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1.
Exp Dermatol ; 30(11): 1686-1692, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33336508

RESUMO

The immunoregulator spleen tyrosine kinase (SYK) is upregulated in cutaneous lupus erythematosus (CLE). This double-blind, multicentre, Phase Ib study evaluated the safety, tolerability, pharmacokinetics, pharmacodynamics and clinical efficacy of the selective SYK inhibitor GSK2646264 in active CLE lesions. Two lesions from each participant (n = 11) were each randomized to topical application of 1% (w/w) GSK2646264 or placebo for 28 days; all participants received GSK2646264 and placebo. The primary endpoint was safety and tolerability of GSK2646264, assessed by adverse event incidence and a skin tolerability test. Secondary endpoints included change from baseline in clinical activity and mRNA expression of interferon-related genes in skin biopsies. Levels of several immune cell markers were evaluated over time. Eight (73%) participants experienced ≥ 1 adverse event (all mild in intensity), and maximal dermal response was similar for GSK2646264 and placebo. The expression of several interferon-related genes, including CXCL10 and OAS1, showed modest decreases from baseline after 28 days of treatment with GSK2646264 compared with placebo. Similar findings were observed for CD3 + T cell and CD11c + dendritic cell levels; however, overall clinical activity remained unchanged with GSK2646264 vs. placebo. Further studies are warranted to assess SYK inhibitors as potential treatment for CLE.


Assuntos
Lúpus Eritematoso Cutâneo/tratamento farmacológico , Piridinas/farmacologia , Piridinas/uso terapêutico , Quinase Syk/antagonistas & inibidores , Administração Tópica , Adulto , Idoso , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Piridinas/administração & dosagem , Piridinas/farmacocinética , Resultado do Tratamento
2.
Recent Results Cancer Res ; 211: 19-44, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30069757

RESUMO

The human epidermal growth factor receptor (HER) family of receptor tyrosine kinases plays an important role in the biology of many cancers. In breast and gastrointestinal cancer, and at lower rates also in additional tumor types, HER2 and its homo- or heterodimerization with HER1 or HER3 are essential for cancer cell growth and survival. Breast cancer patients overexpressing HER2 have a more aggressive course of their disease. The poor prognosis associated with HER2 overexpression can be substantially improved by adding HER2-targeted therapy to standard of care using the monoclonal antibody trastuzumab. Lapatinib, an oral dual tyrosine kinase inhibitor, blocks HER1 and HER2 tyrosine kinase activity by binding to the ATP-binding site of the receptor's intracellular domain, resulting in inhibition of tumor cell growth. Lapatinib is generally well tolerated with diarrhea being the most common adverse effect. However, although being mainly of mild to moderate severity, interruption or discontinuation of treatment has been reported in a substantial proportion of patients in clinical trials. In 2007, lapatinib has been approved in combination with capecitabine in patients with advanced HER2-positive breast cancer upon progressive disease following standard therapy with anthracyclines, taxanes, and trastuzumab. In 2013, the approval was extended to a chemotherapy-free combination with trastuzumab for patients with metastatic HER2-positive, hormone receptor-negative breast cancer progressing on prior trastuzumab and chemotherapy. Since 2010, lapatinib is approved in combination with letrozole in the treatment of postmenopausal women with advanced HER2- and hormone receptor-positive breast cancer. In contrast, in first-line cytotoxic-based therapy of both early and advanced HER2-positive breast cancer, data from clinical trials did not provide evidence of additional benefit of lapatinib compared to trastuzumab. Moreover, over the past few years, novel HER2-targeted drugs, either alone or as a combined anti-HER2 approach, have been extensively evaluated, demonstrating a more favorable outcome. Also, neither in first- nor second-line treatment of advanced gastric cancer, lapatinib has been proven to be superior compared to trastuzumab as hitherto standard of care HER2 blockade. Therefore, lapatinib has become somewhat less important in patients with HER2-positive breast cancer during the past 10 years since its first introduction. Nevertheless, consideration of treatment with lapatinib appears to be reasonable in selected patients not only in the approved applications but also beyond, and further indications such as HER2-positive refractory metastatic colorectal cancer may arise in future. Also, lapatinib may have distinct advantages over antibodies in targeting truncated HER2 and crossing the blood-brain barrier. Finally, the favorable cardiac toxicity profile of lapatinib makes it an attractive alternative to trastuzumab-based regimens in patients at risk for cardiac events.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias Gastrointestinais/tratamento farmacológico , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Feminino , Humanos , Lapatinib , Masculino , Inibidores de Proteínas Quinases/farmacologia
3.
Recent Results Cancer Res ; 201: 125-43, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24756789

