RESUMO
To elucidate their mechanism of action, inhibitors of Bruton tyrosine kinase (BTK) and resistant BTK mutants were employed to dissect target-dependent cellular functions. BTK-C481S and -T474I, expressed in Ramos and NALM-6 cells, maintained BTK auto-phosphorylation under treatment with ibrutinib or dasatinib, respectively, which showed only modest cytotoxicity. Retained activity of BTK-T474 partially rescued cell migration from inhibition by dasatinib. Importantly, resistant BTK mutants reconstituted B cell receptor-triggered chemokine secretion in the presence of corresponding inhibitors, demonstrating that BTK activity is connected with cell-intrinsic functions of malignant B cells with importance for their dialogue with the micro-environment.
Assuntos
Linfócitos B/enzimologia , Leucemia de Células B/genética , Linfoma de Células B/genética , Proteínas Tirosina Quinases/genética , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Antineoplásicos , Quimiocinas/metabolismo , Quimiotaxia/efeitos dos fármacos , Dasatinibe/administração & dosagem , Dasatinibe/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia de Células B/enzimologia , Leucemia de Células B/patologia , Linfoma de Células B/enzimologia , Linfoma de Células B/patologia , Mutação , Fosforilação/efeitos dos fármacos , Piperidinas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Células Tumorais CultivadasRESUMO
Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.
Assuntos
Antígenos Virais de Tumores/genética , Carcinoma de Célula de Merkel/patologia , Transformação Celular Viral , Embrião de Mamíferos/patologia , Células de Merkel/patologia , Poliomavírus das Células de Merkel/fisiologia , Neoplasias Cutâneas/patologia , Anaplasia , Animais , Carcinoma de Célula de Merkel/virologia , Contagem de Células , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Fígado/patologia , Masculino , Poliomavírus das Células de Merkel/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Neoplasias Cutâneas/virologia , Baço/patologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Merkel cell polyomavirus (MCV) is clonally integrated in over 80 % of Merkel cell carcinomas and mediates tumour development through the expression of viral oncoproteins, the large T (LT) and small T antigens (sT). Viral integration is associated with signature mutations in the T-antigen locus that result in deletions of C-terminal replicative functions of the LT antigen. Despite these truncations, the LT LXCXE retinoblastoma (Rb) pocket protein family binding domain is retained, and the entire sT isoform is maintained intact. To investigate the ability of MCV oncoproteins to regulate host gene expression, we performed microarray analysis on cells stably expressing tumour-derived LT, tumour-derived LT along with sT, and tumour-derived LT with a mutated Rb interaction domain. Gene expression alterations in the presence of tumour-derived LT could be classified into three main groups: genes that are involved in the cell cycle (specifically the G1/S transition), genes involved in DNA replication and genes involved in cellular movement. The LXCXE mutant LT largely reversed gene expression alterations detected with the WT tumour-derived LT, while co-expression of sT did not significantly affect these patterns of gene expression. LXCXE-dependent upregulation of cyclin E and CDK2 correlated with increased proliferation in tumour-derived LT-expressing cells. Tumour-derived LT and tumour-derived LT plus sT increased expression of multiple cytokines and chemokines, which resulted in elevated levels of secreted IL-8. We concluded that, in human fibroblasts, the LXCXE motif of tumour-derived LT enhances cellular proliferation and upregulates cell cycle and immune signalling gene transcription.
Assuntos
Antígenos Virais de Tumores/fisiologia , Citocinas/metabolismo , Regulação da Expressão Gênica/imunologia , Inflamação/metabolismo , Poliomavírus das Células de Merkel/imunologia , Antígenos Virais de Tumores/imunologia , Proliferação de Células , Células Cultivadas , Citocinas/genética , Reparo do DNA , Replicação do DNA/fisiologia , Fibroblastos/fisiologia , Fibroblastos/virologia , Regulação da Expressão Gênica/fisiologia , Humanos , Transdução de Sinais/imunologia , Transcrição Gênica , Regulação para CimaRESUMO
Merkel cell carcinoma (MCC) is a neuroendocrine skin cancer associated with high mortality. Merkel cell polyomavirus (MCV), discovered in 2008, is associated with ~80% of MCC. The MCV large tumor (LT) oncoprotein upregulates the cellular oncoprotein survivin through its conserved retinoblastoma protein-binding motif. We confirm here that YM155, a survivin suppressor, is cytotoxic to MCV-positive MCC cells in vitro at nanomolar levels. Mouse survival was significantly improved for NOD-Scid-Gamma mice treated with YM155 in a dose and duration dependent manner for 3 of 4 MCV-positive MCC xenografts. One MCV-positive MCC xenograft (MS-1) failed to significantly respond to YM155, which corresponds with in vitro dose-response activity. Combination treatment of YM155 with other chemotherapeutics resulted in additive but not synergistic cell killing of MCC cell lines in vitro. These results suggest that survivin targeting is a promising therapeutic approach for most but not all MCV-positive MCCs.
