RESUMO
BACKGROUND: Several HER2-targeting antibody-drug conjugates (ADC) have gained market approval for the treatment of HER2-expressing metastasis. Promising responses have been reported with the new generation of ADCs in patients who do not respond well to other HER2-targeting therapeutics. However, these ADCs still face challenges of resistance and/or severe adverse effects associated with their particular payload toxins. Eribulin, a therapeutic agent for the treatment of metastatic breast cancer and liposarcoma, is a new choice of ADC payload with a distinct mechanism of action and safety profile. METHODS: We've generated a novel HER2-tageting eribulin-containing ADC, BB-1701. The potency of BB-1701 was tested in vitro and in vivo against cancer cells where HER2-expressing levels vary in a large range. Bystander killing effect and toxin-induced immunogenic cell death (ICD) of BB-1701 were also tested. RESULTS: In comparison with HER2-targeting ADCs with DM1 and Dxd payload, eribulin-containing ADC demonstrated higher in vitro cytotoxicity in HER2-low cancer cell lines. BB-1701 also effectively suppressed tumors in models resistant to DM1 or Dxd containing ADCs. Mode of action studies showed that BB-1701 had a significant bystander effect on HER2-null cells adjacent to HER2-high cells. In addition, BB-1701 treatment induced ICD. Repeated doses of BB-1701 in nonhuman primates showed favorable pharmacokinetics and safety profiles at the intended clinical dosage, route of administration, and schedule. CONCLUSIONS: The preclinical data support the test of BB-1701 in patients with various HER2-expressing cancers, including those resistant to other HER2-targeting ADCs. A phase I clinical trial of BB-1701 (NCT04257110) in patients is currently underway.
RESUMO
BACKGROUND: The main function of folate receptor α (FOLRα) has been considered to mediate intracellular folate uptake and induce tumor cell proliferation. Given the broad spectrum of expression among malignant tumors, including gastric cancer (GC) but not in normal tissue, FOLRα represents an attractive target for tumor-selective drug delivery. However, the efficacy of anti-FOLRα monoclonal antibodies (mAbs) has not been proved so far, with the reason for this failure remaining unclear, raising the need for a better understanding of FOLRα function. METHODS: The distribution of FOLRα in GC cells was evaluated by immunohistochemistry. The impacts of FOLRα expression on the survival of GC patients and GC cell lines were examined with the Gene Expression Omnibus database and by siRNA of FOLRα. RNA-sequencing and Microarray analysis was conducted to identify the function of FOLRα. Proteins that interact with FOLRα were identified with shotgun LC-MS/MS. The antitumor efficacy of the anti-FOLRα mAb farletuzumab as well as the antibody-drug conjugate (ADC) consists of the farletuzumab and the tublin-depolymerizing agent eribulin (MORAb-202) was evaluated both in vitro and in vivo. RESULTS: FOLRα was detected both at the cell membrane and in the cytoplasm. Shorter overall survival was associated with FOLRα expression in GC patients, whereas reduction of FOLRα attenuated cell proliferation without inducing cell death in GC cell lines. Transcriptomic and proteomic examinations revealed that the FOLRα-expressing cancer cells possess a mechanism of chemotherapy resistance supported by MDM2, and FOLRα indirectly regulates it through a chaperone protein prohibitin2 (PHB2). Although reduction of FOLRα brought about vulnerability for oxaliplatin by diminishing MDM2 expression, farletuzumab did not suppress the MDM2-mediated chemoresistance and cell proliferation in GC cells. On the other hand, MORAb-202 showed significant antitumor efficacy. CONCLUSIONS: The ADC could be a more reasonable choice than mAb as a targeting agent for the FOLRα-expressing tumor.