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1.
Front Microbiol ; 14: 1254999, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38029109

RESUMO

As the name of the genus Pantoea ("of all sorts and sources") suggests, this genus includes bacteria with a wide range of provenances, including plants, animals, soils, components of the water cycle, and humans. Some members of the genus are pathogenic to plants, and some are suspected to be opportunistic human pathogens; while others are used as microbial pesticides or show promise in biotechnological applications. During its taxonomic history, the genus and its species have seen many revisions. However, evolutionary and comparative genomics studies have started to provide a solid foundation for a more stable taxonomy. To move further toward this goal, we have built a 2,509-gene core genome tree of 437 public genome sequences representing the currently known diversity of the genus Pantoea. Clades were evaluated for being evolutionarily and ecologically significant by determining bootstrap support, gene content differences, and recent recombination events. These results were then integrated with genome metadata, published literature, descriptions of named species with standing in nomenclature, and circumscriptions of yet-unnamed species clusters, 15 of which we assigned names under the nascent SeqCode. Finally, genome-based circumscriptions and descriptions of each species and each significant genetic lineage within species were uploaded to the LINbase Web server so that newly sequenced genomes of isolates belonging to any of these groups could be precisely and accurately identified.

2.
J Histotechnol ; 43(4): 182-195, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33245266

RESUMO

The ability to reproduce scientific findings is foundational in research; yet, it is compromised in part by poorly characterized reagents, including antibodies. In this report, we describe the application of complementary validation strategies tailored for use in immunohistochemical assays in the characterization of rabbit monoclonal antibodies against YAP and TAZ, homologous and sequentially similar transcriptional effectors of the Hippo signaling pathway. A lack of antibody reagents rigorously validated for immunohistochemistry has limited the Hippo signaling research community's ability to interrogate YAP and TAZ independently in tissue. In a series of normal and diseased human tissues, we were able to demonstrate differential expression patterns of YAP and TAZ, suggesting the potential for functional differences of these proteins. These differences can now be studied in greater detail with these highly validated tools.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anticorpos Monoclonais/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Humanos , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo
3.
Clin Cancer Res ; 18(16): 4449-57, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22661537

RESUMO

PURPOSE: To deepen our understanding of mutant ROS1 expression, localization, and frequency in non-small cell lung cancer (NSCLC), we developed a highly specific and sensitive immunohistochemistry (IHC)-based assay that is useful for the detection of wild-type and mutant ROS1. EXPERIMENTAL DESIGN: We analyzed 556 tumors with the ROS1 D4D6 rabbit monoclonal antibody IHC assay to assess ROS1 expression levels and localization. A subset of tumors was analyzed by FISH to determine the percentage of these tumors harboring ROS1 translocations. Using specific and sensitive IHC assays, we analyzed the expression of anaplastic lymphoma kinase (ALK), EGFR L858R, and EGFR E746-A750del mutations in a subset of lung tumors, including those expressing ROS1. RESULTS: In our NSCLC cohort of Chinese patients, we identified 9 (1.6%) tumors expressing ROS1 and 22 (4.0%) tumors expressing ALK. FISH identified tumors with ALK or ROS1 rearrangements, and IHC alone was capable of detecting all cases with ALK and ROS1 rearrangements. ROS1 fusion partners were determined by reverse transcriptase PCR identifying CD74-ROS1, SLC34A2-ROS1, and FIG-ROS1 fusions. Some of the ALK and ROS1 rearranged tumors may also harbor coexisting EGFR mutations. CONCLUSIONS: NSCLC tumors with ROS1 rearrangements are uncommon in the Chinese population and represent a distinct entity of carcinomas. The ROS1 IHC assay described here is a valuable tool for identifying patients expressing mutant ROS1 and could be routinely applied in clinical practice to detect lung cancers that may be responsive to targeted therapies.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Quinase do Linfoma Anaplásico , Animais , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Transporte/genética , Linhagem Celular , Proliferação de Células , Expressão Gênica , Genes erbB-1 , Genótipo , Proteínas da Matriz do Complexo de Golgi , Humanos , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Camundongos , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transplante Heterólogo
4.
Cancer Res ; 72(13): 3312-23, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22570254

