RESUMO
SUMMARY: Reverse-Phase Protein Array (RPPA) is a robust high-throughput, cost-effective platform for quantitatively measuring proteins in biological specimens. However, converting raw RPPA data into normalized, analysis-ready data remains a challenging task. Here, we present the RPPA SPACE (RPPA Superposition Analysis and Concentration Evaluation) R package, a substantially improved successor to SuperCurve, to meet that challenge. SuperCurve has been used to normalize over 170 000 samples to date. RPPA SPACE allows exclusion of poor-quality samples from the normalization process to improve the quality of the remaining samples. It also features a novel quality-control metric, 'noise', that estimates the level of random errors present in each RPPA slide. The noise metric can help to determine the quality and reliability of the data. In addition, RPPA SPACE has simpler input requirements and is more flexible than SuperCurve, it is much faster with greatly improved error reporting. AVAILABILITY AND IMPLEMENTATION: The standalone RPPA SPACE R package, tutorials and sample data are available via https://rppa.space/, CRAN (https://cran.r-project.org/web/packages/RPPASPACE/index.html) and GitHub (https://github.com/MD-Anderson-Bioinformatics/RPPASPACE). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Análise Serial de Proteínas , Proteínas , Reprodutibilidade dos Testes , Controle de Qualidade , SoftwareRESUMO
BACKGROUND: Circulating tumour DNA (ctDNA) is known as a tumour-specific personalised biomarker, but the mutation-selection criteria from heterogeneous tumours remain a challenge. METHODS: We conducted multiregional sequencing of 42 specimens from 14 colorectal tumours of 12 patients, including two double-cancer cases, to identify mutational heterogeneity to develop personalised ctDNA assays using 175 plasma samples. RESULTS: "Founder" mutations, defined as a mutation that is present in all regions of the tumour in a binary manner (i.e., present or absent), were identified in 12/14 tumours. In contrast, "truncal" mutations, which are the first mutation that occurs prior to the divergence of branches in the phylogenetic tree using variant allele frequency (VAF) as continuous variables, were identified in 12/14 tumours. Two tumours without founder and truncal mutations were hypermutators. Most founder and truncal mutations exhibited higher VAFs than "non-founder" and "branch" mutations, resulting in a high chance to be detected in ctDNA. In post-operative long-term observation for 10/12 patients, early relapse prediction, treatment efficacy and non-relapse corroboration were achievable from frequent ctDNA monitoring. CONCLUSIONS: A single biopsy is sufficient to develop custom dPCR probes for monitoring tumour burden in most CRC patients. However, it may not be effective for those with hypermutated tumours.
Assuntos
Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Neoplasias Colorretais/genética , Cirurgia Colorretal/mortalidade , Mutação , Recidiva Local de Neoplasia/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Seguimentos , Humanos , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Prognóstico , Taxa de Sobrevida , Carga TumoralRESUMO
Reverse phase protein array (RPPA) is a functional proteomics technology amenable to moderately high throughputs of samples and antibodies. The University of Texas MD Anderson Cancer Center RPPA Core Facility has implemented various processes and techniques to maximize RPPA throughput; key among them are maximizing array configuration and relying on database management and automation. One major tool used by the RPPA Core is a semi-automated RPPA process management system referred to as the RPPA Pipeline. The RPPA Pipeline, developed with the aid of MD Avnderson's Department of Bioinformatics and Computational Biology and InSilico Solutions, has streamlined sample and antibody tracking as well as advanced quality control measures of various RPPA processes. This chapter covers RPPA Core processes associated with the RPPA Pipeline workflow from sample receipt to sample printing to slide staining and RPPA report generation that enables the RPPA Core to process at least 13,000 samples per year with approximately 450 individual RPPA-quality antibodies. Additionally, this chapter will cover results of large-scale clinical sample processing, including The Cancer Genome Atlas Project and The Cancer Proteome Atlas.