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptor 1 de Folato/metabolismo , Furanos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Cetonas/farmacologia , Proibitinas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/química , Neoplasias Gástricas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Feminino , Receptor 1 de Folato/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oxaliplatina/farmacologia , Prognóstico , Proibitinas/genética , Proteoma , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Taxa de Sobrevida , Transcriptoma , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: MORAb-202, an antibody-drug conjugate containing farletuzumab and eribulin with a cathepsin-B cleavable linker, targets folate receptor α (FRα)-expressing tumor cells. The primary objective of this first-in-human study was to evaluate the safety and tolerability of MORAb-202 in patients with solid tumors. PATIENTS AND METHODS: Patients ≥20 years with adequate organ function and FRα-positive solid tumors who failed to respond to standard therapy were eligible. Patients received MORAb-202 intravenously at doses of 0.3 to 1.2 mg/kg once every three weeks. Endpoints included dose-limiting toxicities, safety, tumor responses, pharmacokinetics, and pharmacodynamics. TRIAL REGISTRATION NUMBER: NCT03386942 (ClinicalTrials.gov). RESULTS: Between November 28, 2017 and June 4, 2019, 22 patients (median age, 58.0 years) with advanced solid tumors were enrolled. Treatment-emergent adverse events occurred in 21 (95%) patients, with leukopenia and neutropenia in 10 (45%) patients each. One patient (0.9 mg/kg cohort) experienced two grade 3 dose-limiting toxicities: serum alanine aminotransferase and γ-glutamyl transferase increases. Following review by an independent adjudication committee, grade 1/2 interstitial lung disease thought to be related to MORAb-202 was identified in five (23%) patients. Complete response, partial response, and stable disease were observed in one, nine, and eight patients, respectively. The normalized predose serum FRα tended to be positively correlated with the maximum tumor shrinkage (R 2 = 0.2379; P = 0.0291). CONCLUSIONS: The MTD of MORAb-202 was not reached. MORAb-202 demonstrated promising antitumor activity in FRα-positive solid tumors and was generally well-tolerated at the tested doses. Further investigations are required to establish appropriate dosage and clinical utility of MORAb-202.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Furanos/uso terapêutico , Cetonas/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Adulto , Idoso , Anticorpos Monoclonais Humanizados/efeitos adversos , Feminino , Receptor 1 de Folato/análise , Furanos/efeitos adversos , Humanos , Imunoconjugados/uso terapêutico , Cetonas/efeitos adversos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias/química , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The antibody-drug conjugate (ADC) MORAb-202, consisting of farletuzumab paired with a cathepsin B-cleavable linker and eribulin, targets folate receptor alpha (FRA), which is frequently overexpressed in various tumor types. MORAb-202 was highly cytotoxic to FRA-positive cells in vitro, with limited off-target killing of FRA-negative cells. Furthermore, MORAb-202 showed a clear in vitro bystander cytotoxic effect in coculture with FRA-positive/negative cells. In vivo antitumor efficacy studies of MORAb-202 were conducted with a single administration of MORAb-202 in triple-negative breast cancer (TNBC) patient-derived xenograft (PDx) models expressing low and high levels of FRA. MORAb-202 exhibited durable efficacy proportional to tumor FRA expression. Toxicology studies (Q3Wx2) in nonhuman primates suggested that the major observed toxicity of MORAb-202 is hematologic toxicity. Overall, these findings support the concept that MORAb-202 represents a promising investigational ADC for the treatment of TNBC patients.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Furanos/química , Imunoconjugados/administração & dosagem , Cetonas/química , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas de Transporte Vesicular/metabolismo , Animais , Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Furanos/farmacologia , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/química , Cetonas/farmacologia , Camundongos , Modelagem Computacional Específica para o Paciente , Primatas , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas de Transporte Vesicular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Microtubule-targeting agents (MTA) have been investigated for many years as payloads for antibody-drug conjugates (ADC). In many cases, these ADCs have shown limited benefits due to lack of efficacy or significant toxicity, which has spurred continued investigation into novel MTA payloads for next-generation ADCs. In this study, we have developed ADCs using the MTA eribulin, a derivative of the macrocyclic polyether natural product halichondrin B, as a payload. Eribulin ADCs demonstrated in vitro potency and