RESUMO

Ovarian cancer is the leading cause of death from gynecologic cancer. Improvement in the clinical outcome of patients is likely to be achieved by the identification of molecular events that underlie the oncogenesis of ovarian cancer. Here we show that the anaplastic lymphoma kinase (ALK) is aberrantly activated in ovarian cancer. Using an unbiased and global phosphoproteomic approach, we profiled 69 Chinese primary ovarian tumor tissues and found ALK to be aberrantly expressed and phosphorylated in 4 tumors. Genetic characterization of these ALK-positive tumors indicated that full-length ALK expression in two serous carcinoma patients is consistent with ALK gene copy number gain, whereas a stromal sarcoma patient carries a novel transmembrane ALK fusion gene: FN1-ALK. Biochemical and functional analysis showed that both full-length ALK and FN1-ALK are oncogenic, and tumors expressing ALK or FN1-ALK are sensitive to ALK kinase inhibitors. Furthermore, immunohistochemical analysis of ovarian tumor tissue microarray detected aberrant ALK expression in 2% to 4% serous carcinoma patients. Our findings provide new insights into the pathogenesis of ovarian cancer and identify ALK as a potential therapeutic target in a subset of serous ovarian carcinoma and stromal sarcoma patients.


Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Receptores Proteína Tirosina Quinases/metabolismo , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Quinase do Linfoma Anaplásico , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida , Primers do DNA , Feminino , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosforilação , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Análise Serial de Tecidos
5.
Clin Cancer Res ; 18(6): 1777-89, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22422409

RESUMO

PURPOSE: We sought to determine whether phosphoinositide 3-kinase (PI3K) pathway mutation or activation state and rapamycin-induced feedback loop activation of Akt is associated with rapamycin sensitivity or resistance. EXPERIMENTAL DESIGN: Cancer cell lines were tested for rapamycin sensitivity, Akt phosphorylation, and mTOR target inhibition. Mice injected with breast or neuroendocrine cancer cells and patients with neuroendocrine tumor (NET) were treated with rapalogs and Akt phosphorylation was assessed. RESULTS: Thirty-one cell lines were rapamycin sensitive (RS) and 12 were relatively rapamycin resistant (RR; IC(50) > 100 nmol/L). Cells with PIK3CA and/or PTEN mutations were more likely to be RS (P = 0.0123). Akt phosphorylation (S473 and T308) was significantly higher in RS cells (P < 0.0001). Rapamycin led to a significantly greater pathway inhibition and greater increase in p-Akt T308 (P < 0.0001) and p-Akt S473 (P = 0.0009) in RS cells. Rapamycin and everolimus significantly increased Akt phosphorylation but inhibited growth in an in vivo NET model (BON). In patients with NETs treated with everolimus and octreotide, progression-free survival correlated with p-Akt T308 in pretreatment (R = 0.4762, P = 0.0533) and on-treatment tumor biopsies (R = 0.6041, P = 0.0102). Patients who had a documented partial response were more likely to have an increase in p-Akt T308 with treatment compared with nonresponders (P = 0.0146). CONCLUSION: PIK3CA/PTEN genomic aberrations and high p-Akt levels are associated with rapamycin sensitivity in vitro. Rapamycin-mediated Akt activation is greater in RS cells, with a similar observation in patients with clinical responses on exploratory biomarker analysis; thus feedback loop activation of Akt is not a marker of resistance but rather may function as an indicator of rapamycin activity.


Assuntos
Neoplasias da Mama/genética , Carcinoma Neuroendócrino/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mutação , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/genética , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/análise , Animais , Antibióticos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Everolimo , Feminino , Humanos , Camundongos , Camundongos Nus , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sirolimo/análogos & derivados , Transplante Heterólogo
6.
Sci Transl Med ; 4(120): 120ra17, 2012 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-22277784

RESUMO

Most anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancers (NSCLCs) are highly responsive to treatment with ALK tyrosine kinase inhibitors (TKIs). However, patients with these cancers invariably relapse, typically within 1 year, because of the development of drug resistance. Herein, we report findings from a series of lung cancer patients (n = 18) with acquired resistance to the ALK TKI crizotinib. In about one-fourth of patients, we identified a diverse array of secondary mutations distributed throughout the ALK TK domain, including new resistance mutations located in the solvent-exposed region of the adenosine triphosphate-binding pocket, as well as amplification of the ALK fusion gene. Next-generation ALK inhibitors, developed to overcome crizotinib resistance, had differing potencies against specific resistance mutations. In addition to secondary ALK mutations and ALK gene amplification, we also identified aberrant activation of other kinases including marked amplification of KIT and increased autophosphorylation of epidermal growth factor receptor in drug-resistant tumors from patients. In a subset of patients, we found evidence of multiple resistance mechanisms developing simultaneously. These results highlight the unique features of TKI resistance in ALK-positive NSCLCs and provide the rationale for pursuing combinatorial therapeutics that are tailored to the precise resistance mechanisms identified in patients who relapse on crizotinib treatment.


Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Linhagem Celular Tumoral , Crizotinibe , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/enzimologia , Mutação , Reação em Cadeia da Polimerase
7.
PLoS One ; 6(1): e15640, 2011 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-21253578

RESUMO

Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23) of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.


Assuntos
Neoplasias dos Ductos Biliares/enzimologia , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/enzimologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Humanos , Imunoensaio , Camundongos , Camundongos Nus , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fosforilação , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo
8.
Cancer Cell ; 17(6): 547-59, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20541700

RESUMO

In mice, Lkb1 deletion and activation of Kras(G12D) results in lung tumors with a high penetrance of lymph node and distant metastases. We analyzed these primary and metastatic de novo lung cancers with integrated genomic and proteomic profiles, and have identified gene and phosphoprotein signatures associated with Lkb1 loss and progression to invasive and metastatic lung tumors. These studies revealed that SRC is activated in Lkb1-deficient primary and metastatic lung tumors, and that the combined inhibition of SRC, PI3K, and MEK1/2 resulted in synergistic tumor regression. These studies demonstrate that integrated genomic and proteomic analyses can be used to identify signaling pathways that may be targeted for treatment.


Assuntos
Genômica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Metástase Neoplásica/tratamento farmacológico , Proteínas Serina-Treonina Quinases/deficiência , Proteômica , Transdução de Sinais/efeitos dos fármacos , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Transdiferenciação Celular/genética , Quimioterapia Combinada , Inibidores Enzimáticos/uso terapêutico , Feminino , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/genética , Adesões Focais/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Camundongos , Camundongos Mutantes , Camundongos Nus , Metástase Neoplásica/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , Transdução de Sinais/genética , Serina-Treonina Quinases TOR , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismo
9.
Anticancer Res ; 30(3): 857-65, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20393007

RESUMO

Prostate cancer is the second leading cause of cancer-related deaths in men in Western society. Epidemiological studies suggest that a reduced risk of cancer is associated with the consumption of a phytochemical-rich diet that includes fruits and vegetables. Strategies to delay clinically significant prostate cancer will have a tremendous impact in reducing the overall incidence of prostate cancer as well as improving quality of life for elderly men. Furthermore, the long latency involved in the development of clinically significant prostate cancer provides a plethora of opportunities for its management, especially using prevention approaches. Previous studies from our laboratory show that Nexrutine (bark extract from Phellodendron amurense) prevents prostate tumor development when given prior to the development of high-grade prostatic intraepithelial neoplasia in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. In this study, we investigated the effect on the progression of established tumors in the TRAMP model by administering Nexrutine to 28-week-old TRAMP mice. Efficacy of Nexrutine was determined by histopathological evaluation of the prostate. Our data indicate that Nexrutine inhibited progression of prostate tumors that was correlated with tissue levels of transcription factors nuclear factor kappa B, cyclic-AMP response element-binding protein and phosphorylated CREB. Moreover, Nexrutine intervention resulted in a significant increase in the bone mineral density of the left femur diaphysis (p=0.009) and prevented the development of metastatic lesions. Nexrutine treatment also significantly (p=0.005) inhibited invasion of androgen-independent prostate cancer cells.


