RESUMO
As many tumor cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, frontline cancer therapies often administer anti-VEGF (α-VEGF) antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumor vasculature, we studied the functional tolerance, evasion of neutralization, and induction of α-VEGF antibodies of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the 3-fold capsid spike was replaced by VEGF-blocking peptides: P6L (PQPRPL) and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture, exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti-spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an α-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the 3-fold axis to close up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture into a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow an infectious parvovirus with immune antineovascularization and evasion capacities by replacing an antibody footprint in the capsid 3-fold axis with VEGF-blocking peptides, and it also illustrates the evolutionary capacity of single-stranded DNA (ssDNA) viruses to overcome engineered capsid structural restrictions.IMPORTANCE Targeting the VEGF signaling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumors. VEGF-blocking peptides (VEbp) engineered in the capsid 3-fold axis endowed the infectious parvovirus MVM with the ability to induce α-VEGF antibodies without adjuvant and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restraints that the capsid imposes on the peptide configuration and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp resolved the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. These data show the promise of antineovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses but also raise safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.
Assuntos
Vacinas Anticâncer/imunologia , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Vírus Miúdo do Camundongo/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Proteínas do Capsídeo/genética , Camundongos Endogâmicos BALB C , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/crescimento & desenvolvimento , Vírus Oncolíticos/genética , Vírus Oncolíticos/crescimento & desenvolvimento , Vírus Oncolíticos/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Carga Viral , Montagem de Vírus , Ligação Viral , Internalização do VírusRESUMO
Most E2F-binding sites repress transcription through the recruitment of Retinoblastoma (RB) family members until the end of the G1 cell-cycle phase. Although the MYB promoter contains an E2F-binding site, its transcription is activated shortly after the exit from quiescence, before RB family members inactivation, by unknown mechanisms. We had previously uncovered a nuclear factor distinct from E2F, Myb-sp, whose DNA-binding site overlapped the E2F element and had hypothesized that this factor might overcome the transcriptional repression of MYB by E2F-RB family members. We have purified Myb-sp and discovered that Myc-associated zinc finger proteins (MAZ) are major components. We show that various MAZ isoforms are present in Myb-sp and activate transcription via the MYB-E2F element. Moreover, while forced RB or p130 expression repressed the activity of a luciferase reporter driven by the MYB-E2F element, co-expression of MAZ proteins not only reverted repression, but also activated transcription. Finally, we show that MAZ binds the MYB promoter in vivo, that its binding site is critical for MYB transactivation, and that MAZ knockdown inhibits MYB expression during the exit from quiescence. Together, these data indicate that MAZ is essential to bypass MYB promoter repression by RB family members and to induce MYB expression.
Assuntos
Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F/genética , Fase G1/genética , Regulação da Expressão Gênica , Proteínas Oncogênicas v-myb/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Sítios de Ligação , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F/metabolismo , Genes Reporter , Células HEK293 , Humanos , Células Jurkat , Luciferases/genética , Luciferases/metabolismo , Linfócitos/citologia , Linfócitos/metabolismo , Proteínas Oncogênicas v-myb/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Tumor formation involves the acquisition of numerous capacities along the progression from a normal cell into a malignant cell, including limitless proliferation (immortalization) and anchorage-independent growth, a capacity that correlates extremely well with tumorigenesis. Great efforts have been made to uncover genes involved in tumor formation, but most genes identified participate in processes related to cell proliferation. Accordingly, therapies targeting these genes also affect the proliferation of normal cells. To identify potential targets for therapeutic intervention more specific to tumor cells, we looked for genes implicated in the acquisition of anchorage-independent growth and in vivo tumorigenesis capacity. A transcriptomic analysis identified CDCA7 as a candidate gene. Indeed, CDCA7 protein was upregulated in Burkitt's lymphoma cell lines and human tumor biopsy specimens relative to control cell lines and tissues, respectively. CDCA7 levels were also markedly elevated in numerous T and B-lymphoid tumor cell lines. While CDCA7 was not required for anchorage-dependent growth of normal fibroblasts or non-malignant lymphocytes, it was essential but not sufficient for anchorage-independent growth of lymphoid tumor cells and for lymphomagenesis. These data suggest that therapies aimed at inhibiting CDCA7 expression or function might significantly decrease the growth of lymphoid tumors.