specificity using various linkers and two different conjugation approaches. MORAb-202 is an investigational agent that consists of the humanized anti-human folate receptor alpha (FRA) antibody farletuzumab conjugated via reduced interchain disulfide bonds to maleimido-PEG2-valine-citrulline-p-aminobenzylcarbamyl-eribulin at a drug-to-antibody ratio of 4.0. MORAb-202 displayed preferable biophysical properties and broad potency across a number of FRA-positive tumor cell lines as well as demonstrated improved specificity in vitro compared with farletuzumab conjugated with a number of other MTA payloads, including MMAE, MMAF, and the reducible maytansine linker-payload sulfo-SPDB-DM4. A single-dose administration of MORAb-202 in FRA-positive human tumor cell line xenograft and patient-derived tumor xenograft models elicited a robust and durable antitumor response. These data support further investigation of MORAb-202 as a potential new treatment modality for FRA-positive cancers, using the novel MTA eribulin as a payload.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Receptor 1 de Folato/antagonistas & inibidores , Furanos/farmacologia , Imunoconjugados/farmacologia , Cetonas/farmacologia , Microtúbulos/metabolismo , Animais , Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Receptor 1 de Folato/metabolismo , Furanos/química , Humanos , Imunoconjugados/química , Cetonas/química , Camundongos SCID , Polietilenoglicóis/química , Resultado do TratamentoRESUMO
Over-expression of endosialin/CD248 (herein referred to as CD248) has been associated with increased tumor microvasculature in various tissue origins which makes it an attractive anti-angiogenic target. In an effort to target CD248, we have generated a human CD248 knock-in mouse line and MORAb-004, the humanized version of the mouse anti-human CD248 antibody Fb5. Here, we report that MORAb-004 treatment significantly impacted syngeneic tumor growth and tumor metastasis in the human CD248 knock-in mice. In comparison with untreated tumors, MORAb-004 treated tumors displayed overall shortened and distorted blood vessels. Immunofluorescent staining of tumor sections revealed drastically more small and dysfunctional vessels in the treated tumors. The CD248 levels on cell surfaces of neovasculature pericytes were significantly reduced due to its internalization. This reduction of CD248 was also accompanied by reduced α-SMA expression, depolarization of pericytes and endothelium, and ultimately dysfunctional microvessels. These results suggest that MORAb-004 reduced CD248 on pericytes, impaired tumor microvasculature maturation and ultimately suppressed tumor development.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Microvasos/efeitos dos fármacos , Neovascularização Patológica , Pericitos/efeitos dos fármacos , Actinas/metabolismo , Inibidores da Angiogênese/metabolismo , Animais , Anticorpos Monoclonais Humanizados/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Transporte Biológico , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microvasos/imunologia , Microvasos/metabolismo , Microvasos/patologia , Metástase Neoplásica , Pericitos/imunologia , Pericitos/metabolismo , Pericitos/patologia , Interferência de RNA , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacosRESUMO
Glucocorticoid-induced TNF receptor ligand (GITRL) is a member of the TNF super family (TNFSF). GITRL plays an important role in controlling regulatory T cells. The crystal structure of the mouse GITRL (mGITRL) was determined to 1.8-A resolution. Contrary to the current paradigm that all ligands in the TNFSF are trimeric, mGITRL associates as dimer through a unique C terminus tethering arm. Analytical ultracentrifuge studies revealed that in solution, the recombinant mGITRL exists as monomers at low concentrations and as dimers at high concentrations. Biochemical studies confirmed that the mGITRL dimer is biologically active. Removal of the three terminal residues in the C terminus resulted in enhanced receptor-mediated NF-kappaB activation than by the wild-type receptor complex. However, deletion of the tethering C-terminus arm led to reduced activity. Our studies suggest that the mGITRL may undergo a dynamic population shift among different oligomeric forms via C terminus-mediated conformational changes. We hypothesize that specific oligomeric forms of GITRL may be used as a means to differentially control GITR receptor signaling in diverse cells.
