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1.
Eur J Clin Invest ; 54(6): e14174, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38291340

RESUMO

BACKGROUND: Amplification of HER2, a receptor tyrosine kinase and a breast cancer-linked oncogene, is associated with aggressive disease. HER2 protein is localised mostly at the cell membrane, but a fraction translocates to mitochondria. Whether and how mitochondrial HER2 contributes to tumorigenicity is currently unknown. METHODS: We enriched the mitochondrial (mt-)HER2 fraction in breast cancer cells using an N-terminal mitochondrial targeting sequence and analysed how this manipulation impacts bioenergetics and tumorigenic properties. The role of the tyrosine kinase activity of mt-HER2 was assessed in wild type, kinase-dead (K753M) and kinase-enhanced (V659E) mtHER2 constructs. RESULTS: We document that mt-HER2 associates with the oxidative phosphorylation system, stimulates bioenergetics and promotes larger respiratory supercomplexes. mt-HER2 enhances proliferation and invasiveness in vitro and tumour growth and metastatic potential in vivo, in a kinase activity-dependent manner. On the other hand, constitutively active mt-HER2 provokes excessive mitochondria ROS generation, sensitises to cell death, and restricts growth of primary tumours, suggesting that regulation of HER2 activity in mitochondria is required for the maximal pro-tumorigenic effect. CONCLUSIONS: mt-HER2 promotes tumorigenicity by supporting bioenergetics and optimal redox balance.


Assuntos
Neoplasias da Mama , Mitocôndrias , Receptor ErbB-2 , Mitocôndrias/metabolismo , Humanos , Receptor ErbB-2/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Feminino , Animais , Linhagem Celular Tumoral , Espécies Reativas de Oxigênio/metabolismo , Camundongos , Carcinogênese/metabolismo , Fosforilação Oxidativa , Proliferação de Células , Metabolismo Energético , Respiração Celular/fisiologia
2.
Int J Cancer ; 139(10): 2252-60, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27407063

RESUMO

Richter syndrome represents the transformation of the chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most frequently the diffuse large B-cell lymphoma (DLBCL). In this report we describe a patient with CLL, who developed a clonally-related pleomorphic highly-aggressive mantle cell lymphoma (MCL) after five cycles of a fludarabine-based second-line therapy for the first relapse of CLL. Molecular cytogenetic methods together with whole-exome sequencing revealed numerous gene alterations restricted to the MCL clone (apart from the canonical t(11;14)(q13;q32) translocation) including gain of one copy of ATM gene or emergence of TP53, CREBBP, NUP214, FUBP1 and SF3B1 gene mutations. Similarly, gene expression analysis revealed vast differences between the MCL and CLL transcriptome, including overexpression of cyclin D1, downregulation of cyclins D2 and D3, or downregulation of IL4R in the MCL clone. Backtracking analysis using quantitative PCR specifically detecting an MCL-restricted focal deletion of TP53 revealed that the pre-MCL clone appeared in the bone marrow and peripheral blood of the patient approximately 4 years before the clinical manifestation of MCL. Both molecular cytogenetic and sequencing data support the hypothesis of a slow development of the pre-MCL clone in parallel to CLL over several years, and thereby exclude the possibility that the transformation event occurred at the stage of the CLL relapse clone by mere t(11;14)(q13;q32) acquisition.


Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 14 , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/metabolismo , Perda de Heterozigosidade , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Linfoma de Célula do Manto/metabolismo , Pessoa de Meia-Idade , Translocação Genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
3.
Lab Invest ; 94(7): 806-17, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24862967

RESUMO

Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma (NHL) associated with poor prognosis. Animal models of MCL are scarce. We established and characterized various in vivo models of metastatic human MCL by tail vein injection of either primary cells isolated from patients with MCL or established MCL cell lines (Jeko-1, Mino, Rec-1, Hbl-2, and Granta-519) into immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. MCL infiltration was assessed with immunohistochemistry (tissues) and flow cytometry (peripheral blood). Engraftment of primary MCL cells was observed in 7 out of 12 patient samples. The pattern of engraftment of primary MCL cells varied from isolated involvement of the spleen to multiorgan infiltration. On the other hand, tumor engraftment was achieved in all five MCL cell lines used and lymphoma involvement of murine bone marrow, spleen, liver, and brain was observed. Overall survival of xenografted mice ranged from 22 ± 1 to 54 ± 3 days depending on the cell line used. Subsequently, we compared the gene expression profile (GEP) and phenotype of the engrafted MCL cells compared with the original in vitro growing cell lines (controls). We demonstrated that engrafted MCL cells displayed complex changes of GEP, protein expression, and sensitivity to cytotoxic agents when compared with controls. We further demonstrated that our MCL mouse models could be used to test the therapeutic activity of systemic chemotherapy, monoclonal antibodies, or angiogenesis inhibitors. The characterization of MCL murine models is likely to aid in improving our knowledge in the disease biology and to assist scientists in the preclinical and clinical development of novel agents in relapsed/refractory MCL patients.


