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1.
ACS Chem Biol ; 11(5): 1322-31, 2016 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-26938486

RESUMO

Colorectal cancer (CRC) is a genetic disease, due to progressive accumulation of mutations in oncogenes and tumor suppressor genes. Large scale genomic sequencing projects revealed >100 mutations in any individual CRC. Many of these mutations are likely passenger mutations, and fewer are driver mutations. Of these, activating mutations in RAS proteins are essential for cancer initiation, progression, and/or resistance to therapy. There has been significant interest in developing drugs targeting mutated cancer gene products or downstream signaling pathways. Due to the number of mutations involved and inherent redundancy in intracellular signaling, drugs targeting one mutation or pathway have been either ineffective or led to rapid resistance. We have devised a strategy whereby multiple cancer pathways may be simultaneously targeted for drug discovery. For proof-of-concept, we targeted the oncogenic KRAS and HIF pathways, since oncogenic KRAS has been shown to be required for cancer initiation and progression, and HIF-1α and HIF-2α are induced by the majority of mutated oncogenes and tumor suppressor genes in CRC. We have generated isogenic cell lines defective in either oncogenic KRAS or both HIF-1α and HIF-2α and subjected them to multiplex genomic, siRNA, and high-throughput small molecule screening. We have identified potential drug targets and compounds for preclinical and clinical development. Screening of our marine natural product library led to the rediscovery of the microtubule agent dolastatin 10 and the class I histone deacetylase (HDAC) inhibitor largazole to inhibit oncogenic KRAS and HIF pathways. Largazole was further validated as an antiangiogenic agent in a HIF-dependent manner in human cells and in vivo in zebrafish using a genetic model with activated HIF. Our general strategy, coupling functional genomics with drug susceptibility or chemical-genetic interaction screens, enables the identification of potential drug targets and candidates with requisite selectivity. Molecules prioritized in this manner can easily be validated in suitable zebrafish models due to the genetic tractability of the system. Our multidimensional platform with cellular and organismal components can be extended to larger scale multiplex screens that include other mutations and pathways.


Assuntos
Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , Reto/efeitos dos fármacos , Reto/metabolismo , Reto/patologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Transcriptoma/efeitos dos fármacos , Peixe-Zebra
2.
BMC Cancer ; 13: 517, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24180670

RESUMO

BACKGROUND: CD26/dipeptidyl peptidase IV (DPPIV) is a multifunctional membrane protein with a key role in T-cell biology and also serves as a marker of aggressive cancers, including T-cell malignancies. METHODS: Versican expression was measured by real-time RT-PCR and Western blots. Gene silencing of versican in parental Karpas 299 cells was performed using transduction-ready viral particles. The effect of versican depletion on surface expression of MT1-MMP was monitored by flow cytometry and surface biotinylation. CD44 secretion/cleavage and ERK (1/2) activation was followed by Western blotting. Collagenase I activity was measured by a live cell assay and in vesicles using a liquid-phase assay. Adhesion to collagen I was quantified by an MTS assay. RESULTS: Versican expression was down-regulated in CD26-depleted Karpas 299 cells compared to the parental T-ALCL Karpas 299 cells. Knock down of versican in the parental Karpas 299 cells led to decreased MT1-MMP surface expression as well as decreased CD44 expression and secretion of the cleaved form of CD44. Parental Karpas 299 cells also exhibited higher collagenase I activity and greater adhesion to collagenase I than CD26-knockdown or versican-knockdown cells. ERK activation was also highest in parental Karpas 299 cells compared to CD26-knockdown or versican-knockdown clones. CONCLUSIONS: Our data indicate that CD26 has a key role in cell adhesion and invasion, and potentially in tumorigenesis of T-cell lines, through its association with molecules and signal transduction pathways integral to these processes.


Assuntos
Dipeptidil Peptidase 4/genética , Regulação Neoplásica da Expressão Gênica , Linfoma Anaplásico de Células Grandes/genética , Metaloproteinase 14 da Matriz/genética , Versicanas/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Colagenases/metabolismo , Dipeptidil Peptidase 4/metabolismo , Perfilação da Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Linfoma Anaplásico de Células Grandes/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Transdução de Sinais , Versicanas/metabolismo
3.
BMC Cancer ; 11: 51, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21284881