Assuntos
Adenocarcinoma/tratamento farmacológico , Extratos Vegetais/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Apoptose/efeitos dos fármacos , Reabsorção Óssea/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Progressão da Doença , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 3 da Matriz/biossíntese , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Metástase Neoplásica , Phellodendron/química , Casca de Planta/química , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
10.
Cancer Res ; 70(6): 2485-94, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20215504

RESUMO

ErbB3 is a critical activator of phosphoinositide 3-kinase (PI3K) signaling in epidermal growth factor receptor (EGFR; ErbB1), ErbB2 [human epidermal growth factor receptor 2 (HER2)], and [hepatocyte growth factor receptor (MET)] addicted cancers, and reactivation of ErbB3 is a prominent method for cancers to become resistant to ErbB inhibitors. In this study, we evaluated the in vivo efficacy of a therapeutic anti-ErbB3 antibody, MM-121. We found that MM-121 effectively blocked ligand-dependent activation of ErbB3 induced by either EGFR, HER2, or MET. Assessment of several cancer cell lines revealed that MM-121 reduced basal ErbB3 phosphorylation most effectively in cancers possessing ligand-dependent activation of ErbB3. In those cancers, MM-121 treatment led to decreased ErbB3 phosphorylation and, in some instances, decreased ErbB3 expression. The efficacy of single-agent MM-121 was also examined in xenograft models. A machine learning algorithm found that MM-121 was most effective against xenografts with evidence of ligand-dependent activation of ErbB3. We subsequently investigated whether MM-121 treatment could abrogate resistance to anti-EGFR therapies by preventing reactivation of ErbB3. We observed that an EGFR mutant lung cancer cell line (HCC827), made resistant to gefitinib by exogenous heregulin, was resensitized by MM-121. In addition, we found that a de novo lung cancer mouse model induced by EGFR T790M-L858R rapidly became resistant to cetuximab. Resistance was associated with an increase in heregulin expression and ErbB3 activation. However, concomitant cetuximab treatment with MM-121 blocked reactivation of ErbB3 and resulted in a sustained and durable response. Thus, these results suggest that targeting ErbB3 with MM-121 can be an effective therapeutic strategy for cancers with ligand-dependent activation of ErbB3.


Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias/terapia , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Células CHO , Linhagem Celular Tumoral , Cetuximab , Cricetinae , Cricetulus , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Humanos , Ligantes , Camundongos , Neoplasias/metabolismo , Neuregulina-1/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Am J Surg Pathol ; 33(7): 984-91, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19363441

RESUMO

NUT midline carcinoma (NMC) is a uniformly lethal malignancy that is defined by rearrangement of the nuclear protein in testis (NUT) gene on chromosome 15q14. NMCs are morphologically indistinguishable from other poorly differentiated carcinomas, and the diagnosis is usually made currently by fluorescence in situ hybridization (FISH). As normal NUT expression is confined to testis and ovary, we reasoned that an immunohistochemical (IHC) stain for NUT would be useful in diagnosing NMC. To this end, we raised a highly specific rabbit monoclonal antibody, C52, against a recombinant NUT polypeptide, and developed an IHC staining protocol. The sensitivity and specificity of C52 staining was evaluated in a panel of 1068 tissues, predominantly diverse types of carcinomas (n=906), including 30 NMCs. Split-apart FISH for NUT rearrangement was used as a "gold standard" diagnostic test for NMC. C52 immunoreactivity among carcinomas was confined to NMCs. IHC staining had a sensitivity of 87%, a specificity of 100%, a negative predictive value of 99%, and a positive predictive value of 100%. Two new cases of NMC containing BRD4-NUT fusions were detected by C52 IHC, but missed by conventional FISH. In both instances, these tumors contained cryptic BRD4-NUT rearrangements, as confirmed by FISH using a refined set of probes. Some germ cell tumors, including 64% of dysgerminomas, showed weak NUT immunoreactivity, consistent with the expression of NUT in normal germ cells. We conclude that IHC staining with the C52 monoclonal antibody is a highly sensitive and specific test that reliably distinguishes NMC from other forms of carcinoma. The NUT antibody is being prepared for commercial release and will be available in the near future.


Assuntos
Anticorpos Monoclonais , Carcinoma/diagnóstico , Animais , Especificidade de Anticorpos , Carcinoma/genética , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Proteínas de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Proteínas de Fusão Oncogênica , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
12.
Clin Cancer Res ; 15(9): 3023-8, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19366827

RESUMO

PURPOSE: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non-small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry. EXPERIMENTAL DESIGN: We tested mutation-specific antibodies by Western blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing. RESULTS: These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry-based DNA sequencing. CONCLUSIONS: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.