Assuntos
Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Linfoma de Célula do Manto/genética , Transcriptoma/genética , Idoso , Animais , Medula Óssea/metabolismo , Encéfalo/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Imunofenotipagem , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Estimativa de Kaplan-Meier , Fígado/metabolismo , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Baço/metabolismo , Transplante Heterólogo , Células Tumorais Cultivadas
4.
Haematologica ; 102(4): e152-e155, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28057744
5.
Mol Med Rep ; 26(4)2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36043519

RESUMO

Gene inactivation of the cyclin­dependent kinase inhibitors p16INK4a, p15INK4b and p21WAF is frequently mediated by promoter gene methylation, whereas histone deacetylases (HDACs) control gene expression through their ability to deacetylate proteins. The effect of suberohydroxamic acid (SBHA) and 5­Aza­2'­deoxycytidine (Decitabine) (DAC) treatments on the transcription of CDKN2A, CDKN2B and CDKN1A genes, and their effects on molecular biological behavior were examined in two myeloma cell lines, RPMI8226 and U266, which differ in p53­functionality and IL­6 expression. In both tested myeloma cell lines, a non­methylated state of the CDKN2B gene promoter region was detected with normal gene expression, and the same level of p15INK4b protein was detected by immunocytochemical staining. Furthermore, in myeloma cells treated with SBHA and DAC alone, the expression of both p15INK4b and p21WAF was significantly upregulated in RPMI8226 cells (p53­functional, without IL­6 expression), whereas in the U266 cell line (p53 deleted, expressing IL­6) only p21WAF expression was significantly increased. Moreover, the analysis revealed that treatment with DAC induced DNMT3B enhancement in U266 cells. In conclusion, in myeloma cells with IL­6 expression, significantly increased DNMT3B expression indicated the tumorigenic consequences of 5­Aza­2'deoxycytidine treatment, which requires careful use in diseases involving epigenetic dysregulation, such as multiple myeloma (MM).


Assuntos
DNA (Citosina-5-)-Metiltransferases , Decitabina , Epigênese Genética , Mieloma Múltiplo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Decitabina/farmacologia , Inativação Gênica , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , DNA Metiltransferase 3B
7.
Clin Cancer Res ; 25(14): 4455-4465, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31004002

RESUMO

PURPOSE: Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2. A BCL2-targeting drug, venetoclax, has promising anticancer activity in MCL. We analyzed molecular mechanisms of venetoclax resistance in MCL cells and tested strategies to overcome it. EXPERIMENTAL DESIGN: We confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are, however, differentially expressed in cell lines compared with primary cells. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, deletions of BIM gene harbored by three commonly used MCL cell lines (JEKO-1, MINO, and Z138) were not found by array comparative genomic hybridization using a validation set of 24 primary MCL samples. RESULTS: We demonstrated that MCL1 and NOXA play important roles in mediating resistance to venetoclax. Consequently, we tested an experimental treatment strategy based on cotargeting BCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo on a panel of five patient-derived xenografts established from patients with relapsed MCL with adverse cytogenetics. CONCLUSIONS: Our data strongly support investigation of venetoclax in combination with S63845 as an innovative treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Sinergismo Farmacológico , Linfoma de Célula do Manto/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Recidiva Local de Neoplasia/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/patologia , Camundongos , Camundongos Endogâmicos NOD , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cancer Res ; 66(1): 78-86, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16397219

RESUMO

Gene deregulation is a frequent cause of malignant transformation. Alteration of the gene structure and/or expression leading to cellular transformation and tumor growth can be experimentally achieved by insertion of the retroviral genome into the host DNA. Retrovirus-containing host loci found repeatedly in clonal tumors are called common viral integration sites (cVIS). cVIS are located in genes or chromosomal regions whose alterations participate in cellular transformation. Here, we present the chicken model for the identification of oncogenes and tumor suppressor genes in solid tumors by mapping the cVIS. Using the combination of inverse PCR and long terminal repeat-rapid amplification of cDNA ends technique, we have analyzed 93 myeloblastosis-associated virus type 2-induced clonal nephroblastoma tumors in detail, and mapped >500 independent retroviral integration sites. Eighteen genomic loci were hit repeatedly and thus classified as cVIS, five of these genomic loci have previously been shown to be involved in malignant transformation of different human cell types. The expression levels of selected genes and their human orthologues have been assayed in chicken and selected human renal tumor samples, and their possible correlation with tumor development, has been suggested. We have found that genes associated with cVIS are frequently, but not in all cases, deregulated at the mRNA level as a result of proviral integration. Furthermore, the deregulation of their human orthologues has been observed in the samples of human pediatric renal tumors. Thus, the avian nephroblastoma is a valid source of cancer-associated genes. Moreover, the results bring deeper insight into the molecular background of tumorigenesis in distant species.