RESUMO

BACKGROUND: CD26 (dipeptidyl peptidase IV, DPPIV) is a 110 kDa surface glycoprotein expressed in most normal tissues, and is a potential novel therapeutic target for selected cancers. Our work evaluates the mechanism involved in confluence-dependent CD26 expression in colon cancer. METHODS: Colon adenocarcinoma cells were grown to confluence, and expression of CD26 and transcription factors implicated in its regulation was confirmed by immunofluorescence and Western blotting. Real-time PCR was also performed to evaluate CD26 upregulation at the transcriptional level. The influence of c-Myc on CD26 expression during different growth conditions was further evaluated following transient transfection of a c-Myc-expressing plasmid and a c-Myc specific siRNA. RESULTS: We found that the colon cancer cell lines HCT-116 and HCT-15 exhibited a confluence-dependent increase in CD26 mRNA and protein, associated with decreased expression of c-Myc, increased USF-1 and Cdx 2 levels, and unchanged HNF-1α expression. Meanwhile, ectopic expression of c-Myc in both cell lines led to decreased CD26 expression. In contrast, transfection of a siRNA targeted to Cdx2 resulted in decreased CD26 level. Importantly, culturing of cells in serum-depleted media, but not acidic conditions, upregulated CD26. While HIF-1α level also increased when cells were cultured in serum-depleted media, its expression was required but not sufficient for CD26 upregulation. CONCLUSIONS: CD26 mRNA and protein levels increase in a confluence-dependent manner in colon carcinoma cell lines, with c-Myc acting as a repressor and Cdx2 acting as an enhancer of CD26 expression. The enhanced expression of CD26 in serum-depleted media and a requirement for HIF-1α suggest a role for nutrients or growth factors in the regulation of CD26 protein expression.


Assuntos
Movimento Celular , Proliferação de Células , Dipeptidil Peptidase 4/genética , Regulação Neoplásica da Expressão Gênica/genética , Western Blotting , Fator de Transcrição CDX2 , Adesão Celular , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dipeptidil Peptidase 4/metabolismo , Células HCT116 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Microscopia de Fluorescência , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Transativadores/metabolismo , Transfecção
4.
Front Biosci ; 13: 1634-45, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17981655

RESUMO

CD26/DPPIV is a multifunctional cell surface protein that is widely expressed in most cell types including T lymphocytes, on which it is a marker of activation. It is also present in serum and other body fluids in a truncated form (sCD26/DPPIV). It preferentially cleaves N-terminal dipeptides from polypeptides with proline or alanine in the penultimate position, and in doing so, regulates the activities of a number of cytokines and chemokines. Due in part to this ability to regulate the activity of biopeptides, it can act as a tumor suppressor or activator. It can associate with several proteins, among them fibroblast activating protein-alpha (FAP-alpha), plasminogen, adenosine deaminase (ADA), the tyrosine phosphatase CD45, and the chemokine receptor CXCR4. It can also bind to the extracellular matrix (ECM) and depending on the presence of other ligands, this process can either lead to increased or decreased invasive activity of the cells on which it is expressed. As a result of these characteristics, CD26/DPPIV plays an important role in tumor biology, and is useful as a marker for various cancers, with its levels either on the cell surface or in the serum being increased in some neoplasms and decreased in others. Our group has shown that CD26/DPPIV can be manipulated by such agents as CD26 cDNA-carrying plasmids, siRNA and monoclonal antibodies, resulting in both in vitro and in vivo inhibition of cell growth, enhanced sensitivity to selected chemotherapeutic agents, and enhanced survival of mouse xenograft models. These studies have demonstrated the utility of these tools as potential targeted therapies for specific cancers expressing CD26/DPPIV.


Assuntos
Dipeptidil Peptidase 4/fisiologia , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Dipeptidil Peptidase 4/metabolismo , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Neoplasias Hematológicas/metabolismo , Humanos , Camundongos , Modelos Biológicos , RNA Interferente Pequeno/metabolismo
5.
Cancer Res ; 62(5): 1443-9, 2002 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-11888918

RESUMO

Thiol antioxidants, typified by N-acetyl cysteine, are known to induce p53-dependent apoptosis in transformed mouse embryo fibroblasts but not in normal mouse embryo fibroblasts. We now report that this is also the case for human cells. First, we used an isogenic fibroblast cell lineage exhibiting progressive stages of transformation, from primary derived cells to v-MYC immortalized to tumorigenic. At the immortalization stage, cells became 12- and 480-fold more sensitive to the thiol antioxidants N-acetyl cysteine (NAC) and penicillamine (PEN), respectively. Although immortalization of these cells was associated with v-MYC expression, overexpression of MYC was not sufficient for sensitizing these cells to antioxidants. To test whether sensitivity to antioxidants is a general property of immortalized human cells, including fully transformed cells, 12 tumor-derived cell lines were treated with PEN, the more potent of the two antioxidants. Ten of 11 caspase-proficient tumor cell lines underwent apoptosis after treatment, whereas primary fibroblasts and keratinocytes were resistant. The difference between normal and transformed cells was apparent whether the assay used measured caspase 3 activation, Annexin V binding, or cell viability. Tumor cell lines containing wild-type p53 were more sensitive than p53-null cell lines. The requirement for p53 was tested using the p53 inhibitor, pifithrin-alpha, or using stable transfectants of a v-MYC-immortalized, telomerase-positive cell line that expresses HPV16 E6 to bind and degrade p53. In the latter case, > or = 80% of the PEN-induced apoptosis was dependent on the presence of wild-type p53. These studies suggest that treatment with thiol-containing antioxidants, such as PEN, may offer a useful approach for preferential induction of apoptosis in preneoplastic and neoplastic cells.


Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Penicilamina/farmacologia , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Genes myc , Humanos , Neoplasias/patologia , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/fisiologia
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