RESUMO

The human epidermal growth factor receptor (HER) family of receptor tyrosine kinases plays an important role in the biology of many cancers. In breast and gastric cancer, and maybe also additional tumor types, HER2 and its homo- or heterodimerization with HER1 or HER3 are essential for cancer cell growth and survival. Breast cancer patients overexpressing HER2 have a poor prognosis, which can be substantially improved upon HER2-targeted therapy using the monoclonal antibody trastuzumab. Lapatinib is a dual tyrosine kinase inhibitor (TKI), blocking HER1 and HER2 tyrosine kinase activity by binding to the ATP-binding site of the receptor's intracellular domain. This results in the inhibition of tumor cell growth. In patients, the drug is relatively well tolerated with mostly low-grade adverse effects. In particular and unlike to trastuzumab, it has very little, if any, adverse effects on cardiac function. In 2007, lapatinib has been approved in combination with capecitabine in patients with advanced HER2-positive breast cancer upon progressive disease following standard therapy with anthracyclines, taxanes, and trastuzumab. In 2010, the approval was extended to the treatment of postmenopausal women with advanced, hormone receptor- and HER2-positive breast cancer, for whom hormonal therapy is indicated. Ongoing and future studies will explore its role in the (neo)adjuvant therapy setting, in further drug combinations as well as in the treatment of HER2-positive tumors other than breast cancer.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Quinazolinas/uso terapêutico , Animais , Feminino , Humanos , Lapatinib , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico
4.
Int J Cancer ; 131(2): E10-20, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21953178

RESUMO

Receptor-targeted therapies have become standard in the treatment of various lymphomas. In view of its unparalleled specificity for the malignant B-cell clone, the B-cell receptor (BCR) on B cell lymphoma cells is a potential therapeutic target. We have used two BCR epitope mimicking peptides binding to the Burkitt's lymphoma cell lines CA46 and SUP-B8. We proved their functionality by demonstrating calcium flux and BCR-mediated endocytosis upon peptide receptor binding. Toxicity experiments in vitro via cross-linking of the BCR with tetramerized epitope mimics lead to apoptosis in both cell lines but was far more effective in SUP-B8 cells. We established a SUP-B8-based disseminated Burkitt's lymphoma model in NOD/SCID mice. Treatment of tumor-bearing mice with tetramerized epitope mimics had significant anti-tumor effects in vivo. We conclude that peptide-mediated, BCR-targeted therapy is a promising approach which may be explored and further developed for application in highly aggressive lymphoma.


Assuntos
Linfoma de Burkitt/tratamento farmacológico , Epitopos de Linfócito B/imunologia , Peptídeos/uso terapêutico , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Apoptose , Linfoma de Burkitt/patologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Endocitose , Epitopos de Linfócito B/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mimetismo Molecular , Terapia de Alvo Molecular , Peptídeos/imunologia , Peptídeos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Mol Carcinog ; 51(10): 816-25, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21882255