Assuntos
Galinhas , Neoplasias Renais/genética , Oncogenes/genética , Doenças das Aves Domésticas/genética , Integração Viral/genética , Tumor de Wilms/genética , Animais , Vírus da Mieloblastose Aviária/genética , Proteínas Aviárias/genética , Embrião de Galinha , Mapeamento Cromossômico , Proteínas de Ligação a DNA/genética , Genes Supressores de Tumor , Genes ras/genética , Humanos , Neoplasias Renais/virologia , Proteínas Oncogênicas/genética , Reação em Cadeia da Polimerase , Provírus/genética , Sequências Repetidas Terminais , Proteína 1 Relacionada a Twist/genética , Tumor de Wilms/virologia
9.
Oncoimmunology ; 5(4): e1115940, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27141398

RESUMO

Tumor immunotherapy based on the use of chimeric antigen receptor modified T cells (CAR T cells) is a promising approach for the treatment of refractory hematological malignancies. However, a robust response mediated by CAR T cells is observed only in a minority of patients and the expansion and persistence of CAR T cells in vivo is mostly unpredictable.Lenalidomide (LEN) is an immunomodulatory drug currently approved for the treatment of multiple myeloma (MM) and mantle cell lymphoma, while it is clinically tested in the therapy of diffuse large B-cell lymphoma of activated B cell immunophenotype. LEN was shown to increase antitumor immune responses at least partially by modulating the activity of E3 ubiquitin ligase Cereblon, which leads to increased ubiquitinylation of Ikaros and Aiolos transcription factors, which in turn results in changed expression of various receptors on the surface of tumor cells. In order to enhance the effectiveness of CAR-based immunotherapy, we assessed the anti-lymphoma efficacy of LEN in combination with CAR19 T cells or CAR20 T cells in vitro and in vivo using various murine models of aggressive B-cell non-Hodgkin lymphomas (B-NHL).Immunodeficient NSG mice were transplanted with various human B-NHL cells followed by treatment with CAR19 or CAR20 T cells with or without LEN. Next, CAR19 T cells were subjected to series of tests in vitro to evaluate their response and signaling capacity following recognition of B cell in the presence or absence of LEN.Our data shows that LEN significantly enhances antitumor functions of CAR19 and CAR20 T cells in vivo. Additionally, it enhances production of interferon gamma by CAR19 T cells and augments cell signaling via CAR19 protein in T cells in vitro. Our data further suggests that LEN works through direct effects on T cells but not on B-NHL cells. The biochemical events underlying this costimulatory effect of LEN are currently being investigated. In summary, our data supports the use of LEN for augmentation of CAR-based immunotherapy in the clinical grounds.

10.
Clin Cancer Res ; 22(5): 1138-49, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26467384

RESUMO

PURPOSE: To investigate the roles of BCL2, MCL1, and BCL-XL in the survival of diffuse large B-cell lymphoma (DLBCL). EXPERIMENTAL DESIGNS: Immunohistochemical analysis of 105 primary DLBCL samples, and Western blot analysis of 18 DLBCL cell lines for the expression of BCL2, MCL1, and BCL-XL. Pharmacologic targeting of BCL2, MCL1, and BCL-XL with ABT-199, homoharringtonine (HHT), and ABT-737. Analysis of DLBCL clones with manipulated expressions of BCL2, MCL1, and BCL-XL. Immunoprecipitation of MCL1 complexes in selected DLBCL cell lines. Experimental therapy aimed at inhibition of BCL2 and MCL1 using ABT-199 and HHT, single agent, or in combination, in vitro and in vivo on primary cell-based murine xenograft models of DLBCL. RESULTS: By the pharmacologic targeting of BCL2, MCL1, and BCL-XL, we demonstrated that DLBCL can be divided into BCL2-dependent and MCL1-dependent subgroups with a less pronounced role left for BCL-XL. Derived DLBCL clones with manipulated expressions of BCL2, MCL1, and BCL-XL, as well as the immunoprecipitation experiments, which analyzed MCL1 protein complexes, confirmed these findings at the molecular level. We demonstrated that concurrent inhibition of BCL2 and MCL1 with ABT-199 and HHT induced significant synthetic lethality in most BCL2-expressing DLBCL cell lines. The marked cytotoxic synergy between ABT-199 and HHT was also confirmed in vivo using primary cell-based murine xenograft models of DLBCL. CONCLUSIONS: As homoharringtonine is a clinically approved antileukemia drug, and ABT-199 is in advanced phases of diverse clinical trials, our data might have direct implications for novel concepts of early clinical trials in patients with aggressive DLBCL.