Assuntos
Receptores de Fator de Crescimento Neural/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Animais , Células COS , Chlorocebus aethiops , Cristalografia por Raios X/métodos , Dimerização , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Ligantes , Camundongos , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , NF-kappa B/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de Fator de Crescimento Neural/química , Receptores do Fator de Necrose Tumoral/química , Linfócitos T/metabolismo , Fatores de Necrose Tumoral/metabolismoRESUMO
The transcription factor Foxp3 is involved in the differentiation, function and survival of CD4+CD25+ regulatory T (T(reg)) cells. Details of the mechanism underlying the induction of Foxp3 expression remain unknown, because studies of the transcriptional regulation of the Foxp3 gene are limited by the small number of T(reg) cells in mononuclear cell populations. Here we have generated a model system for analyzing Foxp3 induction and, by using this system with primary T cells, we have identified an enhancer element in this gene. The transcription factors Smad3 and NFAT are required for activity of this Foxp3 enhancer, and both factors are essential for histone acetylation in the enhancer region and induction of Foxp3. These biochemical properties that define Foxp3 expression explain many of the effects of transforming growth factor-beta on the function of Foxp3+ T(reg) cells.
Assuntos
Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Fatores de Transcrição NFATC/metabolismo , Proteína Smad3/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Camundongos , Dados de Sequência MolecularRESUMO
OX40 is a member of the TNFR superfamily (CD134; TNFRSF4) that is expressed on activated T cells and regulates T cell-mediated immune responses. In this study, we have examined the regulation of OX40 gene expression in T cells. Low-level OX40 mRNA expression was detected in both resting T cells and the nonactivated EL4 T cell line, and was up-regulated in both types of T cells upon activation with anti-CD3 Ab. We have shown in this study that basal OX40 promoter activity is regulated by constitutively expressed Sp1/Sp3 and YY1 transcription factors. NF-kappaB (p50 and p65) also binds to the OX40 promoter region, but the level of direct enhancement of the OX40 promoter activity by this transcription factor is not sufficient to account for the observed up-regulation of OX40 mRNA expression associated with activation. We have detected by chromatin immunoprecipitation that histone H4 molecules in the OX40 promoter region are highly acetylated by activation and NF-kappaB binds to the OX40 promoter in vivo. These findings suggest that OX40 gene expression is regulated by chromatin remodeling, and that NF-kappaB might be involved in initiation of chromatin remodeling in the OX40 promoter region in activated T cells. CD4(+)CD25(+) regulatory T (Treg) cells also express OX40 at high levels, and signaling through this receptor can neutralize suppressive activity of this Treg cell. In CD4(+)CD25(+) Treg cells, histone H4 molecules in the OX40 promoter region are also highly acetylated, even in the absence of in vitro activation.
Assuntos
Montagem e Desmontagem da Cromatina/imunologia , Subunidade p50 de NF-kappa B/metabolismo , Regiões Promotoras Genéticas/imunologia , Receptores OX40/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Regulação para Cima/imunologia , Fator de Transcrição YY1/metabolismo , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Complexo CD3/imunologia , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Soros Imunes/farmacologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Linfoma de Células T/genética , Linfoma de Células T/imunologia , Linfoma de Células T/metabolismo , Camundongos , RNA Mensageiro/biossíntese , Receptores OX40/biossíntese , Receptores OX40/metabolismo , Fator de Transcrição Sp1/fisiologia , Fator de Transcrição Sp3/fisiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Transcrição RelA/metabolismo , Regulação para Cima/genética , Fator de Transcrição YY1/fisiologiaRESUMO
mAbs capable of disabling heterodimeric kinase complexes of the epidermal growth factor receptor (EGFR) and human EGFR type 2/neu have therapeutic relevance to various human cancers. In this study, we demonstrate that in addition to the dimer, EGFR and human EGFR type 2 can associate as homo- and heterotetramers. EGF-induced phosphorylation of the tetramers was significantly lower than that of the dimers, indicating that the tetrameric receptor complexes have impaired signaling activity. Targeting v-erb-b2 erythroblastic leukemia viral oncogene homolog (erbB) receptors with mAbs promoted erbB tetrameric assembly, suggesting that a component of the antitumor activity may be mediated by the ability of Abs to shift the equilibrium from active dimeric to impaired tetrameric receptor complex states. This study suggests a novel therapeutic approach to disable signaling of erbB and potentially other receptors in tumors by biologic agents capable of inducing receptor tetramerization.