RESUMO

Activation of Ras and its downstream signaling pathways, likely contribute to the development of hepatocarcinoma. We have previously shown that intraperitoneal injections of the Ras inhibitor S-trans, trans-farnesylthiosalicyclic acid (FTS) blocks Ras activation and prevents heptocarcinoma development in rats receiving weekly injections of the carcinogene diethylnitrosamine (DEN) for 16 wk. Using this in vivo model, we evaluated the relationship between the tumor preventive effect of Ras inhibition and activation of downstream signaling pathways, cell proliferation, cell cycle events, and angiogenesis. Western blotting, quantitative PCR, immunohistochemistry, and transcription factor activity assays were used. DEN-induced activation of NFkB and Stat3 was abrogated by FTS treatment. FTS treatment showed no effect on DEN-induced elevation of TNFα, interleukin 6 and TLR4, known activators of these transcription factors. FTS significantly reduced phosphorylation of the MAPkinase p38 and of the p70S6 kinase, a surrogate marker for mTor activation, without affecting ERK and AKT phosphorylation. These events were associated with reduced c-myc and cyclin D expression as well as reduced cell proliferation in transformed, GSTp-positive hepatocytes. Moreover, FTS treatment shifted cell proliferation from transformed hepatocytes to apparently normal, GSTp negative hepatocytes. FTS treatment did not down-regulate expression of angiogenesis markers HIFα, VEGF, VEGF receptor1, and placenta growth factor. FTS treatment inhibits important signaling pathways involved in cellular proliferation leading to strongly reduced proliferation of transformed hepatocytes without affecting normal hepatocytes. This re-adjustment of the proliferation balance likely contributes to the tumor preventive of FTS in the context of Ras inhibition in hepatocarcinogenesis.


Assuntos
Anticarcinógenos/farmacologia , Farneseno Álcool/análogos & derivados , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/patologia , Neovascularização Patológica/tratamento farmacológico , Salicilatos/farmacologia , Proteínas ras/antagonistas & inibidores , Animais , Carcinógenos/toxicidade , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D/metabolismo , Dietilnitrosamina/toxicidade , Modelos Animais de Doenças , Regulação para Baixo , Farneseno Álcool/farmacologia , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , NF-kappa B/metabolismo , Neovascularização Patológica/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Ratos Wistar , Fator de Transcrição STAT3/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/metabolismo
6.
J Immunol ; 184(1): 512-20, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19949082

RESUMO

Ab-dependent cellular cytotoxicity (ADCC) is usually considered an important mechanism of action for immunotherapy with human IgG1 but not IgG2 Abs. The epidermal growth factor receptor (EGF-R) Ab panitumumab represents the only human IgG2 Ab approved for immunotherapy and inhibition of EGF-R signaling has been described as its principal mechanism of action. In this study, we investigated effector mechanisms of panitumumab compared with zalutumumab, an EGF-R Ab of the human IgG1 isotype. Notably, panitumumab was as effective as zalutumumab in recruiting ADCC by myeloid effector cells (i.e., neutrophils and monocytes) in contrast to NK cell-mediated ADCC, which was only induced by the IgG1 Ab. Neutrophil-mediated tumor cell killing could be stimulated by myeloid growth factors and was triggered via FcgammaRIIa. Panitumumab-mediated ADCC was significantly affected by the functional FcgammaRIIa-R131H polymorphism and was induced more effectively by neutrophils from FcgammaRIIa-131H homozygous donors than from -131R individuals. This polymorphism did not affect neutrophil ADCC induced by the IgG1 Ab zalutumumab. The in vivo activity of both Abs was assessed in two animal models: a high-dose model, in which signaling inhibition is a dominant mechanism of action, and a low-dose model, in which effector cell recruitment plays a prominent role. Zalutumumab was more effective than panitumumab in the high-dose model, reflecting its stronger ability to induce EGF-R downmodulation and growth inhibition. In the low-dose model, zalutumumab and panitumumab similarly prevented tumor growth. Thus, our results identify myeloid cell-mediated ADCC as a potent and additional mechanism of action for EGF-R-directed immunotherapy.