Assuntos
Análise Serial de Proteínas , Proteômica , Estudos Clínicos como Assunto , Humanos , Proteoma , Proteômica/instrumentação , Proteômica/métodos , Proteômica/tendências , Controle de QualidadeRESUMO
MOTIVATION: High-throughput reverse-phase protein array (RPPA) technology allows for the parallel measurement of protein expression levels in approximately 1000 samples. However, the many steps required in the complex protocol (sample lysate preparation, slide printing, hybridization, washing and amplified detection) may create substantial variability in data quality. We are not aware of any other quality control algorithm that is tuned to the special characteristics of RPPAs. RESULTS: We have developed a novel classifier for quality control of RPPA experiments using a generalized linear model and logistic function. The outcome of the classifier, ranging from 0 to 1, is defined as the probability that a slide is of good quality. After training, we tested the classifier using two independent validation datasets. We conclude that the classifier can distinguish RPPA slides of good quality from those of poor quality sufficiently well such that normalization schemes, protein expression patterns and advanced biological analyses will not be drastically impacted by erroneous measurements or systematic variations. AVAILABILITY AND IMPLEMENTATION: The classifier, implemented in the "SuperCurve" R package, can be freely downloaded at http://bioinformatics.mdanderson.org/main/OOMPA:Overview or http://r-forge.r-project.org/projects/supercurve/. The data used to develop and validate the classifier are available at http://bioinformatics.mdanderson.org/MOAR.
Assuntos
Algoritmos , Análise Serial de Proteínas/métodos , Proteômica/métodos , Controle de Qualidade , SoftwareRESUMO
Signaling pathways invoke interplays between forward signaling and feedback to drive robust cellular response. In this study, we address the dynamics of growth factor signaling through profiling of protein phosphorylation and gene expression, demonstrating the presence of a kinetically defined cluster of delayed early genes that function to attenuate the early events of growth factor signaling. Using epidermal growth factor receptor signaling as the major model system and concentrating on regulation of transcription and mRNA stability, we demonstrate that a number of genes within the delayed early gene cluster function as feedback regulators of immediate early genes. Consistent with their role in negative regulation of cell signaling, genes within this cluster are downregulated in diverse tumor types, in correlation with clinical outcome. More generally, our study proposes a mechanistic description of the cellular response to growth factors by defining architectural motifs that underlie the function of signaling networks.
Assuntos
Retroalimentação Fisiológica/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Transdução de Sinais/genética , Fatores de Transcrição/fisiologia , Canais Iônicos Sensíveis a Ácido , Células Cultivadas , Análise por Conglomerados , Canais de Sódio Degenerina , Fator de Crescimento Epidérmico/fisiologia , Canais Epiteliais de Sódio/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Regulação da Expressão Gênica , Células HeLa , Humanos , Fatores de Transcrição Kruppel-Like/fisiologia , Fator de Transcrição MafF/fisiologia , Modelos Biológicos , Proteínas do Tecido Nervoso/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/genética , Tristetraprolina/fisiologiaRESUMO
Primary liver cancer is a heterogeneous disease in terms of its etiology, histology, and therapeutic response. Concurrent proteomic and genomic characterization of a large set of clinical liver cancer samples can help elucidate the molecular basis of heterogeneity and thus serve as a valuable resource for personalized liver cancer treatment. In this study, we perform proteomic profiling of ~300 proteins on 259 primary liver cancer tissues with reverse-phase protein arrays, mutational analysis using whole genome sequencing and transcriptional analysis with RNA-Seq. Patients are of Japanese ethnic background and mainly HBV or HCV positive, providing insight into this important liver cancer subtype. Unsupervised classification of tumors based on protein expression profiles reveal three proteomic subclasses R1, R2, and R3. The R1 subclass is immunologically hot and demonstrated a good prognosis. R2 contains advanced proliferative tumor with TP53 mutations, high expression of VEGF receptor 2 and the worst prognosis. R3 is enriched with CTNNB1 mutations and elevated mTOR signaling pathway activity. Twenty-two proteins, including CDK1 and CDKN2A, are identified as potential prognostic markers. The proteomic classification presented in this study can help guide therapeutic decision making for liver cancer treatment.