Assuntos
Anticorpos Monoclonais/imunologia , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Animais , Catálise , Linhagem Celular , Chlorocebus aethiops , Receptores ErbB/metabolismo , Deleção de Genes , Humanos , Peso Molecular , Ligação Proteica , Receptor ErbB-2/genéticaRESUMO
We have previously shown that transduction of HOXD3, one of homeobox genes, into human lung cancer A549 cells enhances cell motility, invasion and metastasis. In the present study, we examined the roles of integrin beta3 which was up-regulated by HOXD3-overexpression in the HOXD3-induced motility of A549 cells. We first established integrin beta3-transfectants and compared their motile activity to those of the HOXD3-transfected, control-transfected and parental cells by three different assays. The integrin beta3-transfectants as well as the HOXD3-transfectants formed heterodimer with integrin alphav subunit, and showed highly motile activities assessed by haptotaxis or phagokinetic track assay compared to the control transfectants or parental cells. In vitro wound-healing assay revealed that migratory activities were graded as the HOXD3-transfectants > the integrin beta3-transfectants > the control transfectants or parental cells. E-cadherin was expressed in the integrin beta3-transfectants but not expressed in the HOXD3-transfectants. An addition of function-blocking antibody to E-cadherin into the wound-healing assay promoted the migratory activity of the integrin beta3-transfectants, suggesting that E-cadherin prevented the cells from dissociating from the wound edges. These results indicate that increased expression of integrin alphav beta3 and loss of E-cadherin by HOXD3-overexpression are responsible for the enhanced motility and dissociation.
Assuntos
Caderinas/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Homeodomínio/fisiologia , Integrina alfaVbeta3/fisiologia , Neoplasias Pulmonares/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Humanos , Camundongos , Metástase Neoplásica , Transdução de Sinais , Fatores de Transcrição , Fator de Crescimento Transformador beta/farmacologia , CicatrizaçãoRESUMO
Our recent studies have identified dynamic protein ensembles containing forkhead box protein 3 (FOXP3) that provide insight into the molecular complexity of suppressor T-cell activities, and it is our goal to determine how these ensembles regulate FOXP3's transcriptional activity in vivo. In this review, we summarize our current understanding of how FOXP3 expression is induced and how FOXP3 functions in vivo as a transcriptional regulator by assembling a multisubunit complex involved in histone modification as well as chromatin remodeling.
Assuntos
Montagem e Desmontagem da Cromatina , Fatores de Transcrição Forkhead/metabolismo , Histonas/metabolismo , Linfócitos T Reguladores/imunologia , Transcrição Gênica , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/terapia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Fatores de Transcrição Forkhead/análise , Fatores de Transcrição Forkhead/genética , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Imunoterapia , Camundongos , Linfócitos T Reguladores/química , Linfócitos T Reguladores/enzimologiaRESUMO
The degree of E- and P-cadherin expressions inversely correlate with the progression stage of human melanoma. In the present study, we analyzed mechanisms of down-regulation of E- and P-cadherin gene expressions in 8 human melanoma cell lines. In 5 of the 8 melanoma cell lines, E-cadherin expression was lost or markedly decreased compared to that in normal melanocytes, and 4 of the 5 melanoma cell lines lost P-cadherin expression. All of the melanoma cell lines expressed snail, which is known to encode a transcription repressor for E-cadherin, at a higher level than melanocytes whereas expression levels of the snail varied among cell lines. Transduction of snail gene into MMAc cells which expressed a high level of E-cadherin and an extremely low level of snail decreased expression of E-cadherin but not P-cadherin. In contrast, transduction of antisense-snail gene into A375M cells which expressed no E-cadherin and a high level of snail restored expression of E-cadherin but not P-cadherin. Methylation-specific PCR analysis revealed CpG methylation in the promoter region of E-cadherin of MeWo and AKI cells. Further, the treatment with a demethylating agent, 5-azacytidine led AKI and A375M cells to re-express both E- and P-cadherin. The results show E-cadherin gene is silenced by at least two distinct mechanisms (methylation and transrepression by Snail) in human melanoma cell lines whereas P-cadherin gene seems to be silenced by methylation but not by snail.