Assuntos
Linfoma Difuso de Grandes Células B/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína bcl-X/biossíntese , Animais , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Harringtoninas/administração & dosagem , Mepesuccinato de Omacetaxina , Humanos , Linfoma Difuso de Grandes Células B/classificação , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Nitrofenóis/administração & dosagem , Piperazinas/administração & dosagem , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cancer Res ; 69(11): 4605-12, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19487282

RESUMO

A tumor cell is formed when a critical amount of endogenous and/or exogenous tumorigenic stimuli is exceeded. We have shown that the transient presence of nontumorigenic stray cells in tissues of experimental animals that contain cells with a subcritical set of genetic mutations can act as a tumor-promoting stimulus. To induce somatic mutations in all chicken tissues, we have used the MAV-2 retroviral insertion system that almost exclusively generates nephroblastomas. MAV-2 mutagenized animals i.v. inoculated with nonmalignant cells developed early clonal lung tumors before nephroblastomas. Importantly, the injected cells did not become a component of resultant tumors. Lung tumors displayed specific mutational signature characterized by an insertion of MAV-2 provirus into the fyn-related kinase (frk) promoter that results in the overexpression of the frk gene. In contrast, plag1, foxP, and twist genes were most often mutagenized in nephroblastomas. Based on such observations, we propose the mechanism termed industasis, a promotion of fully malignant phenotype of incipient tumor cell by stray cells, and hypothesize that it might be the underlying cause of human multiple primary tumors.


Assuntos
Transformação Celular Neoplásica/patologia , Células/patologia , Animais , Movimento Celular/fisiologia , Células/virologia , Células Cultivadas , Embrião de Galinha , Galinhas , Neoplasias Renais/patologia , Neoplasias Renais/virologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/virologia , Modelos Biológicos , Mutagênese Insercional/fisiologia , Invasividade Neoplásica , Neoplasias Primárias Múltiplas/etiologia , Provírus/crescimento & desenvolvimento , Provírus/fisiologia , Fenômenos Fisiológicos Virais , Tumor de Wilms/patologia , Tumor de Wilms/virologia
13.
Avian Pathol ; 36(1): 15-27, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17364506

RESUMO

An efficient induction of wasting disease in chickens by avian leukosis virus (ALV), particularly ALV subgroup C, requires >102 infectious units virus inoculated in mid embryogenesis. The most conspicuous symptoms of the disease were induced by ALV subgroup C; however, significant differences in the occurrence of wasting disease were found among individual members of this subgroup. Almost comparable pathogenicity was exhibited by ALV subgroup D, whereas viruses of subgroups B and A proved to be moderately and almost non-pathogenic, respectively. Using antibodies to cellular antigens, tissue alterations were shown clearly in ALV-C-infected chickens. An essential feature was depletion of lymphocytes in the thymus, bursa and spleen. While the number of dendritic cells in the bursa was increased, their representation in the thymus and spleen was reduced. In the spleen, however, the reduction of dendritic cells concerned only an ellipsoid compartment, which in itself was also markedly reduced. An increased number of macrophages in the thymus and spleen corresponded with the observed general activation of the monocyte-macrophage system. In the spleen, CD4+ T cells were reduced while CD8+ T cells were increased. In agreement with this finding was a failure of chickens to respond to Brucella antigen and an inability of their splenocytes to respond to Concanavalin A, both of which pointed to the damage of immune reactivity. Variation in the pathogenicity among individual ALV strains provides ground for depicting gene sequences playing an important role in ALV acute pathogenicity.


Assuntos
Vírus da Leucose Aviária/classificação , Vírus da Leucose Aviária/patogenicidade , Leucose Aviária/virologia , Galinhas/virologia , Doenças das Aves Domésticas/virologia , Animais , Bolsa de Fabricius/citologia , Baço/citologia , Timo/patologia , Virulência
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