RESUMO
Hypoxia stimulates neoangiogenesis, promoting tumor outgrowth, and triggers the epithelial-mesenchymal transition (EMT), which bestows cells with mesenchymal traits and multi-lineage differentiation potential. Here, we investigated whether EMT can confer endothelial attributes upon carcinoma cells, augmenting tumor growth and vascularization. Following orthotopic implantation of MCF-7 human epithelial breast cancer cells into mice, tumors of different sizes were immunostained for markers of hypoxia and EMT. Larger tumors were well-vascularized with CD31-positive cells of human origin. Hypoxic regions, demarcated by HIF-1α staining, exhibited focal areas of E-cadherin loss and elevated levels of vimentin and the EMT-mediator FOXC2. Implantation of MCF-7 cells, co-mixed with human mammary epithelial (HMLE) cells overexpressing the EMT-inducer Snail, markedly potentiated tumor growth and vascularization, compared with MCF-7 cells injected alone or co-mixed with HMLE-vector cells. Intra-tumoral vessels contained CD31-positive cells derived from either donor cell type. FOXC2 knockdown abrogated the potentiating effects of HMLE-Snail cells on MCF-7 tumor growth and vascularization, and compromised endothelial transdifferentiation of mesenchymal cells cultured in endothelial growth medium. Hence, cells that have undergone EMT can promote tumor growth and neovascularization either indirectly, by promoting endothelial transdifferentiation of carcinoma cells, or directly, by acquiring an endothelial phenotype, with FOXC2 playing key roles in these processes.
RESUMO
BACKGROUND: Triple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC. METHODS: A library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3ß inhibitors were identified as EMT inhibitors. The effects of GSK3ß inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3ß correlates with the overall survival of breast cancer patients. RESULTS: We identified a GSK3ß inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3ß inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3ß reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3ß inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24- in cells after exposure to GSK3ß inhibitors. We found that GSK3ß inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3ß inhibitors and found that GSK3ß inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3ß correlated with poorer overall patient survival. CONCLUSIONS: Taken together, our data demonstrate that GSK3ß is a potential target for TNBCs and suggest that GSK3ß inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3ß inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Conjuntos de Dados como Assunto , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Humanos , Concentração Inibidora 50 , Cloreto de Lítio/farmacologia , Cloreto de Lítio/uso terapêutico , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirróis/farmacologia , Pirróis/uso terapêutico , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/mortalidade , Via de Sinalização WntRESUMO
Resistance to chemotherapy and metastases are the major causes of breast cancer-related mortality. Moreover, cancer stem cells (CSC) play critical roles in cancer progression and treatment resistance. Previously, it was found that CSC-like cells can be generated by aberrant activation of epithelial-mesenchymal transition (EMT), thereby making anti-EMT strategies a novel therapeutic option for treatment of aggressive breast cancers. Here, we report that the transcription factor FOXC2 induced in response to multiple EMT signaling pathways as well as elevated in stem cell-enriched factions is a critical determinant of mesenchymal and stem cell properties, in cells induced to undergo EMT- and CSC-enriched breast cancer cell lines. More specifically, attenuation of FOXC2 expression using lentiviral short hairpin RNA led to inhibition of the mesenchymal phenotype and associated invasive and stem cell properties, which included reduced mammosphere-forming ability and tumor initiation. Whereas, overexpression of FOXC2 was sufficient to induce CSC properties and spontaneous metastasis in transformed human mammary epithelial cells. Furthermore, a FOXC2-induced gene expression signature was enriched in the claudin-low/basal B breast tumor subtype that contains EMT and CSC features. Having identified PDGFR-ß to be regulated by FOXC2, we show that the U.S. Food and Drug Administration-approved PDGFR inhibitor, sunitinib, targets FOXC2-expressing tumor cells leading to reduced CSC and metastatic properties. Thus, FOXC2 or its associated gene expression program may provide an effective target for anti-EMT-based therapies for the treatment of claudin-low/basal B breast tumors or other EMT-/CSC-enriched tumors.