Assuntos
Caderinas/biossíntese , Perfilação da Expressão Gênica , Inativação Gênica , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Metilação de DNA , Proteínas de Ligação a DNA/farmacologia , Regulação para Baixo , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição da Família Snail , Fatores de Transcrição/farmacologia , Células Tumorais Cultivadas , Dedos de ZincoRESUMO
The centrosome is a unique organelle that functions as the microtubule organizing center in most animal cells. During cell division, the centrosomes form the poles of the bipolar mitotic spindle. In addition, the centrosomes are also needed for cytokinesis. Each mammalian somatic cell typically contains one centrosome, which is duplicated in coordination with DNA replication. Just like the chromosomes, the centrosome is precisely reproduced once and only once during each cell cycle. However, it remains a mystery how this protein-based structure undergoes accurate duplication in a semiconservative manner. Intriguingly, amplification of the centrosome has been found in numerous forms of cancers. Cells with multiple centrosomes tend to form multipolar spindles, which result in abnormal chromosome segregation during mitosis. It has therefore been postulated that centrosome aberration may compromise the fidelity of cell division and cause chromosome instability. Here we review the current understanding of how the centrosome is assembled and duplicated. We also discuss the possible mechanisms by which centrosome abnormality contributes to the development of malignant phenotype.
Assuntos
Centrossomo/fisiologia , Mitose/fisiologia , Modelos Biológicos , Fuso Acromático/fisiologia , Aneuploidia , Aurora Quinases , Quinases relacionadas a CDC2 e CDC28/fisiologia , Proteínas de Ciclo Celular , Divisão Celular/fisiologia , Transformação Celular Neoplásica/patologia , Instabilidade Cromossômica/fisiologia , Quinase 2 Dependente de Ciclina , Replicação do DNA/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Fosfatases , Proteínas de Saccharomyces cerevisiaeRESUMO
AIE-75 has been known as a 75-kDa autoantigen detected in the serum of autoimmune enteropathy (AIE) and as a colon cancer-related antigen, and now designated as a gene causative of Usher syndrome type 1C hereditary syndromic hearing loss. It binds to a novel putative tumor suppressor MCC2 that is homologous to MCC (mutated in colon cancer) through a PSD-95/Dlg/ZO-1 (PDZ) domain. To clarify the functional role in colon cancer cells, we transfected AIE-75 gene into SW480 colon cancer cells which do not express AIE-75. Expression of AIE-75 suppressed growth of SW480 cells in vitro in correlation with the expression levels. It was due mainly to G2/M phase cell cycle arrest associated with mitotic slippage, resulting in emergence of hyperploid giant-nucleated or multi-nucleated cells. Screening of proteins that bound to PDZ domains of AIE-75 by a yeast two hybrid system showed that three serine/threonine phosphatase catalytic subunits (PP2AC-alpha, PP2AC-beta, and PPP6C) could bind to AIE-75. Since PP2AC is known to regulate G2/M checkpoint, we suggest that AIE-75 interacts with PP2AC and prevent cells to transit mitotic phase.