Assuntos
Anticorpos Monoclonais , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutação/imunologia , Animais , Bioensaio , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/secundário , Análise Mutacional de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Coelhos , Sensibilidade e Especificidade , Deleção de Sequência , Transplante Heterólogo , Células Tumorais Cultivadas
13.
Cancer Cell ; 15(2): 148-59, 2009 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-19185849

RESUMO

mTOR complex 2 (mTORC2) contains the mammalian target of rapamycin (mTOR) kinase and the Rictor regulatory protein and phosphorylates Akt. Whether this function of mTORC2 is critical for cancer progression is unknown. Here, we show that transformed human prostate epithelial cells lacking PTEN require mTORC2 to form tumors when injected into nude mice. Furthermore, we find that Rictor is a haploinsufficient gene and that deleting one copy protects Pten heterozygous mice from prostate cancer. Finally, we show that the development of prostate cancer caused by Pten deletion specifically in prostate epithelium requires mTORC2, but that for normal prostate epithelial cells, mTORC2 activity is nonessential. The selective requirement for mTORC2 in tumor development suggests that mTORC2 inhibitors may be of substantial clinical utility.


Assuntos
Proteínas de Transporte/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Neoplasias da Próstata/fisiopatologia , Proteínas Quinases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Transporte/genética , Transformação Celular Neoplásica , Células Cultivadas , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , PTEN Fosfo-Hidrolase/genética , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Próstata/citologia , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/patologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Interferência de RNA , Proteína Companheira de mTOR Insensível à Rapamicina , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR , Fatores de Transcrição/genética , Transplante Heterólogo
14.
Nat Med ; 14(12): 1351-6, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19029981

RESUMO

Somatic mutations that activate phosphoinositide 3-kinase (PI3K) have been identified in the p110-alpha catalytic subunit (encoded by PIK3CA). They are most frequently observed in two hotspots: the helical domain (E545K and E542K) and the kinase domain (H1047R). Although the p110-alpha mutants are transforming in vitro, their oncogenic potential has not been assessed in genetically engineered mouse models. Furthermore, clinical trials with PI3K inhibitors have recently been initiated, and it is unknown if their efficacy will be restricted to specific, genetically defined malignancies. In this study, we engineered a mouse model of lung adenocarcinomas initiated and maintained by expression of p110-alpha H1047R. Treatment of these tumors with NVP-BEZ235, a dual pan-PI3K and mammalian target of rapamycin (mTOR) inhibitor in clinical development, led to marked tumor regression as shown by positron emission tomography-computed tomography, magnetic resonance imaging and microscopic examination. In contrast, mouse lung cancers driven by mutant Kras did not substantially respond to single-agent NVP-BEZ235. However, when NVP-BEZ235 was combined with a mitogen-activated protein kinase kinase (MEK) inhibitor, ARRY-142886, there was marked synergy in shrinking these Kras-mutant cancers. These in vivo studies suggest that inhibitors of the PI3K-mTOR pathway may be active in cancers with PIK3CA mutations and, when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers.


Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Classe I de Fosfatidilinositol 3-Quinases , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética
15.
RNA ; 14(7): 1318-27, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18515545

RESUMO

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/análise , Proteínas de Ciclo Celular , Linhagem Celular , Núcleo Celular/química , Células Cultivadas , Embrião de Mamíferos/citologia , Fator de Iniciação 4E em Eucariotos/análise , Fatores de Iniciação em Eucariotos , Fibroblastos/química , Fibroblastos/citologia , Camundongos , Fosfoproteínas/análise , Fosforilação , RNA Mensageiro/metabolismo
16.
Neoplasia ; 9(11): 893-9, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18030357