Assuntos
Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal , Fatores de Transcrição Forkhead/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Transformada , Feminino , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Invitrogen's Gateway technology is a recombination-based cloning method that allows for rapid transfer of numerous open reading frames (ORFs) into multiple plasmid vectors, making it useful for diverse high-throughput applications. Gateway technology has been utilized to create an ORF library for Myxoma virus (MYXV), a member of the Poxviridae family of DNA viruses. MYXV is the prototype virus for the genus Leporipoxvirus, and is pathogenic only in European rabbits. MYXV replicates exclusively in the host cell cytoplasm, and its genome encodes 171 ORFs. A number of these ORFs encode proteins that interfere with or modulate host defense mechanisms, particularly the inflammatory responses. Furthermore, MYXV is able to productively infect a variety of human cancer cell lines and is being developed as an oncolytic virus for treating human cancers. MYXV is therefore an excellent model for studying poxvirus biology, pathogenesis, and host tropism, and a good candidate for ORFeome development.
Assuntos
Expressão Gênica , Myxoma virus/genética , Proteínas Virais/biossíntese , Animais , Clonagem Molecular/métodos , Biblioteca Gênica , Vetores Genéticos , Humanos , Myxoma virus/patogenicidade , Vírus Oncolíticos/genética , Vírus Oncolíticos/patogenicidade , Plasmídeos , Coelhos , Recombinação GenéticaRESUMO
Aberrant activation of a latent embryonic program - known as the epithelial-mesenchymal transition (EMT) - can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence. The induction of EMT entails the loss of epithelial characteristics and the de novo acquisition of a mesenchymal phenotype. In breast cancer, the EMT state has been associated with cancer stem cell properties including expression of the stem cell-associated CD44+/CD24-/low antigenic profile, self-renewal capabilities and resistance to conventional therapies. Intriguingly, EMT features are also associated with stem cells isolated from the normal mouse mammary gland and human breast reduction tissues as well as the highly aggressive metaplastic and claudin-low breast tumor subtypes. This has implications for the origin of these breast tumors as it remains unclear whether they derive from cells that have undergone EMT or whether they represent an expansion of a pre-existing stem cell population that expresses EMT-associated markers to begin with. In the present review, we consider the current evidence connecting EMT and stem cell attributes and discuss the ramifications of these newly recognized links for our understanding of the emergence of distinct breast cancer subtypes and breast cancer progression.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Transição Epitelial-Mesenquimal/genética , Células-Tronco Neoplásicas/metabolismo , Animais , Biomarcadores/metabolismo , Neoplasias da Mama/tratamento farmacológico , Desdiferenciação Celular , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Humanas/citologia , Camundongos , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/patologia , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Viruses have evolved an assortment of mechanisms for regulating the Akt signaling pathway to establish a cellular environment more favorable for viral replication. Myxoma virus (MYXV) is a rabbit-specific poxvirus that encodes many immunomodulatory factors, including an ankyrin repeat-containing host range protein termed M-T5 that functions to regulate tropism of MYXV for rabbit lymphocytes and certain human cancer cells. MYXV permissiveness in these human cancer cells is dependent upon the direct interaction between M-T5 and Akt, which has been shown to induce the kinase activity of Akt. In this study, an array of compounds that selectively manipulate Akt signaling was screened and we show that only a subset of Akt inhibitors significantly decreased the ability of MYXV to replicate in previously permissive human cancer cells. Furthermore, reduced viral replication efficiency was correlated with lower levels of phosphorylated Akt. In contrast, the PP2A-specific phosphatase inhibitor okadaic acid promoted increased Akt kinase activation and rescued MYXV replication in human cancer cells that did not previously support viral replication. Finally, phosphorylation of Akt at residue Thr308 was shown to dictate the physical interaction between Akt and M-T5, which then leads to phosphorylation of Ser473 and permits productive MYXV replication in these human cancer cells. The results of this study further characterize the mechanism by which M-T5 exploits the Akt signaling cascade and affirms this interaction as a major tropism determinant that regulates the replication efficiency of MYXV in human cancer cells.
Assuntos
Myxoma virus/fisiologia , Neoplasias/virologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/fisiologia , Tropismo Viral , Replicação Viral , Linhagem Celular Tumoral , Cloridrato de Fingolimode , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Ácido Okadáico/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Fosforilação , Propilenoglicóis/farmacologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Sirolimo/farmacologia , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Serina-Treonina Quinases TOR , Proteínas Virais/fisiologiaRESUMO
Most poxviruses express multiple proteins containing ankyrin (ANK) repeats accounting for a large superfamily of related but unique determinants of poxviral tropism. Recently, select members of this novel family of poxvirus proteins have drawn considerable attention for their potential roles in modulating intracellular signaling networks during viral infection. The rabbit-specific poxvirus, myxoma virus (MYXV), encodes four unique ANK repeat proteins, termed M-T5, M148, M149, and M150, all of which include a carboxy-terminal PRANC domain which closely resembles a cellular protein motif called the F-box domain. Here, we show that each MYXV-encoded ANK repeat protein, including M-T5, interacts directly with the Skp1 component of the host SCF ubiquitin ligase complex, and that the binding of M-T5 to cullin 1 is indirect via binding to Skp1 in the host SCF complex. To understand the significance of these virus-host protein interactions, the various binding domains of M-T5 were mapped. The N-terminal ANK repeats I and II were identified as being important for interaction with Akt, whereas the C-terminal PRANC/F-box-like domain was essential for binding to Skp1. We also report that M-T5 can bind Akt and the host SCF complex (via Skp1) simultaneously in MYXV-infected cells. Finally, we report that M-T5 specifically mediates the relocalization of Akt from the nucleus to the cytoplasm during infection with the wild-type MYXV, but not the M-T5 knockout version of the virus. These results indicate that ANK/PRANC proteins play a critical role in reprogramming disparate cellular signaling cascades to establish a new cellular environment more favorable for virus replication.
Assuntos
Myxoma virus/patogenicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais , Proteínas Virais/fisiologia , Animais , Linhagem Celular , Núcleo Celular/química , Chlorocebus aethiops , Proteínas Culina/metabolismo , Citoplasma/química , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Ligases SKP Culina F-Box/metabolismoRESUMO
Myxoma virus (MV) is a highly lethal, rabbit-specific poxvirus that induces a disease called myxomatosis in European rabbits. In an effort to understand the function of predicted immunomodulatory genes we have deleted various viral genes from MV and tested the ability of these knockout viruses to induce lethal myxomatosis. MV encodes a unique 15 kD cytoplasmic protein (M130R) that is expressed late (12h post infection) during infection. M130R is a non-essential gene for MV replication in rabbit, monkey or human cell lines. Construction of a targeted gene knockout virus (vMyx130KO) and infection of susceptible rabbits demonstrate that the M130R knockout virus is attenuated and that loss of M130R expression allows the rabbit host immune system to effectively respond to and control the lethal effects of MV. M130R expression is a bona fide poxviral virulence factor necessary for full and lethal development of myxomatosis.
Assuntos
Myxoma virus/patogenicidade , Proteínas Virais/fisiologia , Fatores de Virulência/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Deleção de Genes , Técnicas de Inativação de Genes , Ordem dos Genes , Haplorrinos , Humanos , Masculino , Dados de Sequência Molecular , Myxoma virus/genética , Filogenia , Coelhos , Alinhamento de Sequência , Índice de Gravidade de Doença , Proteínas Virais/genética , Virulência , Fatores de Virulência/genéticaRESUMO
As a family of viruses, poxviruses collectively exhibit a broad host range and most of the individual members are capable of replicating in a wide array of cell types from various host species, at least in vitro. At the cellular level, poxvirus tropism is dependent not upon specific cell surface receptors, but rather upon: (1) the ability of the cell to provide intracellular complementing factors needed for productive virus replication, and (2) the ability of the specific virus to successfully manipulate intracellular signaling networks that regulate cellular antiviral processes downstream of virus entry. The large genomic coding capacity of poxviruses enables the virus to express a unique collection of viral proteins that function as host range factors, which specifically target and manipulate host signaling pathways to establish optimal cellular conditions for viral replication. Functionally, the known host range factors from poxviruses have been associated with manipulation of a diverse array of cellular targets, which includes cellular kinases and phosphatases, apoptosis, and various antiviral pathways. To date, only a small number of poxvirus host range genes have been identified and studied, and only a handful of these have been functionally characterized. For this reason, poxvirus host range factors represent a potential gold mine for the discovery of novel pathogen-host protein interactions. This review summarizes our current understanding of the mechanisms by which the known poxvirus host range genes, and their encoded factors, expand tropism through the manipulation of host cell intracellular signaling pathways.
Assuntos
Interações Hospedeiro-Patógeno , Myxoma virus/fisiologia , Orthopoxvirus/fisiologia , Infecções por Poxviridae/virologia , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Regulação Viral da Expressão Gênica , Humanos , Myxoma virus/química , Myxoma virus/genética , Orthopoxvirus/química , Orthopoxvirus/genética , Infecções por Poxviridae/imunologia , Infecções por Poxviridae/fisiopatologia , Transdução de Sinais , Tropismo , Proteínas Virais/química , Proteínas Virais/genética , Replicação ViralRESUMO
Poxviruses demonstrate strict species specificity in vivo that range from narrow to broad, however the fundamental factors that mediate the basis of poxvirus tropism remain poorly understood. It is generally believed that most, if not all, poxviruses can efficiently bind and enter a wide range of mammalian cells and all of the known host anti-viral pathways that block viral replication in nonpremissive cells operate downstream of virus entry. A productive poxvirus infection is heavily dependent upon the production of a vast array of host modulatory products that specifically target and manipulate both extracellular immune response pathways of the host, as well as intracellular signal transduction pathways of the individually infected cells. The unique pathogenesis and host tropism of specific poxviruses can be attributed to the broad diversity of host modulatory proteins they express. Myxoma virus (MV) is a rabbit-specific poxviruses that encodes multiple host range factors, including an ankyrin-repeat protein M-T5, which functions to regulate tropism of MV for rabbit lymphocytes and some human cancer cells. At the molecular level, M-T5 binds and alters at least two distinct cellular proteins: Akt and cullin-1. The direct interaction between M-T5 and Akt was shown to be a key restriction determinant for MV tropism in a spectrum of human cancer cells making MV an excellent oncolytic candidate. Thus, the intricate relationship between viral encoded proteins and components of the host cell signaling networks can have profound impact on poxvirus tropism. The lessons we continue to learn from poxvirus host range factors like M-T5 will provide further insights into the factors that regulate poxvirus tropism and the mechanisms by which poxviruses micromanipulate the signaling pathways of the infected cell.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Myxoma virus/fisiologia , Transdução de Sinais , Proteínas Virais/metabolismo , Animais , Repetição de Anquirina , Antibióticos Antineoplásicos/farmacologia , Motivos F-Box , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Myxoma virus/genética , Myxoma virus/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Neoplasias/virologia , Terapia Viral Oncolítica , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Coelhos , Sirolimo/farmacologia , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Myxoma virus (MV) is a rabbit-specific poxvirus, whose unexpected tropism to human cancer cells has led to studies exploring its potential use in oncolytic therapy. MV infects a wide range of human cancer cells in vitro, in a manner intricately linked to the cellular activation of Akt kinase. MV has also been successfully used for treating human glioma xenografts in immunodeficient mice. This study examines the effectiveness of MV in treating primary and metastatic mouse tumors in immunocompetent C57BL6 mice. We have found that several mouse tumor cell lines, including B16 melanomas, are permissive to MV infection. B16F10 cells were used for assessing MV replication and efficacy in syngeneic primary tumor and metastatic models in vivo. Multiple intratumoral injections of MV resulted in dramatic inhibition of tumor growth. Systemic administration of MV in a lung metastasis model with B16F10LacZ cells was dramatically effective in reducing lung tumor burden. Combination therapy of MV with rapamycin reduced both size and number of lung metastases, and also reduced the induced antiviral neutralizing antibody titres, but did not affect tumor tropism. These results show MV to be a promising virotherapeutic agent in immunocompetent animal tumor models, with good efficacy in combination with rapamycin.
Assuntos
Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Melanoma Experimental/terapia , Melanoma Experimental/virologia , Myxoma virus , Terapia Viral Oncolítica , Adjuvantes Farmacêuticos/uso terapêutico , Animais , Antibióticos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/virologia , Melanoma Experimental/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Myxoma virus/efeitos dos fármacos , Myxoma virus/genética , Terapia Viral Oncolítica/métodos , Coelhos , Sirolimo/uso terapêuticoRESUMO
Interleukin-18 (IL-18) is a critical proinflammatory cytokine whose extracellular bioactivity is regulated by a cellular IL-18 binding protein (IL-18BP). Many poxviruses have acquired variants of this IL-18BP gene, some of which have been shown to act as viral virulence factors. Yaba monkey tumor virus (YMTV) encodes a related family member, 14L, which is similar to the orthopoxvirus IL-18BPs. YMTV 14L was expressed from a baculovirus system and tested for its ability to bind and inhibit IL-18. We found that YMTV 14L bound both human IL-18 (hIL-18) and murine IL-18 with high affinity, at 4.1 nM and 6.5 nM, respectively. YMTV 14L was able to fully sequester hIL-18 but could only partially inhibit the biological activity of hIL-18 as measured by gamma interferon secretion from KG-1 cells. Additionally, 17 hIL-18 point mutants were tested by surface plasmon resonance for their ability to bind to YMTV 14L. Two clusters of hIL-18 surface residues were found to be important for the hIL-18-YMTV 14L interaction, in contrast to results for the Variola virus IL-18BP, which has been shown to primarily interact with a single cluster of three amino acids. The altered binding specificity of YMTV 14L most likely represents an adaptation resulting in increased fitness of the virus and affirms the plasticity of poxviral inhibitor domains that target cytokines like IL-18.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Interleucina-18/antagonistas & inibidores , Proteínas Virais/fisiologia , Vírus do Tumor do Macaco de Yaba/imunologia , Linhagem Celular , Humanos , Interferon gama/biossíntese , Mutagênese Sítio-Dirigida , Mutação Puntual , Ligação Proteica , Mapeamento de Interação de Proteínas , Ressonância de Plasmônio de SuperfícieRESUMO
Myxoma virus (MV) is a poxvirus that evolved in Sylvilagus lagomorphs, and is the causative agent of myxomatosis in European rabbits (Oryctolagus cuniculus). This virus is not a natural pathogen of O. cuniculus, yet is able to subvert the host rabbit immune system defenses and cause a highly lethal systemic infection. The interaction of MV proteins and the rabbit immune system has been an ideal model to help elucidate host/poxvirus interactions, and has led to a greater understanding of how other poxvirus pathogens are able to cause disease in their respective hosts. This review will examine how MV causes myxomatosis, by examining a selection of the identified immunomodulatory proteins that this virus expresses to subvert the immune and inflammatory pathways of infected rabbit hosts.
Assuntos
Myxoma virus/isolamento & purificação , Mixomatose Infecciosa/virologia , Coelhos/virologia , Animais , Apoptose , Europa (Continente)/epidemiologia , Leucócitos/imunologia , Myxoma virus/imunologia , Mixomatose Infecciosa/epidemiologia , Mixomatose Infecciosa/imunologia , Mixomatose Infecciosa/patologiaRESUMO
The myxoma virus (MV) ankyrin repeat, host range factor M-T5 has the ability to bind and activate cellular Akt, leading to permissive MV replication in a variety of diverse human cancer cell lines (G. Wang, J. W. Barrett, M. Stanford, S. J. Werden, J. B. Johnston, X. Gao, M. Sun, J. Q. Cheng, and G. McFadden, Proc. Natl. Acad. Sci. USA 103:4640-4645, 2006). The susceptibility of permissive human cancer cells to MV infection is directly correlated with the basal or induced levels of phosphorylated Akt. When M-T5 is deleted from MV, the knockout virus, vMyxT5KO, can no longer productively infect a subset of human cancer cells (designated type II) that exhibit little or no endogenous phosphorylated Akt. In searching for a host counterpart of M-T5, we noted sequence similarity of M-T5 to a recently identified ankyrin repeat cellular binding protein of Akt called PIKE-A. PIKE-A binds and activates the kinase activity of Akt in a GTP-dependent manner and promotes the invasiveness of human cancer cell lines. Here, we demonstrate that transfected PIKE-A is able to rescue the ability of vMyxT5KO to productively infect type II human cancer cells that were previously resistant to infection. Also, cancer cells that were completely nonpermissive for both wild-type and vMyxT5KO infection (called type III) were rendered fully permissive following ectopic expression of PIKE-A. We conclude that the MV M-T5 host range protein is functionally interchangeable with the host PIKE-A protein and that the activation of host Akt by either M-T5 or PIKE-A is critical for the permissiveness of human cancer cells for MV.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Myxoma virus/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Repetição de Anquirina , Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Ativação Enzimática , Proteínas de Ligação ao GTP/genética , Proteínas Ativadoras de GTPase/genética , Expressão Gênica , Humanos , Dados de Sequência Molecular , Myxoma virus/genética , Myxoma virus/patogenicidade , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Replicação ViralRESUMO
The myxoma virus M063R gene product exhibits some sequence similarity to the poxvirus host range gene, C7L, of vaccinia virus. To address the potential host range function of the M063R gene product in rabbits, a deletion mutant of myxoma virus (vMyx63KO) was generated and characterized. vMyx63KO replicated to normal titre levels and produced foci that were indistinguishable from those produced by MV in vitro in a monkey kidney cell line (BGMK) that are permissive for wild type MV. However, vMyx63KO failed to replicate in all rabbit cell lines tested, including both primary and established cells lines, as well as cells derived from a variety of tissues. M063R expression was not required for myxoma virus binding, entry or early gene expression, whereas DNA replication was aborted and late genes were not expressed in vMyx63KO infected rabbit cells. Thus, the replication block for vMyx63KO in rabbit cells preceded the stage of late gene expression and DNA replication. Finally, an in vivo pathogenesis study indicated that vMyx63KO failed to cause any signs of classic myxomatosis in infected rabbits, but functioned as a non-replicating vaccine and provided protection for subsequent challenge by wild type myxoma virus. Altogether, these observations demonstrate that M063R plays a critical role in determining the host specificity of myxoma virus in rabbit cells.
Assuntos
Myxoma virus/fisiologia , Mixomatose Infecciosa/virologia , Fases de Leitura Aberta/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Feminino , Humanos , Dados de Sequência Molecular , Mutação , Myxoma virus/patogenicidade , Coelhos , Alinhamento de Sequência , Especificidade da Espécie , Virulência , Replicação ViralRESUMO
We demonstrate that the susceptibility of human cancer cells to be infected and killed by an oncolytic poxvirus, myxoma virus (MV), is related to the basal level of endogenous phosphorylated Akt. We further demonstrate that nonpermissive tumor cells will switch from resistant to susceptible for MV infection after expression of ectopically active Akt (Myr-Akt) and that permissive cancer cells can be rendered nonpermissive by blocking Akt activation with a dominant-negative inhibitor of Akt. Finally, the activation of Akt by MV involves the formation of a complex between the viral host range ankyrin-repeat protein, M-T5, and Akt. We conclude that the Akt pathway is a key restriction determinant for permissiveness of human cancer cells by MV.