Assuntos
Adenocarcinoma/genética , Proteínas de Transporte/genética , Ciclo Celular , Neoplasias do Colo/genética , Perfilação da Expressão Gênica , Proteínas Adaptadoras de Transdução de Sinal , Adenocarcinoma/patologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Neoplasias do Colo/patologia , Proteínas do Citoesqueleto , Humanos , Fosfoproteínas Fosfatases/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais CultivadasRESUMO
The APC (adenomatous polyposis coli) gene status in a familial adenomatous polyposis (FAP) patient who developed hepatoblastoma was analyzed by the yeast color assay. Although a single base insertion at codon 462 resulting in truncation of its product was documented in hepatoblastoma cells, no additional somatic mutation was detectable in the whole coding sequence of the APC gene. The nuclear accumulation of beta-catenin without mutation in the exons 2-4 of the beta-catenin gene, however, was observed in the tumor cells by immunohistochemistry. The similar nuclear accumulation of beta-catenin without an additional somatic mutation in its gene, in the absence of somatic mutation in cluster region of the APC gene, has been previously reported in the single FAP case. Moreover, review in the hepatoblastoma cases in the FAP families showed a relatively later onset of the disease when compared with the sporadic cases. These observations suggest that accumulation of beta-catenin without an additional somatic mutation in the APC gene might be a possible mechanism for tumorigenesis of hepatoblastoma in the FAP families.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/complicações , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Hepatoblastoma/complicações , Neoplasias Hepáticas/complicações , Transativadores/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA/métodos , Saúde da Família , Hepatoblastoma/genética , Hepatoblastoma/metabolismo , Humanos , Imuno-Histoquímica , Lactente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Mutação , Transativadores/genética , Proteína Supressora de Tumor p53/genética , beta CateninaRESUMO
In human genetics and molecular oncology, mutation research is necessary not only to identify mutations in nucleic acid sequences, but also to analyze the loss of function caused by mutant proteins. We reconstructed a protein-protein network system of human beta-catenin and TCF4, in Saccharomyces cerevisiae. beta-Catenin and TCF4 proteins form a complex and transactivate reporter genes. Co-expressed wild-type APC with beta-catenin and TCF4 inhibit the transcriptional activity of the beta-catenin/TCF4 complex in yeast, as well as in mammals. This unique method in which the beta-catenin/TCF4 signaling pathway is reconstructed in vivo may prove useful for the functional evaluation of APC mutants, including a type of APC truncated and missense mutants influenced to the ability of binding to beta-catenin.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/fisiologia , Genes APC , Mutação/fisiologia , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Neoplasias Colorretais/diagnóstico , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica , Humanos , Mutação de Sentido Incorreto/fisiologia , Fatores de Transcrição TCF , Transativadores/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , Transcrição Gênica/fisiologia , beta CateninaRESUMO
We investigated the assembly and activation of the epidermal growth factor receptor (EGFR)-p185c-neu heterodimer by using a sequential immunoprecipitation methodology. Using this approach we detected heterodimers and also higher-ordered oligomeric complexes. Phosphorylated EGFR-p185c-neu heterodimeric forms were detected in the absence of EGF, but the species became highly phosphorylated after EGF stimulation. To evaluate heterodimer formation and additional transactivation by EGF, we investigated the roles of the four extracellular subdomains of p185c-neu and the EGFR. Subdomains I-IV of the EGFR dimerized with subdomains I-IV of p185c-neu, respectively, in a parallel manner. In addition, subdomains I-IV of the EGFR also associated with p185c-neu subdomains III, IV, I, and II, respectively. A lack of one of the p185c-neu cysteine-rich domains (subdomains II or IV) resulted in a loss of EGF-induced transactivation. These data suggest that two cysteine-rich domains play defining roles in ligand-dependent transactivation and that both of these cysteine-rich extracellular subdomains as well as non-cysteine-rich extracellular subdomains are involved in ligand-independent interactions with the EGFR. Our studies provide biochemical evidence of the role of the cysteine-rich domains of p185c-neu in assembly and transactivation of erbB complexes and also indicate that these subdomains might be useful clinical targets.
