. Purified and reconstituted CDK6/cyclin complexes phosphorylated p65 in vitro and in transfected cells. The physiological role of CDK6 for basal as well as cytokine-induced p65 phosphorylation or NF-?B activation was revealed upon RNAi-mediated suppression of CDK6. Inhibition of CDK6 catalytic activity by PD332991 suppressed activation of NF-?B and TNF-induced gene expression. In complex with a constitutively active viral cyclin CDK6 stimulated NF-?B p65-mediated transcription in a target gene specific manner and this effect was partially dependent on its ability to phosphorylate p65 at serine 536. Tumor formation in thymi and spleens of v-cyclin transgenic mice correlated with increased levels of p65 Ser536 phosphorylation, increased expression of CDK6 and upregulaton of the NF-?B target cyclin D3. These results suggest that aberrant CDK6 expression or activation that is frequently observed in human tumors can contribute through NF-?B to chronic inflammation and neoplasia.

Nuclear factor kappa-B (NF-kappaB) activates multiple genes with overlapping roles in cell proliferation, inflammation and cancer. Using an unbiased approach we identified human CDK6 as a novel kinase phosphorylating NF-kappaB p65 at serine 536. Purified and reconstituted CDK6/cyclin complexes phosphorylated p65 in vitro and in transfected cells. The physiological role of CDK6 for basal as well as cytokine-induced p65 phosphorylation or NF-?B activation was revealed upon RNAi-mediated suppression of CDK6. Inhibition of CDK6 catalytic activity by PD332991 suppressed activation of NF-?B and TNF-induced gene expression. In complex with a constitutively active viral cyclin CDK6 stimulated NF-?B p65-mediated transcription in a target gene specific manner and this effect was partially dependent on its ability to phosphorylate p65 at serine 536. Tumor formation in thymi and spleens of v-cyclin transgenic mice correlated with increased levels of p65 Ser536 phosphorylation, increased expression of CDK6 and upregulaton of the NF-?B target cyclin D3. These results suggest that aberrant CDK6 expression or activation that is frequently observed in human tumors can contribute through NF-?B to chronic inflammation and neoplasia.

Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that in vivo expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis.

Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that in vivo expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis.

Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that in vivo expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis.