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1.
Nucleic Acids Res ; 48(8): 4013-4027, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31989173

RESUMO

Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC-MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Exossomos/metabolismo , Neoplasias da Próstata/química , Aptâmeros de Nucleotídeos , Linhagem Celular Tumoral , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Masculino , Neoplasias da Próstata/classificação , Neoplasias da Próstata/metabolismo , Técnica de Seleção de Aptâmeros , Proteína 1 de Ligação a Y-Box/metabolismo
2.
Cell Chem Biol ; 26(5): 756-764.e6, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30930163

RESUMO

Spliceosomal dysregulation dramatically affects many cellular processes, notably signal transduction, metabolism, and proliferation, and has led to the concept of targeting intracellular spliceosomal proteins to combat cancer. Here we show that a subset of lymphoma cells displays a spliceosomal complex on their surface, which we term surface spliceosomal complex (SSC). The SSC consists of at least 13 core components and was discovered as the binding target of the non-Hodgkin's lymphoma-specific aptamer C10.36. The aptamer triggers SSC internalization, causing global changes in alternative splicing patterns that eventually lead to necrotic cell death. Our study reveals an exceptional spatial arrangement of a spliceosomal complex and defines it not only as a potential target of anti-cancer drugs, but also suggests that its localization plays a fundamental role in cell survival.


Assuntos
Processamento Alternativo , Spliceossomos/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Humanos , Linfoma/metabolismo , Linfoma/patologia , Espectrometria de Massas em Tandem
3.
Nat Commun ; 9(1): 1219, 2018 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-29572535

RESUMO

Assessing the phenotypic diversity underlying tumour progression requires the identification of variations in the respective molecular interaction networks. Here we report proof-of-concept for a platform called poly-ligand profiling (PLP) that surveys these system states and distinguishes breast cancer patients who did or did not derive benefit from trastuzumab. We perform tissue-SELEX on breast cancer specimens to enrich single-stranded DNA (ssDNA) libraries that preferentially interact with molecular components associated with the two clinical phenotypes. Testing of independent sample sets verifies the ability of PLP to classify trastuzumab-treated patients according to their clinical outcomes with ROC-AUC of 0.78. Standard HER2 testing of the same patients gives a ROC-AUC of 0.47. Kaplan-Meier analysis reveals a median increase in benefit from trastuzumab-containing treatments of 300 days for PLP-positive compared to PLP-negative patients. If prospectively validated, PLP may increase success rates in precision oncology and clinical trials, thus improving both patient care and drug development.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Trastuzumab/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Área Sob a Curva , Biomarcadores Tumorais/análise , Neoplasias da Mama/genética , DNA de Cadeia Simples/análise , Progressão da Doença , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Ligantes , Pessoa de Meia-Idade , Fenótipo , Medicina de Precisão , Técnica de Seleção de Aptâmeros , Análise de Sequência de DNA , Resultado do Tratamento
4.
Sci Rep ; 7: 42741, 2017 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-28218293

RESUMO

Technologies capable of characterizing the full breadth of cellular systems need to be able to measure millions of proteins, isoforms, and complexes simultaneously. We describe an approach that fulfils this criterion: Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT). ADAPT employs an enriched library of single-stranded oligodeoxynucleotides (ssODNs) to profile complex biological samples, thus achieving an unprecedented coverage of system-wide, native biomolecules. We used ADAPT as a highly specific profiling tool that distinguishes women with or without breast cancer based on circulating exosomes in their blood. To develop ADAPT, we enriched a library of ~1011 ssODNs for those associating with exosomes from breast cancer patients or controls. The resulting 106 enriched ssODNs were then profiled against plasma from independent groups of healthy and breast cancer-positive women. ssODN-mediated affinity purification and mass spectrometry identified low-abundance exosome-associated proteins and protein complexes, some with known significance in both normal homeostasis and disease. Sequencing of the recovered ssODNs provided quantitative measures that were used to build highly accurate multi-analyte signatures for patient classification. Probing plasma from 500 subjects with a smaller subset of 2000 resynthesized ssODNs stratified healthy, breast biopsy-negative, and -positive women. An AUC of 0.73 was obtained when comparing healthy donors with biopsy-positive patients.


Assuntos
Neoplasias da Mama/sangue , Exossomos/genética , Oligodesoxirribonucleotídeos/metabolismo , Biologia de Sistemas/métodos , Área Sob a Curva , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnica de Seleção de Aptâmeros , Análise de Sequência de DNA
5.
Mol Cancer Ther ; 11(2): 503-13, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22161861

RESUMO

A growing body of data indicates that inhibiting the type 1 insulin-like growth factor receptor (IGF-1R) might be an effective treatment strategy for hepatocellular carcinoma (HCC). OSI-906 is a dual IGF-1R/IR kinase inhibitor currently in phase II clinical development for HCC. However, biomarkers are lacking to help identify patients with HCC who are more likely to benefit from OSI-906 treatment. We sought to determine the effect of OSI-906 on proliferation against a panel of 21 HCC cell lines and to investigate molecular determinants of responsiveness to OSI-906. We identified a subset of HCC cell lines that was sensitive to OSI-906, and sensitivity is associated with elevated phosphorylation levels of IGF-1R and IR and greater inhibition of AKT signaling. Dual targeting of both receptors seems to be important for maximal inhibition as treatment with a selective IGF-1R-neutralizing antibody was associated with increased IR signaling, whereas OSI-906 fully inhibited both phosphorylated IR and IGF-1R and resulted in greater inhibition of the IRS/AKT pathway. Epithelial-mesenchymal transition (EMT) seems to predict HCC cell sensitivity to OSI-906, as the epithelial phenotype is strongly associated with expression of IGF-2 and IR, activation of IGF-1R and IR, and sensitivity to OSI-906, alone or in combination with erlotinib. Induction of EMT upon treatment with TGFß reduced sensitivity to OSI-906. Collectively, these data support the concept for dual IGF-1R/IR targeting in HCC, where EMT status and expressions of IGF-2 and IR may be used to identify those patients who are most likely to benefit from treatment with an IGF-1R/IR dual inhibitor.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Imidazóis/farmacologia , Pirazinas/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/genética , Cloridrato de Erlotinib , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/farmacologia , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos
6.
Cancer Res ; 71(5): 1573-83, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21363918

RESUMO

The mammalian target of rapamycin (mTOR) pathway is implicated widely in cancer pathophysiology. Dual inhibition of the mTOR kinase complexes mTORC1 and mTORC2 decreases tumor xenograft growth in vivo and VEGF secretion in vitro, but the relationship between these two effects are unclear. In this study, we examined the effects of mTORC1/2 dual inhibition on VEGF production, tumor angiogenesis, vascular regression, and vascular regrowth, and we compared the effects of dual inhibition to mTORC1 inhibition alone. ATP-competitive inhibitors OSI-027 and OXA-01 targeted both mTORC1 and mTORC2 signaling in vitro and in vivo, unlike rapamycin that only inhibited mTORC1 signaling. OXA-01 reduced VEGF production in tumors in a manner associated with decreased vessel sprouting but little vascular regression. In contrast, rapamycin exerted less effect on tumoral production of VEGF. Treatment with the selective VEGFR inhibitor OSI-930 reduced vessel sprouting and caused substantial vascular regression in tumors. However, following discontinuation of OSI-930 administration tumor regrowth could be slowed by OXA-01 treatment. Combining dual inhibitors of mTORC1 and mTORC2 with a VEGFR2 inhibitor decreased tumor growth more than either inhibitor alone. Together, these results indicate that dual inhibition of mTORC1/2 exerts antiangiogenic and antitumoral effects that are even more efficacious when combined with a VEGFR antagonist.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Proteínas/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Nus , Microscopia Confocal , Complexos Multiproteicos , Neoplasias Experimentais/metabolismo , Neovascularização Patológica/metabolismo , Quinolinas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Tiofenos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Mol Cancer Ther ; 9(10): 2652-64, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20924128

RESUMO

Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase-AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti-IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti-IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone.


Assuntos
Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Imidazóis/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Fosforilação , Reação em Cadeia da Polimerase , Pirazinas/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Transdução de Sinais
8.
Cancer Res ; 68(20): 8322-32, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18922904

RESUMO

Epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR) can cooperate to regulate tumor growth and survival, and synergistic growth inhibition has been reported for combined blockade of EGFR and IGF-IR. However, in preclinical models, only a subset of tumors exhibit high sensitivity to this combination, highlighting the potential need for patient selection to optimize clinical efficacy. Herein, we have characterized the molecular basis for cooperative growth inhibition upon dual EGFR and IGF-IR blockade and provide biomarkers that seem to differentiate response. We find for epithelial, but not for mesenchymal-like, tumor cells that Akt is controlled cooperatively by EGFR and IGF-IR. This correlates with synergistic apoptosis and growth inhibition in vitro and growth regression in vivo upon combined blockade of both receptors. We identified two molecular aspects contributing to synergy: (a) inhibition of EGFR or IGF-IR individually promotes activation of the reciprocal receptor; (b) inhibition of EGFR-directed mitogen-activated protein kinase (MAPK) shifts regulation of Akt from EGFR toward IGF-IR. Targeting the MAPK pathway through downstream MAPK/extracellular signal-regulated kinase kinase (MEK) antagonism similarly promoted IGF-driven pAkt and synergism with IGF-IR inhibition. Mechanistically, we find that inhibition of the MAPK pathway circumvents a negative feedback loop imposed on the IGF-IR- insulin receptor substrate 1 (IRS-1) signaling complex, a molecular scenario that parallels the negative feedback loop between mTOR-p70S6K and IRS-1 that mediates rapamycin-directed IGF-IR signaling. Collectively, these data show that resistance to inhibition of MEK, mTOR, and EGFR is associated with enhanced IGF-IR-directed Akt signaling, where all affect feedback loops converging at the level of IRS-1.


Assuntos
Antineoplásicos/farmacologia , Receptores ErbB/antagonistas & inibidores , Imidazóis/farmacologia , Pirazinas/farmacologia , Quinazolinas/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Receptores ErbB/fisiologia , Cloridrato de Erlotinib , Retroalimentação Fisiológica , Feminino , Humanos , Proteínas Substratos do Receptor de Insulina , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/efeitos dos fármacos
9.
Clin Exp Metastasis ; 25(6): 685-93, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18236164

RESUMO

Over 90% of all cancers are carcinomas, malignancies derived from cells of epithelial origin. As carcinomas progress, these tumors may lose epithelial morphology and acquire mesenchymal characteristics which contribute to metastatic potential. An epithelial-to-mesenchymal transition (EMT) similar to the process critical for embryonic development is thought to be an important mechanism for promoting cancer invasion and metastasis. Epithelial-to-mesenchymal transitions have been induced in vitro by transient or unregulated activation of receptor tyrosine kinase signaling pathways, oncogene signaling and disruption of homotypic cell adhesion. These cellular models attempt to mimic the complexity of human carcinomas which respond to autocrine and paracrine signals from both the tumor and its microenvironment. Activation of the epidermal growth factor receptor (EGFR) has been implicated in the neoplastic transformation of solid tumors and overexpression of EGFR has been shown to correlate with poor survival. Notably, epithelial tumor cells have been shown to be significantly more sensitive to EGFR inhibitors than tumor cells which have undergone an EMT-like transition and acquired mesenchymal characteristics, including non-small cell lung (NSCLC), head and neck (HN), bladder, colorectal, pancreas and breast carcinomas. EGFR blockade has also been shown to inhibit cellular migration, suggesting a role for EGFR inhibitors in the control of metastasis. The interaction between EGFR and the multiple signaling nodes which regulate EMT suggest that the combination of an EGFR inhibitor and other molecular targeted agents may offer a novel approach to controlling metastasis.


Assuntos
Carcinoma/metabolismo , Carcinoma/patologia , Epitélio/patologia , Receptores ErbB/metabolismo , Mesoderma/patologia , Invasividade Neoplásica/patologia , Animais , Humanos
10.
Bioorg Med Chem Lett ; 17(15): 4378-81, 2007 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-17574417

RESUMO

Novel anthranilamides were surprisingly found to exert additional activity on B-RAF. Corresponding thiophene, pyrazole, and thiazole core analogs were prepared as VEGFR-2 inhibitors with c-KIT, and B-RAF activity. Compounds in the phenyl, thiophene, and thiazole series are in vivo active.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Camundongos , Inibidores de Proteínas Quinases/uso terapêutico , Relação Estrutura-Atividade
11.
Expert Opin Investig Drugs ; 15(11): 1411-25, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17040200

RESUMO

Targeting cellular proliferation persists as a mainstay of cancer therapeutic strategy. Although microtubule-targeting drugs (such as taxanes and vinca alkaloids) have been used successfully in the clinic to treat a variety of cancers, they carry substantial liabilities that have spurred drug companies to aggressively pursue new tubulin-targeting drug candidates with improved efficacy and toxicity profiles. The recent discoveries of new mitotic targets for cancer therapy (such as kinesin spindle protein, Aurora kinases and Polo-like kinase-1) have also stimulated intense work focused on identifying novel antimitotic drugs directed at these new targets. A number of novel antimitotic drugs have demonstrated encouraging activity in preclinical models and have progressed into clinical development. This review focuses on selected new antimitotic drugs under evaluation in clinical trials.


Assuntos
Antimitóticos/uso terapêutico , Antineoplásicos/uso terapêutico , Drogas em Investigação/uso terapêutico , Animais , Antineoplásicos/farmacologia , Ensaios Clínicos como Assunto , Drogas em Investigação/farmacologia , Humanos , Moduladores de Tubulina/uso terapêutico
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