Assuntos
Neoplasias Hepáticas , Proteômica , Humanos , Neoplasias Hepáticas/genética , Genômica , Mutação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Circulating tumor DNA (ctDNA) is released from tumor cells into blood in advanced cancer patients. Although gene mutations in individual tumors can be diverse and heterogenous, ctDNA has the potential to provide comprehensive biomarker information. Here, we performed multi-region sampling (three sites) per resected specimen from 10 gastric cancer patients followed by targeted sequencing and proteomic profiling using reverse-phase protein arrays. A total of 126 non-synonymous mutations were identified from 30 samples from 10 tumors. Of these, 16 (12.7%) were present in all three regions and were designated as founder mutations. Variant allele frequencies (VAFs) of founder mutations were significantly higher than those of non-founder mutations. Phylogenetic analysis also demonstrated a good concordance between founder and truncal mutations, defined as mutations shared by all simulated clones at the trunk of the tumor phylogenetic tree. These findings led us to prioritize founder mutations for quantitative ctDNA monitoring by digital PCR with individually-designed primer/probe sets. In preoperative plasma, the average ctDNA VAF of founder mutations was significantly higher than that of non-founder mutations (p = 0.039). Proteomic heterogeneity was present across the tumor regions both within and between patients independent of mutational status. Our results suggest that, in practice, mutations having high VAF identified without multi-regional sequencing may be immediately useful for quantitative ctDNA monitoring but do not provide sufficient information to predict the proteomic composition of tumors.
Assuntos
Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/sangue , Neoplasias Gástricas , Carga Tumoral , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteômica , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
Our clinically relevant finding is that glucocorticoids block estrogen (E2)-induced apoptosis in long-term E2-deprived (LTED) breast cancer cells. However, the mechanism remains unclear. Here, we demonstrated that E2 widely activated adipose inflammatory factors such as fatty acid desaturase 1 (FADS1), IL6, and TNFα in LTED breast cancer cells. Activation of glucocorticoid receptor (GR) by the synthetic glucocorticoid dexamethasone upregulated FADS1 and IL6, but downregulated TNFα expression. Furthermore, dexamethasone was synergistic or additive with E2 in upregulating FADS1 and IL6 expression, whereas it selectively and constantly suppressed TNFα expression induced by E2 in LTED breast cancer cells. Regarding regulation of endoplasmic reticulum stress, dexamethasone effectively blocked activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) by E2, but it had no inhibitory effects on inositol-requiring protein 1 alpha (IRE1α) expression increased by E2 Consistently, results from reverse-phase protein array (RPPA) analysis demonstrated that dexamethasone could not reverse IRE1α-mediated degradation of PI3K/Akt-associated signal pathways activated by E2 Unexpectedly, activated GR preferentially repressed nuclear factor-κB (NF-κB) DNA-binding activity and expression of NF-κB-dependent gene TNFα induced by E2, leading to the blockade of E2-induced apoptosis. Together, these data suggest that trans-suppression of NF-κB by GR in the nucleus is a fundamental mechanism thereby blocking E2-induced apoptosis in LTED breast cancer cells. This study provided an important rationale for restricting the clinical use of glucocorticoids, which will undermine the beneficial effects of E2-induced apoptosis in patients with aromatase inhibitor-resistant breast cancer.