Assuntos
Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Receptores ErbB/imunologia , Imunoglobulina G/imunologia , Animais , Anticorpos Monoclonais Humanizados , Linhagem da Célula , Citometria de Fluxo , Humanos , Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , Camundongos , Monócitos/imunologia , Neutrófilos/imunologia , Panitumumabe
7.
Proc Natl Acad Sci U S A ; 105(16): 6109-14, 2008 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-18427122

RESUMO

The epidermal growth factor receptor (EGFR) activates cellular pathways controlling cell proliferation, differentiation, migration, and survival. It thus represents a valid therapeutic target for treating solid cancers. Here, we used an electron microscopy-based technique (Protein Tomography) to study the structural rearrangement accompanying activation and inhibition of native, individual, EGFR molecules. Reconstructed tomograms (3D density maps) showed a level of detail that allowed individual domains to be discerned. Monomeric, resting EGFR ectodomains demonstrated large flexibility, and a number of distinct conformations were observed. In contrast, ligand-activated EGFR complexes were detected only as receptor dimers with ring-like conformations. Zalutumumab, a therapeutic inhibitory EGFR antibody directed against domain III, locked EGFR molecules into a very compact, inactive conformation. Biochemical analyses showed bivalent binding of zalutumumab to provide potent inhibition of EGFR signaling. The structure of EGFR-zalutumumab complexes on the cell surface visualized by Protein Tomography indicates that the cross-linking spatially separates the EGFR molecules' intracellular kinase domains to an extent that appears incompatible with the induction of signaling. These insights into the mechanisms of action of receptor inhibition may also apply to other cell-surface tyrosine kinase receptors of the ErbB family.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Animais , Anticorpos Monoclonais Humanizados , Sítios de Ligação , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/farmacologia , Mapeamento de Epitopos , Receptores ErbB/genética , Humanos , Ligantes , Camundongos , Microscopia Eletrônica , Mutação , Conformação Proteica , Transdução de Sinais/efeitos dos fármacos
8.
Cancer Sci ; 101(5): 1080-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20331636

RESUMO

Oncogenic mutations of the KRAS gene have emerged as a common mechanism of resistance against epidermal growth factor receptor (EGF-R)-directed tumor therapy. Mutated KRAS leads to ligand-independent activation of signaling pathways downstream of EGF-R. Thereby, direct effector mechanisms of EGF-R antibodies, such as blockade of ligand binding and inhibition of signaling, are bypassed. Thus, a humanized variant of the approved EGF-R antibody Cetuximab inhibited growth of wild-type KRAS-expressing A431 cells, but did not inhibit KRAS-mutated A549 tumor cells. We then investigated whether killing of tumor cells harboring mutated KRAS can be improved by enhancing antibody-dependent cellular cytotoxicity (ADCC). Protein- and glyco-engineering of antibodies' Fc region are established technologies to enhance ADCC by increasing antibodies' affinity to activating Fcgamma receptors. Thus, EGF-R antibody variants with increased affinity for the natural killer (NK) cell-expressed FcgammaRIIIa (CD16) were generated and analyzed. These variants triggered significantly enhanced mononuclear cell (MNC)-mediated killing of KRAS-mutated tumor cells compared to wild-type antibodies. Additionally, cells transfected with mutated KRAS were killed as effectively by ADCC as vector-transfected control cells. Together, these data demonstrate that KRAS mutations are not sufficient to render tumor cells resistant to ADCC. Consequently Fc-engineered EGF-R antibodies may prove effective against KRAS-mutated tumors, which are not susceptible to signaling inhibition by EGF-R antibodies.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Receptores ErbB/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Mutação , Neoplasias/genética , Engenharia de Proteínas , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Leucócitos Mononucleares/imunologia , Neoplasias/imunologia , Proteínas Proto-Oncogênicas p21(ras) , Receptores de IgG/imunologia
9.
Blood ; 112(6): 2390-9, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18566325