RESUMO

We recently showed that Nexrutine, a Phellodendron amurense bark extract, suppresses proliferation of prostate cancer cell lines and tumor development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Our data also indicate that the anti-proliferative effects of Nexrutine are emediated in part by Akt and Cyclic AMP response element binding protein (CREB). Cyclooxygenase (Cox-2), a pro-inflammatory mediator, is a CREB target that induces prostaglandin E(2) (PGE(2)) and suppresses apoptosis. Treatment of LNCaP cells with Nexrutine reduced tumor necrosis factor alpha-induced enzymatic as well as promoter activities of Cox-2. Nexrutine also reduced the expression and promoter activity of Cox-2 in PC-3 cells that express high constitutive levels of Cox-2. Deletion analysis coupled with mutational analysis of the Cox-2 promoter identified CRE as being sufficient for mediating Nexrutine response. Immunohistochemical analysis of human prostate tumors show increased expression of CREB and DNA binding activity in high-grade tumors (three-fold higher in human prostate tumors compared to normal prostate; P = .01). We have identified CREB-mediated activation of Cox-2 as a potential signaling pathway in prostate cancer which can be blocked with a nontoxic, cost-effective dietary supplement like Nexrutine, demonstrating a prospective for development of Nexrutine for prostate cancer management.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Ciclo-Oxigenase 2/metabolismo , Extratos Vegetais/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/análise , Ciclo-Oxigenase 2/genética , DNA/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia
17.
Clin Cancer Res ; 13(9): 2784-94, 2007 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17473212

RESUMO

PURPOSE: Development of prostate cancer prevention strategies is an important priority to overcome high incidence, morbidity, and mortality. Recently, we showed that Nexrutine, an herbal extract, inhibits prostate cancer cell proliferation through modulation of Akt and cAMP-responsive element binding protein (CREB)-mediated signaling pathways. However, it is unknown if Nexrutine can be developed as a dietary supplement for the prevention of prostate cancer. In this study, we used the transgenic adenocarcinoma of mouse prostate (TRAMP) model to examine the ability of Nexrutine to protect TRAMP mice from developing prostate cancer. EXPERIMENTAL DESIGN: Eight-week-old TRAMP mice were fed with pelleted diet containing 300 and 600 mg/kg Nexrutine for 20 weeks. Efficacy of Nexrutine was evaluated by magnetic resonance imaging at 18 and 28 weeks of progression and histologic analysis of prostate tumor or tissue at the termination of the experiment. Tumor tissue was analyzed for modulation of various signaling molecules. RESULTS: We show that Nexrutine significantly suppressed palpable tumors and progression of cancer in the TRAMP model. Expression of total and phosphorylated Akt, CREB, and cyclin D1 was significantly reduced in prostate tissue from Nexrutine intervention group compared with tumors from control animals. Nexrutine also inhibited cyclin D1 transcriptional activity in androgen-independent PC-3 cells. Overexpression of kinase dead Akt mutant or phosphorylation-defective CREB inhibited cyclin D1 transcriptional activity. CONCLUSIONS: The current study shows that Nexrutine-mediated targeting of Akt/CREB-induced activation of cyclin D1 prevents the progression of prostate cancer. Expression of CREB and phosphorylated CREB increased in human prostate tumors compared with normal tissue, suggesting their potential use as prognostic markers.


Assuntos
Adenocarcinoma/tratamento farmacológico , Suplementos Nutricionais , Extratos Vegetais/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Adenocarcinoma/patologia , Adenocarcinoma/prevenção & controle , Animais , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/análise , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ciclina D1/análise , Ciclina D1/antagonistas & inibidores , Ciclina D1/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação , Neoplasias da Próstata/patologia , Neoplasias da Próstata/prevenção & controle , Proteínas Proto-Oncogênicas c-akt/análise , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética
18.
J Clin Oncol ; 23(5): 982-9, 2005 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-15572730

RESUMO

PURPOSE: Activation of the platelet-derived growth factor (PDGF) and c-kit receptors has been proposed as important in mediating the growth of AIDS-related Kaposi's sarcoma (KS). We investigated the response of KS to the PDGF receptor (PDGFR)/c-kit inhibitor, imatinib mesylate, and investigated the effect of this therapy on critical signal transduction intermediates. PATIENTS AND METHODS: Ten male patients with AIDS-related cutaneous KS, which progressed despite chemotherapy and/or highly active antiretroviral therapy, received imatinib mesylate administered orally, 300 mg twice daily. Clinical response was determined by serial tumor measurements. To determine biologic and histologic response, skin lesion biopsies were obtained at baseline and following 4 weeks of therapy. RESULTS: Five of 10 participants had a partial response by tumor measurements. Biopsies after 4 weeks of therapy demonstrated histologic regression in four of six patients. Four patients' tumor biopsies were assessable for immunohistochemistry end points pre- and post-therapy. These demonstrated inhibition of PDGFR and its downstream effector, extracellular receptor kinase, which is a member of the mitogen-activated protein kinase family. The most common adverse event was diarrhea, which led to dose reduction in six patients. CONCLUSION: Imatinib mesylate administered orally twice daily for AIDS-related KS results in clinical and histologic regression of cutaneous KS lesions within 4 weeks. These promising results demonstrate that inhibition of the c-kit and/or PDGF receptors may represent an effective strategy for treating KS.


