Cell plasticity in cancer : Cues from virus-host interactions

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

KSHV-Initiated Notch Activation Leads to Membrane-Type-1 Matrix Metalloproteinase-Dependent Lymphatic Endothelial-to-Mesenchymal Transition

SummaryKaposi sarcoma (KS), an angioproliferative disease associated with Kaposi sarcoma herpesvirus (KSHV) infection, harbors a diversity of cell types ranging from endothelial to mesenchymal cells of unclear origin. We developed a three-dimensional cell model for KSHV infection and used it to demonstrate that KSHV induces transcriptional reprogramming of lymphatic endothelial cells to mesenchymal cells via endothelial-to-mesenchymal transition (EndMT). KSHV-induced EndMT was initiated by the viral proteins vFLIP and vGPCR through Notch pathway activation, leading to gain of membrane-type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive properties and concomitant changes in viral gene expression. Mesenchymal markers and MT1-MMP were found codistributed with a KSHV marker in the same cells from primary KS biopsies. Our data explain the heterogeneity of cell types within KS lesions and suggest that KSHV-induced EndMT may contribute to KS development by giving rise to infected, invasive cells while providing the virus a permissive cellular microenvironment for efficient spread

Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and beta 1-integrin activation

Lymphatic invasion and lymph node metastasis correlate with poor clinical outcome in melanoma. However, the mechanisms of lymphatic dissemination in distant metastasis remain incompletely understood. We show here that exposure of expansively growing human WM852 melanoma cells, but not singly invasive Bowes cells, to lymphatic endothelial cells (LEC) in 3D co-culture facilitates melanoma distant organ metastasis in mice. To dissect the underlying molecular mechanisms, we established LEC co-cultures with different melanoma cells originating from primary tumors or metastases. Notably, the expansively growing metastatic melanoma cells adopted an invasively sprouting phenotype in 3D matrix that was dependent on MMP14, Notch3 and beta 1-integrin. Unexpectedly, MMP14 was necessary for LEC-induced Notch3 induction and coincident beta 1-integrin activation. Moreover, MMP14 and Notch3 were required for LEC-mediated metastasis of zebrafish xenografts. This study uncovers a unique mechanism whereby LEC contact promotes melanoma metastasis by inducing a reversible switch from 3D growth to invasively sprouting cell phenotype.Peer reviewe

Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and beta 1-integrin activation

Lymphatic invasion and lymph node metastasis correlate with poor clinical outcome in melanoma. However, the mechanisms of lymphatic dissemination in distant metastasis remain incompletely understood. We show here that exposure of expansively growing human WM852 melanoma cells, but not singly invasive Bowes cells, to lymphatic endothelial cells (LEC) in 3D co-culture facilitates melanoma distant organ metastasis in mice. To dissect the underlying molecular mechanisms, we established LEC co-cultures with different melanoma cells originating from primary tumors or metastases. Notably, the expansively growing metastatic melanoma cells adopted an invasively sprouting phenotype in 3D matrix that was dependent on MMP14, Notch3 and beta 1-integrin. Unexpectedly, MMP14 was necessary for LEC-induced Notch3 induction and coincident beta 1-integrin activation. Moreover, MMP14 and Notch3 were required for LEC-mediated metastasis of zebrafish xenografts. This study uncovers a unique mechanism whereby LEC contact promotes melanoma metastasis by inducing a reversible switch from 3D growth to invasively sprouting cell phenotype

Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication

Kaposi's sarcoma herpesvirus (KSHV) causes Kaposi's sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication.Peer reviewe

Cyclin-Dependent Kinase 6 Phosphorylates NF-kappa B P65 at Serine 536 and Contributes to the Regulation of Inflammatory Gene Expression

Peer reviewe

KSHV viral cyclin interferes with T-cell development and induces lymphoma through Cdk6 and Notch activation in vivo

<div><p>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 <i>in vivo</i> 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.</p></div

A purified and reconstituted CDK6/cyclin complex phosphorylates NF-κB p65 Ser 536.