RESUMO

Glycosylation of the antibody Fc fragment is essential for Fc receptor-mediated activity. Carbohydrate heterogeneity is known to modulate the activity of effector cells in the blood, in which fucosylation particularly affects NK cell-mediated killing. Here, we investigated how the glycosylation profile of 2F8, a human IgG(1) monoclonal antibody against epidermal growth factor receptor in clinical development, impacted effector function. Various 2F8 batches differing in fucosylation, galactosylation, and sialylation of the complex-type oligosaccharides in the Fc fragment were investigated. Our results confirmed that low fucose levels enhance mononuclear cell-mediated antibody-mediated cellular cytotoxicity (ADCC). In contrast, polymorphonuclear cells were found to preferentially kill via high-fucosylated antibody. Whole blood ADCC assays, containing both types of effector cells, revealed little differences in tumor cell killing between both batches. Significantly, however, high-fucose antibody induced superior ADCC in blood from granulocyte colony-stimulating factor-primed donors containing higher numbers of activated polymorphonuclear cells. In conclusion, our data demonstrated for the first time that lack of fucose does not generally increase the ADCC activity of therapeutic antibodies and that the impact of Fc glycosylation on ADCC is critically dependent on the recruited effector cell type.


Assuntos
Anticorpos/metabolismo , Citotoxicidade Celular Dependente de Anticorpos , Fucose/imunologia , Células Matadoras Naturais/imunologia , Neutrófilos/imunologia , Anticorpos/imunologia , Linhagem Celular , Fucose/metabolismo , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo
10.
Recent Results Cancer Res ; 184: 45-59, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20072830

RESUMO

The human epidermal growth factor receptor (HER) family of growth factor receptor tyrosine kinases (RTKs) plays an important role in the biology of many cancers. In breast cancer, HER2 and its homo- or heterodimerization with HER1 or HER3 are essential for cancer cell growth and survival. Patients overexpressing HER2 have a poor prognosis, which can be substantially improved upon HER2-targeted therapy using the monoclonal antibody trastuzumab. Lapatinib is a novel dual tyrosine kinase inhibitor, blocking HER1 and HER2 tyrosine kinase activity by binding to the ATP-binding site of the receptor's intracellular domain. This results in inhibition of tumor cell growth. The drug is relatively well tolerated in patients, with few and mostly low-grade adverse effects. In particular and unlike to trastuzumab, it has very little, if any, adverse effects on cardiac function. In patients with advanced HER2-positive breast cancer, lapatinib has shown substantial antitumor activity, particularly in combination with capecitabine upon progressive disease following standard therapy with antracyclines, taxanes, and trastuzumab. Ongoing and future studies will explore its role in the adjuvant therapy setting, in drug combinations other than capecitabine, and in the treatment of HER2-positive tumors other than breast cancer.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Animais , Ensaios Clínicos como Assunto , Receptores ErbB/análise , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/fisiologia , Humanos , Lapatinib , Quinazolinas/efeitos adversos , Quinazolinas/farmacologia , Receptor ErbB-2/análise , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/fisiologia
12.
J Clin Endocrinol Metab ; 89(10): 5233-44, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15472230

RESUMO

Decidualization of the endometrial stromal compartment is critical for embryo implantation. Initiation of this differentiation process requires elevated intracellular cAMP levels. We now report a massive and sustained up-regulation of p53 tumor suppressor protein during cAMP-induced decidualization of cultured endometrial stromal cells. Nuclear accumulation of p53 was not accompanied by increased mRNA expression, suggesting stabilization of the protein as the underlying mechanism. Proteasomal degradation of p53 is known to be mediated by nuclear Mdm2. Nuclear translocation of Mdm2, in turn, is dependent on phosphorylation by protein kinase B/Akt (PKB/Akt). In cAMP-treated decidualized cells, p53 accumulation was associated with decreased nuclear Mdm2 and cytoplasmic PKB/Akt levels. Conversely, withdrawal of the decidualization stimulus resulted in morphological and biochemical dedifferentiation, disappearance of p53, but increased abundance of PKB/Akt. Furthermore, Western blot and immunohistochemical analyses of endometrial biopsies confirmed that p53 is expressed in vivo in the stromal compartment during the late secretory phase of the cycle. The observation that p53 protein expression is closely associated with decidual transformation indicates a novel role for this tumor suppressor in regulating human endometrial function.