Assuntos
Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Antineoplásicos/uso terapêutico , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/uso terapêutico , Sarcoma de Kaposi/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Administração Oral , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Benzamidas , Biópsia , Diarreia/induzido quimicamente , Seguimentos , Humanos , Mesilato de Imatinib , Masculino , Projetos Piloto , Piperazinas/administração & dosagem , Piperazinas/efeitos adversos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Proteínas Proto-Oncogênicas c-kit/efeitos dos fármacos , Pirimidinas/administração & dosagem , Pirimidinas/efeitos adversos , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Indução de Remissão , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/sangue
19.
Clin Cancer Res ; 10(24): 8351-6, 2004 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-15623612

RESUMO

PURPOSE: As kinase inhibitors transition from the laboratory to patients, it is imperative to develop biomarkers that can be used in the clinic. The primary objectives are to identify patients most likely to benefit from molecularly targeted therapies and to document modulation of the drug target. Constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway and its downstream effectors, as a result of PTEN loss or by other mechanisms, occurs in a high proportion of prostate cancers, making it an ideal template for the design of clinical trials involving PI3K pathway inhibitors. Prostate cancers also present unique organ-specific challenges, in that tumors are heterogeneous and diagnostic tissue is extremely limited. EXPERIMENTAL DESIGN: Working within these limitations, we have developed a set of immunohistochemical assays that define activation of the PI3K pathway in clinical samples. RESULTS AND CONCLUSIONS: Using both univariate and multivariate analyses, we show that loss of PTEN is highly correlated with the activation of AKT, and this, in turn, is associated with the phosphorylation of S6, one of its main effectors. These three antibodies are potentially able to define a molecular signature of PTEN loss and/or AKT pathway activation in prostate cancer.


Assuntos
Anticorpos Monoclonais , Regulação Neoplásica da Expressão Gênica , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/enzimologia , Transdução de Sinais , Regulação para Baixo , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Próstata/enzimologia , Próstata/imunologia , Próstata/patologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo
20.
Transplantation ; 77(8): 1288-94, 2004 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-15114100

RESUMO

BACKGROUND: Human CD58 is an adhesion molecule that interacts with CD2 on lymphocytes. We describe here an antibody that blocks responses of human peripheral blood mononuclear cells (PBMCs) to porcine cells and reacts with a porcine protein with homology to CD58. METHODS: Antibodies were isolated with a screen for inhibition of the human antiporcine response. One of these antibodies was used for immunoaffinity purification of a protein that was identified by molecular weight determination, endoglycosidase sensitivity, and microsequencing analysis as a porcine homologue of CD58. RESULTS: The antigen recognized by this antibody was a cell surface protein of relative molecular mass (Mr)=45,000 containing N-linked carbohydrate chains. Immunoaffinity purification of this protein and microsequencing revealed homology to sheep CD58 as well as sequences that were common to this protein and both sheep and human CD58. The protein was widely distributed on porcine cells, including lymphocytes, endothelial cells, muscle cells, and neuronal cells. This antibody efficiently inhibited lysis of porcine targets by human PBMCs in addition to preventing proliferation of the human PBMCs in response to the porcine cells. CONCLUSIONS: The CD2 interaction with porcine cells is important for the efficient recognition of porcine tissue, and inhibition of the human antiporcine immune response with the antibody is likely to be caused by the disruption of the human CD2 interaction with this porcine homologue of CD58. The antibody may prove to be useful for the blocking of this interaction without interfering with other functions of T cells.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD58/isolamento & purificação , Imunossupressores/farmacologia , Sus scrofa/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD58/química , Antígenos CD58/genética , Cromatografia de Afinidade , Testes Imunológicos de Citotoxicidade , Humanos , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Ovinos , Especificidade da Espécie , Sus scrofa/genética , Linfócitos T/imunologia , Distribuição Tecidual
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