Assuntos
Linfoma de Burkitt/metabolismo , Carcinogênese/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares/biossíntese , Regulação para Cima , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Carcinogênese/genética , Carcinogênese/patologia , Feminino , Células HCT116 , Células HeLa , Humanos , Células Jurkat , Células K562 , Masculino , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Células U937RESUMO
Lymphoid tumor patients often exhibit resistance to standard therapies or experience relapse post-remission. Relapse is driven by Tumor Initiating Cells (TICs), a subset of tumor cells capable of regrowing the tumor and highly resistant to therapy. Growing cells in 3D gels is a method to discern tumorigenic cells because it strongly correlates with tumorigenicity. The finding that TICs, rather than differentiated tumor cells, grow in 3D gels offers a unique opportunity to unveil TIC-specific signaling pathways and therapeutic targets common to various cancer types. Here, we show that culturing lymphoid cells in 3D gels triggers reactive oxygen species (ROS) production, leading to non-tumor lymphoid cell death while enabling the survival and proliferation of a subset of lymphoma/leukemia cells, TICs or TIC-like cells. Treatment with the antioxidant N-acetylcysteine inhibits this lethality and promotes the growth of primary non-tumor lymphoid cells in 3D gels. A subset of lymphoma cells, characterized by an increased abundance of the antioxidant glutathione, escape ROS-induced lethality, a response not seen in non-tumor cells. Reducing glutathione production in lymphoma cells, either through pharmacological inhibition of glutamate cysteine ligase (GCL), the enzyme catalyzing the rate-limiting step in glutathione biosynthesis, or via knockdown of GCLC, the GCL catalytic subunit, sharply decreased cell growth in 3D gels and xenografts. Tumor cells from B-cell lymphoma/leukemia patients and λ-MYC mice, a B-cell lymphoma mouse model, overproduce glutathione. Importantly, pharmacological GCL inhibition hindered lymphoma growth in female λ-MYC mice, suggesting that this treatment holds promise as a therapeutic strategy for female lymphoma/leukemia patients.
Assuntos
Glutationa , Linfoma , Células-Tronco Neoplásicas , Espécies Reativas de Oxigênio , Animais , Humanos , Feminino , Linfoma/patologia , Linfoma/metabolismo , Linfoma/tratamento farmacológico , Glutationa/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Masculino , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Carcinogênese/patologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismoRESUMO
Photodynamic therapy (PDT) is widely used to treat non-melanoma skin cancer. However, some patients affected with squamous cell carcinoma (SCC) do not respond adequately to PDT with methyl-δ-aminolevulinic acid (MAL-PDT) and the tumors acquire an infiltrative phenotype and became histologically more aggressive, less differentiated, and more fibroblastic. To search for potential factors implicated in SCC resistance to PDT, we have used the SCC-13 cell line (parental) and resistant SCC-13 cells obtained by repeated MAL-PDT treatments (5th and 10th PDT-resistant generations). Xenografts assays in immunodeficient mice showed that the tumors generated by resistant cells were bigger than those induced by parental cells. Comparative genomic hybridization array (aCGH) showed that the three cell types presented amplicons in 3p12.1 CADM2, 7p11.2 EFGR, and 11q13.3 CCND1 genes. The 5th and 10th PDT-resistant cells showed an amplicon in 5q11.2 MAP3K1, which was not present in parental cells. The changes detected by aCGH on CCND1, EFGR, and MAP3K1 were confirmed in extracts of SCC-13 cells by reverse-transcriptase PCR and by western blot, and by immunohistochemistry in human biopsies from persistent tumors after MAL-PDT. Our data suggest that genomic imbalances related to CCND1, EFGR, and particularly MAP3K1 seem to be involved in the development of the resistance of SCC to PDT.