Assuntos
AMP Cíclico/metabolismo , Decídua/citologia , Decídua/fisiologia , Células Estromais/fisiologia , Proteína Supressora de Tumor p53/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Humanos , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Células Estromais/citologia , Proteína Supressora de Tumor p53/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Regulação para Cima , Leveduras
13.
Neoplasia ; 14(3): 190-205, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22496619

RESUMO

Oncogenic KRAS mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR)-directed antibody (Ab) therapy. However, the mechanisms by which constitutively activated KRAS (KRAS(G12V)) impairs effector mechanisms of EGFR-Abs are incompletely understood. Here, we established isogenic cell line models to systematically investigate the impact of KRAS(G12V) on tumor growth in mouse A431 xenograft models as well as on various modes of action triggered by EGFR-Abs in vitro. KRAS(G12V) impaired EGFR-Ab-mediated growth inhibition by stimulating receptor-independent downstream signaling. KRAS(G12V) also rendered tumor cells less responsive to Fc-mediated effector mechanisms of EGFR-Abs-such as complement-dependent cytotoxicity (CDC) and Ab-dependent cell-mediated cytotoxicity (ADCC). Impaired CDC and ADCC activities could be linked to reduced EGFR expression in KRAS-mutated versus wild-type (wt) cells, which was restored by small interfering RNA (siRNA)-mediated knockdown of KRAS4b. Immunohistochemistry experiments also revealed lower EGFR expression in KRAS-mutated versus KRAS-wt harboring CRC samples. Analyses of potential mechanisms by which KRAS(G12V) downregulated EGFR expression demonstrated significantly decreased activity of six distinct transcription factors. Additional experiments suggested the CCAAT/enhancer-binding protein (C/EBP) family to be implicated in the regulation of EGFR promoter activity in KRAS-mutated tumor cells by suppressing EGFR transcription through up-regulation of the inhibitory family member C/EBPß-LIP. Thus, siRNA-mediated knockdown of C/EBPß led to enhanced EGFR expression and Ab-mediated cytotoxicity against KRAS-mutated cells. Together, these results demonstrate that KRAS(G12V) signaling induced C/EBPß-dependent suppression of EGFR expression, thereby impairing Fc-mediated effector mechanisms of EGFR-Abs and rendering KRAS-mutated tumor cells less sensitive to these therapeutic agents.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas ras/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/genética , Antineoplásicos/farmacologia , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cetuximab , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica , Proteínas ras/genética
14.
J Immunol ; 180(6): 4338-45, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18322248

RESUMO

The epidermal growth factor receptor (EGFR) serves as a molecular target for novel cancer therapeutics such as tyrosine kinase inhibitors (TKI) and EGFR Abs. Recently, specific mutations in the EGFR kinase domain of lung cancers were identified, which altered the signaling capacity of the receptor and which correlated with clinical response or resistance to TKI therapy. In the present study, we investigated the impact of such EGFR mutations on antitumor cell activity of EGFR Abs. Thus, an EGFR-responsive cell line model was established, in which cells with tumor-derived EGFR mutations (L858R, G719S, delE746-A750) were significantly more sensitive to TKI than wild-type EGFR-expressing cells. A clinically relevant secondary mutation (T790M) abolished TKI sensitivity. Significantly, antitumor effects of EGFR Abs, including signaling and growth inhibition and Ab-dependent cellular cytotoxicity, were not affected by any of these mutations. Somatic tumor-associated EGFR kinase mutations, which modulate growth inhibition by TKI, therefore do not impact the activity of therapeutic Abs in vitro.


Assuntos
Anticorpos Antineoplásicos/fisiologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Receptores ErbB/genética , Receptores ErbB/imunologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/imunologia , Mutação , Animais , Anticorpos Antineoplásicos/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/genética , Morte Celular/genética , Morte Celular/imunologia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/fisiologia , Regulação Enzimológica da Expressão Gênica/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Inibidores do Crescimento/fisiologia , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Mutação/imunologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/genética , Transdução de Sinais/imunologia
15.
Cancer Res ; 68(13): 4998-5003, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18593896