Assuntos
Receptores ErbB/química , Receptor ErbB-2/química , Animais , Células COS , Cisteína/química , Dimerização , Receptores ErbB/metabolismo , Vetores Genéticos , Ligantes , Camundongos , Modelos Químicos , Estrutura Terciária de Proteína , Receptor ErbB-2/metabolismo , Ativação Transcricional , TransfecçãoRESUMO
Homeobox genes regulate sets of genes that determine cellular fates in embryonic morphogenesis and maintenance of adult tissue architecture by regulating cellular motility and cell-cell interactions. Our previous studies showed that a specific member, HOXD3, when overexpressed, enhanced cell motility and invasiveness of human lung cancer A549 cells (Hamada et al. Int. J. Cancer 2001; 93: 516-25 [19]). In the present study, we investigated the roles of HOXD3 in motile and invasive behavior of human malignant melanoma cells. Of seven melanoma cell lines examined here, six cell lines expressed the HOXD3 gene, whereas normal melanocytes did not. We transduced the HOXD3-antisense gene expression vector into two cell lines (A375M and MMIV). The cell transduced with the HOXD3-antisense gene showed reduced in vitro invasion of Matrigel. The transduction of the HOXD3-antisense gene also decreased cell spreading, haptotactic activity to vitronectin and laminin-1, and phagokinetic activity. To find the difference of gene expression between the HOXD3-antisense-transduced A375M cells and the control A375MNeo2 cells, we carried out cDNA microarray analysis. The results of the microarray analysis indicated that the increased expression of cdc42-interacting protein 4, KIAA0554 and tropomyosin 1, which are all associated with the cytoskeletal system, may be involved in the reduction of motile and invasive activity by the HOXD3-antisense gene transduction.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Homeodomínio/genética , Melanoma/patologia , Transdução Genética/métodos , Animais , Sequência de Bases , Linhagem Celular , Movimento Celular , Quimiotaxia , Primers do DNA , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação Neoplásica da Expressão Gênica , Genes Homeobox , Humanos , Melanoma/fisiopatologia , Camundongos , Invasividade Neoplásica , Reação em Cadeia da Polimerase , Fatores de Transcrição , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND: beta-Catenin has been shown to function as a Wnt signaling molecule to stimulate cyclin D1 expression and cell growth in several kinds of tumors. METHODS: The authors immunohistochemically examined specimens of 217 surgically resected primary nonsmall cell lung carcinomas (NSCLCs) for beta-catenin expression and classified them semiquantitatively into three categories, including those with high, moderate, and low scores of expression. RESULTS: High, moderate, and low scores of expression were found in 37 (17.1%), 145 (66.8%), and 35 (16.1%) tumors, respectively. beta-Catenin expression was not correlated with cyclin D1 expression, but was positively correlated with the Ki-67 cell growth fraction (P = 0.04). The direct sequencing analysis for the beta-catenin gene mutation of 13 specimens of 217 tumors for the current study revealed no mutations. The relation between survival and beta-catenin expression was evaluated in 148 potentially curatively resected tumors with pathologic Stages I-IIIA. A trend toward better survival was found in patients with tumors having higher scores. In multivariate analysis, high beta-catenin expression was a significant and independent favorable prognostic factor (hazards ratio, 0.31; P = 0.007) as was pathologic stage. Analyzed by cell type, in nonsquamous cell carcinomas, patients with tumors having high scores survived a significantly longer time than those with tumors having moderate or low scores (5-year survival rates, 84%, 55%, and 32%, respectively; P = 0.02), and high beta-catenin expression tended to be a favorable prognostic factor (hazards ratio, 0.32; P = 0.052). CONCLUSIONS: These results indicate that, in NSCLCs, increased expression of beta-catenin can predict favorable prognosis of patients with resected tumors, suggesting that accumulation of beta-catenin has no or little oncogenic effect via activation of the Wnt pathway, unlike in colon carcinomas or hepatomas.