<p>(<b>A</b>) GST fusion proteins of cyclins D1 to D3, viral (v)-cyclin and CDK6 were expressed and purified in <i>E.coli</i>. Comparable amounts of GST, GST-cyclins and GST-CDK6 as judged from immunoblotting analysis (upper panel) were mixed with HeLa cell extract to provide CDK-activating kinase CAK as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051847#pone.0051847-Phelps1" target="_blank">[37]</a>. Kinase reactions were performed by addition of ATP. Then, GST alone or activated CDK6/cyclin complexes were purified by addition of GSH-beads, washed and used to phosphorylate the GST-p65_{354- 551} substrate <i>in vitro</i>. Phosphorylation of p65 Ser536 was detected by immunoblotting of kinase reaction mixtures (lower panel). Recombinant GST protein or kinase reactions without GST-CDK6/cyclin complexes (labeled no kinase) were used to determine background signals. (<b>B</b>) Increasing amounts of a recombinant CDK6/cyclin D1 complex purified to homogeneity from baculovirus-infected insect cells was mixed with GST-p65_{354- 551} and the kinase reaction was allowed to proceed over time. Phosphorylation of p65 at Ser536 was detected by immunoblotting. (<b>C</b>) HeLa cells were transfected with the expression vectors for wild type (wt) CDK6, a gain of function mutant (CDK6-S178P) and cyclin D3 or viral (v)- cyclin. 24 h later cells were lysed and phosphorylation of p65 and of Rb and expression of transfected proteins was analyzed by immunoblotting of cell extracts using the indicated antibodies.</p

Gene-specific regulation of NF-κB target genes by CDK6.

<p>(<b>A</b>) HEK293IL-1R cells were transiently transfected with a vector directing the synthesis of two different shRNA duplexes directed at CDK6 or an empty vector control along with a NF-κB-dependent reporter gene and the SV40-ß gal vector to allow for normalization. After 24 h, cells were stimulated for 4 h with IL-1α(10 ng/ml, black bars) or left untreated (white bars). Cells were lysed and luciferase activity was normalized for co-transfected SV40-promoter driven ß-galactosidase. The graph shows the mean luciferase activity +/− s.e.m. from three independent experiments performed in duplicates relative to the vector control. (<b>B</b>) HEK293IL-1R cells were transiently transfected with empty vector, NF-κB (3) luc and SV40-ß gal. After 24 h cells were pretretreated with 10 µM PD332991 and then further stimulated for 4 h with IL-1α(10 ng/ml) or left untreated. Shown is the mean luciferase activity +/− s.e.m. from two independent experiments. (<b>C</b>) HeLa Fucci cells were arrested for two days and then left untreated or were treated with 10 µM PD332991 (hatched bars) for 30 min followed by 30 min TNFα (20 ng/ml) as indicated. Thereafter, total RNA was prepared and analysed for the expression of the indicated mRNAs by RT-qPCR. Data show the mean –fold change +/− s.e.m. from two independent experiments performed in duplicate. The asterisk indicates significant differences (p< = 0.05) as determined by paired t-test.</p

Phosphorylation of Ser536 of p65 by CDK6/cyclin complexes contributes to activation of NF-κB-dependent transcription.

<p>(<b>A</b>) HEK293IL-1R cells were transiently transfected with the indicated amounts (µg) of expression vectors and a NF-κB-dependent luciferase gene. After 24 h cells were lyzed and luciferase activity was normalized for co-transfected SV40-promoter driven ß-galactosidase. The graph shows the mean luciferase activity +/− s.e.m. from three independent experiments performed in duplicates relative to the vector control. Expression of cotransfected MYC-cyclin D1, v-cyclin derived from Kaposi’s sarcoma herpesvirus (MYC-v-cyclin) and HA-CDK6 was analyzed by immunoblotting using anti MYC or anti CDK6 antibodies for all experiments in parallel. One representative blot is shown. (<b>B</b>) NF-κB p65-deficient MEFs stably reconstituted with wild type p65 (−/−/wt) or the Ser536A mutant (−/−/Ser536A) as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051847#pone.0051847-Okazaki1" target="_blank">[64]</a> were transiently transfected with empty vector or HA-CDK6 plus v-cyclin expression vectors. Cotransfected luciferase reporter genes were either controlled by three NF-κB binding sites (upper graph) or alternatively by the cyclin D1 promoter (lower graph). After 24 h cells were lyzed, luciferase activity was determined and normalized for co-transfected SV40-promoter driven ß-galactosidase activity. Shown is the mean luc. activity +/− s.e.m. from three (lower graph) or four (upper graph) independent experiments performed in duplicates relative to the vector control. The lower panel shows a representative immunoblot for the detection of p65 wt and Ser536Ala mutant, p65 Ser536 phosphorylation and ectopically expressed CDK6. Asterisks indicate significant changes of reporter gene activity.</p