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Estrogênios/farmacologia , NF-kappa B/metabolismo , Receptores de Glucocorticoides/metabolismo , Neoplasias da Mama/genética , DNA de Neoplasias/metabolismo , Dessaturase de Ácido Graxo Delta-5 , Dexametasona/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/patologia , Células MCF-7 , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Glycoprotein Nmb (GPNMB) is overexpressed in triple-negative and basal-like breast cancers and its expression is predictive of poor prognosis within this aggressive breast cancer subtype. GPNMB promotes breast cancer growth, invasion, and metastasis; however, its role in mammary tumor initiation remains unknown. To address this question, we overexpressed GPNMB in the mammary epithelium to generate MMTV/GPNMB transgenic mice and crossed these animals to the MMTV/Wnt-1 mouse model, which is known to recapitulate features of human basal breast cancers. We show that GPNMB alone does not display oncogenic properties; however, its expression dramatically accelerates tumor onset in MMTV/Wnt-1 mice. MMTV/Wnt-1 × MMTV/GPNMB bigenic mice also exhibit a significant increase in the growth rate of established primary tumors, which is attributable to increased proliferation and decreased apoptosis. To elucidate molecular mechanisms underpinning the tumor-promoting effects of GPNMB in this context, we interrogated activated pathways in tumors derived from the MMTV/Wnt-1 and MMTV/Wnt-1 × MMTV/GPNMB mice using RPPA analysis. These data revealed that MMTV/Wnt-1 × MMTV/GPNMB bigenic tumors exhibit a pro-growth signature characterized by elevated PI3K/AKT/mTOR signaling and increased ß-catenin activity. Furthermore, we extended these observations to an independent Wnt-1 expressing model of aggressive breast cancer, and confirmed that GPNMB enhances canonical Wnt pathway activation, as evidenced by increased ß-catenin transcriptional activity, in breast cancer cells and tumors co-expressing Wnt-1 and GPNMB. GPNMB-dependent engagement of ß-catenin occurred, in part, through AKT activation. Taken together, these data ascribe a novel, pro-growth role for GPNMB in Wnt-1 expressing basal breast cancers.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/genética , Glicoproteínas de Membrana/fisiologia , Proteína Wnt1/genética , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/genética , Via de Sinalização Wnt/genética , Proteína Wnt1/metabolismo , beta Catenina/metabolismoRESUMO
The current study analyzed reverse phase protein arrays (RPPA) as a means to experimentally validate biomarkers in blood samples. One microliter samples of sera (n = 71), and plasma (n = 78) were serially diluted and printed on NC-coated slides. CA19-9 levels from RPPA results were compared with identical patient samples as measured by ELISA. There was a strong correlation between RPPA and ELISA (r = 0.87) as determined by scatter plots. Sample reproducibility of CA19-9 levels was excellent (interslide correlation r = 0.88; intraslide correlation r = 0.83). The ability of RPPA to accurately distinguish CA19-9 levels between cancer and noncancer samples were determined using receiver operating characteristic curves and compared with ELISA. The AUC for RPPA and ELISA was comparable (0.87 and 0.86, respectively). When the mean CA19-9 levels of normal samples was used as a cutoff for RPPA and compared with the standard clinical ELISA cutoff, comparable specificities (71% for both) were observed. Notably, RPPA samples normalized to albumin showed increased sensitivity compared to ELISA (90% vs. 75%). As RPPA is a high-throughput method that shows results comparable to that of ELISA, we propose that RPPA is a viable technique for rapid experimental screening and validation of candidate biomarkers in blood samples.
Assuntos
Biomarcadores/sangue , Proteínas Sanguíneas/análise , Neoplasias Pancreáticas/sangue , Análise Serial de Proteínas/métodos , Antígeno CA-19-9/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , Plasma/química , Proteômica/métodos , Reprodutibilidade dos Testes , Soro/químicaRESUMO
Telomere 3' overhang-specific DNA oligonucleotides (T-oligos) induce cell death in cancer cells, presumably by mimicking telomere loop disruption. Therefore, T-oligos are considered an exciting new therapeutic strategy. The purpose of this study was to elucidate how T-oligos exert antitumor effects on human malignant glioma cells in vitro and in vivo. We demonstrated that T-oligos inhibited the proliferation of malignant glioma cells through induction of nonapoptotic cell death and mitochondria hyperpolarization, whereas normal astrocytes were resistant to T-oligos. Tumor cells treated with T-oligos developed features compatible with autophagy, with development of autophagic vacuoles and conversion of an autophagy-related protein, microtubule-associated protein 1 light chain 3 from type I (cytoplasmic form) to type II (membrane form of autophagic vacuoles). A reverse-phase protein microarray analysis and Western blotting revealed that treatment with T-oligos inhibited the mammalian target of the rapamycin (mTOR) and the signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment with T-oligos significantly prolonged the survival time of mice inoculated intracranially with malignant glioma cells compared with that of untreated mice and those treated with control oligonucleotides (P=0.0065 and P=0.043, respectively). These results indicate that T-oligos stimulate the induction of nonapoptotic autophagic also known as type II programmed cell death and are thus promising in the treatment of malignant glioma.
Assuntos
Autofagia , Neoplasias Encefálicas/terapia , DNA/farmacologia , Glioma/terapia , Oligonucleotídeos/farmacologia , Telômero/genética , Animais , Apoptose , Astrócitos/metabolismo , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Sobrevivência Celular , Células Cultivadas , Feminino , Citometria de Fluxo , Glioma/genética , Glioma/patologia , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos , Mitocôndrias/metabolismo , Análise Serial de Proteínas , Proteínas Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Sirolimo/farmacologia , Taxa de Sobrevida , Serina-Treonina Quinases TOR , Telomerase/metabolismo , Telômero/metabolismoRESUMO
Head and neck squamous cell carcinoma (HNSCC) is characterized by epidermal growth factor receptor (EGFR) overexpression, where EGFR levels correlate with survival. To date, EGFR targeting has shown limited antitumor effects in head and neck cancer when administrated as monotherapy. We previously identified a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) aurocrine regulatory pathway in HNSCC, where GRP stimulates Src-dependent cleavage of EGFR proligands with subsequent EGFR phosphorylation and mitogen-activated protein kinase (MAPK) activation. To determine whether GRPR targeting can enhance the antitumor efficacy of EGFR inhibition, we investigated the effects of a GRPR antagonist (PD176252) in conjunction with an EGFR tyrosine kinase inhibitor (erlotinib). Combined blockade of GRPR and EGFR pathways significantly inhibited HNSCC, but not immortalized mucosal epithelial cell, proliferation, invasion, and colony formation. In addition, the percentage of apoptotic cells increased upon combined inhibition. The enhanced antitumor efficacy was accompanied by increased expression of cleaved poly(ADP-ribose) polymerase (PARP) and decreased phospho-EGFR, phospho-MAPK, and proliferating cell nuclear antigen (PCNA). Using reverse-phase protein microarray (RPPA), we further detected decreased expression of phospho-c-Jun, phospho-p70S6K, and phospho-p38 with combined targeting. Cumulatively, these results suggest that GRPR targeting can enhance the antitumor effects of EGFR inhibitors in head and neck cancer.
Assuntos
Antineoplásicos/farmacologia , Receptores ErbB/antagonistas & inibidores , Neoplasias de Cabeça e Pescoço/patologia , Indóis/farmacologia , Quinazolinas/farmacologia , Receptores da Bombesina/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Cloridrato de Erlotinib , Fase G1/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Invasividade Neoplásica , Transdução de Sinais/efeitos dos fármacos , Ensaio Tumoral de Célula-TroncoRESUMO
Src is a non-receptor protein tyrosine kinase, the expression and activity of which is increased in >80% of human colon cancers with respect to normal colonic epithelium. Previous studies from this and other laboratories have demonstrated that Src activity contributes to tumorigenicity of established colon adenocarcinoma cell lines. Src participates in the regulation of many signal transduction pathways, among which are those leading to cellular survival. In this study, we addressed the potential role of Src activation to a specific aspect of tumor cell survival, resistance to detachment-induced apoptosis (anoikis). Using five colon tumor cell lines with different biologic properties and genetic alterations, we demonstrate that expression and activity of Src corresponds with resistance to anoikis. Enforced expression of activated Src in subclones of SW480 cells (of low intrinsic Src expression and activity) increases resistance to anoikis; whereas decreased Src expression in HT29 cells (of high Src expression and activity) by transfection with anti-sense Src expression vectors increases susceptibility to anoikis. In contrast, increasing or decreasing Src expression had no effect on susceptibility to staurosporine-induced apoptosis in attached cells. PD173955, a Src family-specific tyrosine kinase inhibitor, increases the susceptibility of HT29 cells to anoikis in a dose- and time-dependent manner. Increasing Src expression and activity led to increased phosphorylation of Akt, a mediator of cellular survival pathways, whereas decreasing Src activity led to decreased Akt phosphorylation. In colon tumor cells with high Src activity, the PI3 kinase inhibitor LY 294002 sensitized cells to anoikis. These results suggest that Src activation may contribute to colon tumor progression and metastasis in part by activating Akt-mediated survival pathways that decrease sensitivity of detached cells to anoikis.
Assuntos
Adenocarcinoma/patologia , Anoikis/fisiologia , Neoplasias do Colo/patologia , Proteínas de Neoplasias/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/fisiologia , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Substituição de Aminoácidos , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Adesão Celular , Ciclo Celular , Cromonas/farmacologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Progressão da Doença , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Genes src , Humanos , Morfolinas/farmacologia , Mutação de Sentido Incorreto , Metástase Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Piridonas/farmacologia , Pirimidinas/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Estaurosporina/farmacologia , Transfecção , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Proteína bcl-XRESUMO
Breast cancer metastasis is directly associated with breast cancer cell motility. Using a cell culture wounding model, we have demonstrated that keratinocyte growth factor (KGF) enhanced the motility of estrogen receptor-positive breast cancer cells. However, the mechanisms by which KGF enhanced motility of breast cancer cells are not known. In the present study, we report that KGF-induced motility requires intact tyrosine kinase signaling since genistein, a tyrosine kinase inhibitor, led to decreased motility of breast cancer cells mediated by KGF. Using cDNA microarrays, we previously found that KGF increased the expression of Grb2 mRNA by 2 3-fold. Since Grb2 plays an important role in tyrosine kinase signaling, we examined the involvement of Grb2 in KGF-induced motility. Down-regulation of Grb2 protein expression inhibited KGF-induced motility. Since Grb2 is known to regulate Erk1,2 and Akt kinase activities we determined whether these downstream proteins may be vital to KGF-induced motility. Inhibiting the activation of Erk1,2 by PD98059 suppressed KGF-induced motility whereas inhibiting the activation of Akt by wortmannin did not affect KGF-induced motility. In conclusion, these results indicate that KGF mediated signal transduction employs Grb2 to transduce the tyrosine kinase signals resulting in the activation of Erk1,2 and breast cancer cell motility.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Lovastatina/análogos & derivados , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Antifúngicos/farmacologia , Neoplasias da Mama/metabolismo , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Fator 7 de Crescimento de Fibroblastos , Proteína Adaptadora GRB2 , Genisteína/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Lovastatina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
We previously demonstrated that HER2/neu prevents all trans-retinoic acid (ATRA) from inducing growth inhibition in MDA-MB-453 breast cancer cells. For ATRA to induce growth inhibition, it needs to bind to retinoic acid receptors and modulate gene transcription via retinoic acid response elements (RAREs). We hypothesized that HER2/neu suppresses RARE binding activity to prevent ATRA from inducing growth arrest in breast cancer cells. Electrophoretic mobility shift assays showed that when HER2/neu was inhibited by the trastuzumab antibody, RARE binding activity increased, indicating that HER2/neu suppresses RARE binding. Since trastuzumab also decreased Akt activity, we determined whether Akt regulates RARE binding activity. Compared to parental MDA-MB-453 cells, MDA-MB-453 cells transfected with a dominant negative Akt mutant (MDA-MB-453/DN-Akt) had higher RARE binding activity. However, trastuzumab did not further increase RARE binding activity in MDA-MB-453/DN-Akt cells. These data indicate that HER2/neu predominantly uses Akt to suppress RARE binding activity, which may be one mechanism by which HER2/neu induces ATRA resistance in breast cancer cells.
Assuntos
Receptor ErbB-2/metabolismo , Tretinoína/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Regulação para Baixo , Genes Dominantes , Humanos , Lipossomos/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Elementos de Resposta , Transdução de Sinais , Transfecção , TrastuzumabRESUMO
We showed that the HER2/Grb2/Akt pathway induces all-trans retinoic acid (ATRA) resistance in breast cancer cells by suppressing the DNA binding activity of retinoic acid receptors (RAR). AP-1 activation was shown to induce ATRA resistance. Here, we determined whether AP-1 binding activity is correlated with ATRA resistance in HER2-overexpressing cells. Inhibition of HER2/Grb2/Akt decreased AP-1 binding activity in HER2-transfected cells, but increased AP-1 activity in cells that are naturally HER2-overexpressing. Since HER2/Grb2/Akt inhibition sensitized both cell types to ATRA, our results indicate that, unlike RAR, AP-1 binding activity is not correlated with ATRA sensitivity in HER2-overexpressing breast cancer cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor ErbB-2/metabolismo , Fator de Transcrição AP-1/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Antineoplásicos/uso terapêutico , Neoplasias da Mama/genética , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Proteína Adaptadora GRB2 , Genes jun/fisiologia , Humanos , Oligonucleotídeos Antissenso/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt , Receptor ErbB-2/antagonistas & inibidores , Receptores do Ácido Retinoico/metabolismo , Fator de Transcrição AP-1/metabolismo , Tretinoína/uso terapêutico , Células Tumorais Cultivadas , Domínios de Homologia de srcRESUMO
BACKGROUND: Germline and somatic mutations in STK11, the gene encoding the serine/threonine kinase LKB1, are strongly associated with tumorigenesis. While loss of LKB1 expression has been linked to breast cancer, the mechanistic role of LKB1 in regulating breast cancer development, metastasis, and tumor metabolism has remained unclear. METHODS: We have generated and analyzed transgenic mice expressing ErbB2 in the mammary epithelium of LKB1 wild-type or LKB1-deficient mice. We have also utilized ErbB2-expressing breast cancer cells in which LKB1 levels have been reduced using shRNA approaches. These transgenic and xenograft models were characterized for the effects of LKB1 loss on tumor initiation, growth, metastasis and tumor cell metabolism. RESULTS: We demonstrate that loss of LKB1 promotes tumor initiation and induces a characteristic shift to aerobic glycolysis ('Warburg effect') in a model of ErbB2-mediated breast cancer. LKB1-deficient breast cancer cells display enhanced early tumor growth coupled with increased cell migratory and invasive properties in vitro. We show that ErbB2-positive tumors deficient for LKB1 display a pro-growth molecular and phenotypic signature characterized by elevated Akt/mTOR signaling, increased glycolytic metabolism, as well as increased bioenergetic markers both in vitro and in vivo. We also demonstrate that mTOR contributes to the metabolic reprogramming of LKB1-deficient breast cancer, and is required to drive glycolytic metabolism in these tumors; however, LKB1-deficient breast cancer cells display reduced metabolic flexibility and increased apoptosis in response to metabolic perturbations. CONCLUSIONS: Together, our data suggest that LKB1 functions as a tumor suppressor in breast cancer. Loss of LKB1 collaborates with activated ErbB2 signaling to drive breast tumorigenesis and pro-growth metabolism in the resulting tumors.
RESUMO
The presence of the Philadelphia chromosome in patients with acute lymphoblastic leukemia (Ph(+)ALL) is a negative prognostic indicator. Tyrosine kinase inhibitors (TKI) that target BCR/ABL, such as imatinib, have improved treatment of Ph(+)ALL and are generally incorporated into induction regimens. This approach has improved clinical responses, but molecular remissions are seen in less than 50% of patients leaving few treatment options in the event of relapse. Thus, identification of additional targets for therapeutic intervention has potential to improve outcomes for Ph+ALL. The human epidermal growth factor receptor 2 (ErbB2) is expressed in ~30% of B-ALLs, and numerous small molecule inhibitors are available to prevent its activation. We analyzed a cohort of 129 ALL patient samples using reverse phase protein array (RPPA) with ErbB2 and phospho-ErbB2 antibodies and found that activity of ErbB2 was elevated in 56% of Ph(+)ALL as compared to just 4.8% of Ph(-)ALL. In two human Ph+ALL cell lines, inhibition of ErbB kinase activity with canertinib resulted in a dose-dependent decrease in the phosphorylation of an ErbB kinase signaling target p70S6-kinase T389 (by 60% in Z119 and 39% in Z181 cells at 3 µM). Downstream, phosphorylation of S6-kinase was also diminished in both cell lines in a dose-dependent manner (by 91% in both cell lines at 3 µM). Canertinib treatment increased expression of the pro-apoptotic protein Bim by as much as 144% in Z119 cells and 49% in Z181 cells, and further produced caspase-3 activation and consequent apoptotic cell death. Both canertinib and the FDA-approved ErbB1/2-directed TKI lapatinib abrogated proliferation and increased sensitivity to BCR/ABL-directed TKIs at clinically relevant doses. Our results suggest that ErbB signaling is an additional molecular target in Ph(+)ALL and encourage the development of clinical strategies combining ErbB and BCR/ABL kinase inhibitors for this subset of ALL patients.
Assuntos
Apoptose/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Cromossomo Filadélfia/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Proteínas de Fusão bcr-abl/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Adulto JovemRESUMO
PURPOSE: To assess the prognostic value of epidermal growth factor receptor (EGFR) molecular characteristics of head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: HNSCC tumors from patients prospectively enrolled in either an Early Detection Research Network (EDRN) study and treated with surgery without an EGFR-targeted agent (N = 154) or enrolled in a chemoradiation trial involving the EGFR-targeted antibody cetuximab (N = 39) were evaluated for EGFR gene amplification by FISH and EGFR protein by immunohistochemical staining. Fresh-frozen tumors (EDRN) were also evaluated for EGFR protein and site-specific phosphorylation at Y992 and Y1068 using reverse-phase protein array (n = 67). Tumor (n = 50) EGFR and EGFRvIII mRNA levels were quantified using real-time PCR. RESULTS: EGFR expression by immunohistochemistry (IHC) was significantly higher in the EDRN tumors with EGFR gene amplification (P < 0.001), and a similar trend was noted in the cetuximab-treated cohort. In the EDRN and cetuximab-treated cohorts elevated EGFR by IHC was associated with reduced survival (P = 0.019 and P = 0.06, respectively). Elevated expression of total EGFR and EGFR PY1068 were independently significantly associated with reduced progression-free survival in the EDRN cohort [HR = 2.75; 95% confidence interval (CI) = 1.26-6.00 and HR = 3.29; 95% CI = 1.34-8.14, respectively]. CONCLUSIONS: In two independent HNSCC cohorts treated with or without cetuximab, tumor EGFR levels were indicative of survival. Tumor EGFR PY1068 levels provided prognostic information independent of total EGFR.