Assuntos
Carcinoma de Célula de Merkel/metabolismo , Carcinoma de Célula de Merkel/virologia , Transformação Celular Viral , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Poliomavírus das Células de Merkel/genética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Carcinoma de Célula de Merkel/tratamento farmacológico , Carcinoma de Célula de Merkel/mortalidade , Carcinoma de Célula de Merkel/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Viral/genética , Modelos Animais de Doenças , Feminino , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Naftoquinonas/administração & dosagem , Naftoquinonas/farmacologia , Metástase Neoplásica , Infecções por Polyomavirus/complicações , Survivina , Carga Tumoral/efeitos dos fármacos , Infecções Tumorais por Vírus/complicações , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high mortality rate. The majority of MCC (70-80%) harbor clonally integrated Merkel cell polyomavirus (MCV) in the tumor genome and express viral T antigen oncoproteins. The characterization of an early passage MCV-positive MCC cell line MS-1 is described, and its cellular, immunohistochemical, and virological features to MCV-negative (UISO, MCC13, and MCC26) and MCV-positive cell lines (MKL-1 and MKL-2) were compared. The MS-1 cellular genome harbors integrated MCV, which preserves an identical viral sequence from its parental tumor. Neither VP2 gene transcripts nor VP1 protein are detectable in MS-1 or other MCV-positive MCC cell lines tested. Mapping of viral and cellular integration sites in MS-1 and MCC tumor samples demonstrates no consistent viral or cellular gene integration locus. All MCV-positive cell lines show cytokeratin 20 positivity and grow in suspension. When injected subcutaneously into NOD scid gamma (NSG) mice, MS-1 forms a discrete macroscopic tumor. Immunophenotypic analysis of the MS-1 cell line and xenografts in mice show identical profiles to the parental tumor biopsy. Hence, MS-1 is an early passage cell line that provides a useful in vitro model to characterize MCV-positive MCC.
Assuntos
Carcinoma de Célula de Merkel/virologia , Poliomavírus das Células de Merkel/crescimento & desenvolvimento , Neoplasias Cutâneas/virologia , Animais , Antígenos Virais de Tumores/análise , Linhagem Celular Tumoral/virologia , Linhagem da Célula , Humanos , Queratina-20/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Infecções por Polyomavirus/virologia , Transplante Heterólogo , Infecções Tumorais por Vírus/virologia , Proteínas Estruturais Virais/análise , Integração Viral/genéticaRESUMO
Merkel cell polyomavirus (MCV) causes ~80% of primary and metastatic Merkel cell carcinomas (MCCs). By comparing digital transcriptome subtraction deep-sequencing profiles, we found that transcripts of the cellular survivin oncoprotein [BIRC5a (baculoviral inhibitor of apoptosis repeat-containing 5)] were up-regulated sevenfold in virus-positive compared to virus-negative MCC tumors. Knockdown of MCV large T antigen in MCV-positive MCC cell lines decreased survivin mRNA and protein expression. Exogenously expressed MCV large T antigen increased survivin protein expression in non-MCC primary cells. This required an intact retinoblastoma protein-targeting domain that activated survivin gene transcription as well as expression of other G(1)-S-phase proteins including E2F1 and cyclin E. Survivin expression is critical to the survival of MCV-positive MCC cells. A small-molecule survivin inhibitor, YM155, potently and selectively initiates irreversible, nonapoptotic, programmed MCV-positive MCC cell death. Of 1360 other chemotherapeutic and pharmacologically active compounds screened in vitro, only bortezomib (Velcade) was found to be similarly potent, but was not selective in killing MCV-positive MCC cells. YM155 halted the growth of MCV-positive MCC xenograft tumors and was nontoxic in mice, whereas bortezomib was not active in vivo and mice displayed serious morbidity. Xenograft tumors resumed growth once YM155 treatment was stopped, suggesting that YM155 may be cytostatic rather than cytotoxic in vivo. Identifying the cellular pathways, such as those involving survivin, that are targeted by tumor viruses can lead to rapid and rational identification of drug candidates for treating virus-induced cancers.
Assuntos
Carcinoma de Célula de Merkel/tratamento farmacológico , Carcinoma de Célula de Merkel/metabolismo , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/metabolismo , Infecções por Polyomavirus/metabolismo , Infecções Tumorais por Vírus/metabolismo , Animais , Antígenos Virais de Tumores/genética , Antineoplásicos/farmacologia , Sequência de Bases , Ácidos Borônicos/farmacologia , Bortezomib , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/virologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Proteínas Inibidoras de Apoptose/genética , Poliomavírus das Células de Merkel/genética , Poliomavírus das Células de Merkel/patogenicidade , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Naftoquinonas/farmacologia , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/terapia , Infecções por Polyomavirus/virologia , Pirazinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Survivina , Pesquisa Translacional Biomédica , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/terapia , Infecções Tumorais por Vírus/virologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Merkel cell polyomavirus (MCV) is a recently discovered human polyomavirus causing the majority of human Merkel cell carcinomas. We mapped a 71-bp minimal MCV replication core origin sufficient for initiating eukaryotic DNA replication in the presence of wild-type MCV large T protein (LT). The origin includes a poly(T)-rich tract and eight variably oriented, GAGGC-like pentanucleotide sequences (PS) that serve as LT recognition sites. Mutation analysis shows that only four of the eight PS are required for origin replication. A single point mutation in one origin PS from a naturally occurring, tumor-derived virus reduces LT assembly on the origin and eliminates viral DNA replication. Tumor-derived LT having mutations truncating either the origin-binding domain or the helicase domain also prevent LT-origin assembly. Optimal MCV replication requires coexpression of MCV small T protein (sT), together with LT. An intact DnaJ domain on the LT is required for replication but is dispensable on the sT. In contrast, PP2A targeting by sT is required for enhanced replication. The MCV origin provides a novel model for eukaryotic replication from a defined DNA element and illustrates the selective pressure within tumors to abrogate independent MCV replication.