RESUMO

Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) have advanced the treatment of colon and head and neck cancer, and show great promise for the development of treatments for other solid cancers. Antibodies against EGFR have been shown to act via inhibition of receptor signaling and induction of antibody-dependent cellular cytoxicity. However, complement-dependent cytotoxicity, which is considered one of the most powerful cell killing mechanisms of antibodies, seems inactive for such antibodies. Here, we show a remarkable synergy for EGFR antibodies. Combinations of antibodies against EGFR were identified, which resulted in potent complement activation via the classic pathway and effective lysis of tumor cells. Studies on a large panel of antibodies indicated that the observed synergy is a general mechanism, which can be activated by combining human IgG1 antibodies recognizing different, nonoverlapping epitopes. Our findings show an unexpected quality of therapeutic EGFR antibodies, which may be exploited to develop novel and more effective treatments for solid cancers.


Assuntos
Anticorpos/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas do Sistema Complemento/fisiologia , Receptores ErbB/imunologia , Neoplasias/tratamento farmacológico , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Células CACO-2 , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Cetuximab , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Mapeamento de Epitopos , Humanos , Imunoglobulina A/administração & dosagem , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Modelos Biológicos , Neoplasias/imunologia , Panitumumabe , Células Tumorais Cultivadas
16.
J Immunol ; 179(5): 2936-43, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17709508

RESUMO

IgA is the most abundantly produced Ab isotype in humans, but its potential as immunotherapeutic reagent has hardly been explored. In this study, we describe anti-tumor mechanisms of mouse/human chimeric IgA Abs against the epidermal growth factor receptor (EGF-R). EGF-R Abs of IgG isotype are currently approved for the treatment of colon or head and neck cancers. As expected, the human IgG1, IgA(1), and IgA(2) variants of the 225 Ab demonstrated similar binding to EGF-R. Furthermore, IgA Abs were as effective as IgG in mediating direct effector mechanisms such as blockade of EGF binding, inhibition of EGF-R phosphorylation, and induction of growth inhibition. None of the three variants induced complement-mediated lysis. Human IgG1 effectively recruited MNC for ADCC, but activated PMN only weakly, whereas both IgA isoforms proved to be effective in triggering neutrophils. Interestingly, the IgA(2) isoform was significantly superior to its IgA(1) counterpart in recruiting PMN as effector cells. Because neutrophils constitute the most abundant effector cell population in human blood, this enhanced neutrophil recruitment lead to increased killing of EGF-R expressing tumor cells in whole blood assays. This killing was further enhanced when blood from G-CSF-primed donors was compared with healthy donor blood. Together, these data suggest EGF-R Abs of human IgA isotype to bear promise for therapeutic use in cancer.


Assuntos
Anticorpos Biespecíficos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Receptores ErbB/imunologia , Imunoglobulina A/imunologia , Neoplasias/imunologia , Animais , Anticorpos Biespecíficos/farmacologia , Humanos , Imunoglobulina A/farmacologia , Imunoglobulina G/imunologia , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia
17.
J Biol Chem ; 281(1): 269-78, 2006 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-16227626

RESUMO

The tumor suppressor protein p53 is not only involved in defending cells against genotoxic insults but is also implicated in differentiation processes, a function that it shares with the CCAAT/enhancer-binding protein beta (C/EBPbeta). We previously reported an up-regulation of both factors in the cycle-dependent differentiation process of human endometrial stromal cells, termed decidualization. C/EBPbeta-mediated activation of a decidualization marker, the decidual prolactin promoter, was antagonized by p53. Here we report that C/EBPbeta in turn represses the transcriptional activity of p53. Competition for limiting amounts of coactivator CREB-binding protein/p300 was ruled out as the underlying mechanism of transrepression. Physical interaction between p53 and C/EBPbeta was demonstrated in vitro and in vivo and shown to depend on the C-terminal domains of both proteins. In gel shift experiments, C/EBPbeta reduced complex formation between p53 and its response element. Conversely, p53 strongly inhibited binding of endogenous C/EBPbeta from endometrial stromal cells to the C/EBP-responsive region in the decidual prolactin promoter. The observed negative cross-talk between p53 and C/EBPbeta is likely to impact expression of their respective target genes.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica/fisiologia , Células Estromais/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Ensaio de Desvio de Mobilidade Eletroforética , Endométrio/citologia , Feminino , Humanos , Osteossarcoma , Elementos de Resposta/fisiologia , Células Estromais/citologia , Fatores de Transcrição de p300-CBP/metabolismo
18.
Mol Hum Reprod ; 9(10): 611-23, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12970399

RESUMO

The promyelocytic leukaemia zinc finger (PLZF) protein belongs to the family of Krüppel-like zinc finger proteins. It is a transcriptional repressor involved in cell cycle control and has been implicated in limb development, differentiation of myeloid cells, and spermatogenesis. Little is known about the regulation of PLZF expression. In search for mediators of progesterone signalling in the female reproductive tract, we discovered induction of PLZF mRNA in primary cultures of human endometrial stromal cells and myometrial smooth muscle cells (SMC) in response to progesterone. Surprisingly, dexamethasone was a more potent inducer of PLZF expression than progesterone and elicited a sustained up-regulation of PLZF mRNA levels within 2 h. Immunofluorescence showed localization of PLZF to the nuclei of dexamethasone-treated SMC. In uterine biopsies, nuclear staining for PLZF was found in myometrial cells and endometrial stromal cells of the secretory phase. The transcriptional start site of the PLZF gene was located to position -5801 in SMC. Transfected promoter constructs containing up to 4.1 kb of 5'-flanking DNA were not induced by activated glucocorticoid or progesterone receptor. In contrast, co-transfection of c-jun and c-fos expression vectors resulted in stimulation of reporter gene activity, indicating an involvement of AP-1 transcription factors in PLZF expression.


Assuntos
Proteínas de Ligação a DNA/genética , Endométrio/citologia , Glucocorticoides/farmacologia , Miócitos de Músculo Liso/metabolismo , Miométrio/citologia , Progesterona/farmacologia , Células Estromais/metabolismo , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacos , Sequência de Bases , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Dexametasona/farmacologia , Feminino , Humanos , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Miócitos de Músculo Liso/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Alinhamento de Sequência , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Tretinoína/farmacologia
19.
J Biol Chem ; 277(23): 20825-32, 2002 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-11893744

RESUMO

Decidual transformation of human endometrial stromal (ES) cells requires sustained activation of the protein kinase A (PKA) pathway. In a search for novel transcriptional mediators of this process, we used differential display PCR analysis of undifferentiated primary ES cells and cells stimulated with 8-bromo-cAMP (8-Br-cAMP). We now report on the role of forkhead homologue in rhabdomyosarcoma (FKHR), a recently described member of the forkhead/winged-helix transcription factor family, as a mediator of endometrial differentiation. Sustained 8-Br-cAMP stimulation resulted in the induction and nuclear accumulation of FKHR in differentiating ES cells. Immunohistochemical studies revealed that endometrial stromal expression of FKHR in vivo is confined to decidualizing cells during the late secretory phase of the cycle and coincides with the expression of CCAAT/enhancer-binding protein beta (C/EBPbeta). Reporter gene studies showed that FKHR potently enhances PKA-dependent activation of the tissue-specific decidual prolactin (dPRL) promoter, a major differentiation marker in human ES cells. Transcriptional augmentation by FKHR was effected through functional cooperation with C/EBPbeta and binding to a composite FKHR-C/EBPbeta response unit in the proximal promoter region. Furthermore, FKHR and C/EBPbeta were shown to interact directly in a glutathione S-transferase pull-down assay. These results provide the first evidence of regulated expression of FKHR and demonstrate that FKHR has an integral role in PKA-dependent endometrial differentiation through its ability to bind and functionally cooperate with C/EBPbeta.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular , AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endométrio/metabolismo , Células Estromais/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Primers do DNA , Proteínas de Ligação a DNA/biossíntese , Endométrio/citologia , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Humanos , Imuno-Histoquímica , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Células Estromais/citologia , Fatores de Transcrição/biossíntese
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