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1.
Mod Pathol ; 34(4): 748-757, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33299109

RESUMO

Alveolar Rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with about 80% of cases characterized by either a t(1;13)(p36;q14) or t(2;13)(q35;q14), which results in the formation of the fusion oncogenes PAX7-FOXO1 and PAX3-FOXO1, respectively. Since patients with fusion-positive ARMS (FP-RMS) have a poor prognosis and are treated with an aggressive therapeutic regimen, correct classification is of clinical importance. Detection of the translocation by different molecular methods is used for diagnostics, including fluorescence in situ hybridization and RT-PCR or NGS based approaches. Since these methods are complex and time consuming, we developed specific monoclonal antibodies (mAbs) directed to the junction region on the PAX3-FOXO1 fusion protein. Two mAbs, PFM.1 and PFM.2, were developed and able to immunoprecipitate in vitro-translated PAX3-FOXO1 and cellular PAX3-FOXO1 from FP-RMS cells. Furthermore, the mAbs recognized a 105 kDa band in PAX3-FOXO1-transfected cells and in FP-RMS cell lines. The mAbs did not recognize proteins in fusion-negative embryonal rhabdomyosarcoma cell lines, nor did they recognize PAX3 or FOXO1 alone when compared to anti-PAX3 and anti-FOXO1 antibodies. We next evaluated the ability of mAb PFM.2 to detect the fusion protein by immunohistochemistry. Both PAX3-FOXO1 and PAX7-FOXO1 were detected in HEK293 cells transfected with the corresponding cDNAs. Subsequently, we stained 26 primary tumor sections and a rhabdomyosarcoma tissue array and detected both fusion proteins with a positive predictive value of 100%, negative predictive value of 98%, specificity of 100% and a sensitivity of 91%. While tumors are stained homogenously in PAX3-FOXO1 cases, the staining pattern is heterogenous with scattered positive cells only in tumors expressing PAX7-FOXO1. No staining was observed in stromal cells, embryonal rhabdomyosarcoma, and fusion-negative rhabdomyosarcoma. These results demonstrate that mAbs specific for the chimeric oncoproteins PAX3-FOXO1 and PAX7-FOXO1 can be used efficiently for simple and fast subclassification of rhabdomyosarcoma in routine diagnostics via immunohistochemical detection.


Assuntos
Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/análise , Imuno-Histoquímica , Proteínas de Fusão Oncogênica/análise , Fatores de Transcrição Box Pareados/análise , Rabdomiossarcoma Alveolar/imunologia , Adolescente , Adulto , Animais , Especificidade de Anticorpos , Criança , Pré-Escolar , Feminino , Células HEK293 , Células HeLa , Humanos , Lactente , Masculino , Camundongos , Pessoa de Meia-Idade , Células NIH 3T3 , Proteínas de Fusão Oncogênica/imunologia , Fatores de Transcrição Box Pareados/imunologia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Rabdomiossarcoma Alveolar/patologia , Adulto Jovem
2.
BMC Cancer ; 19(1): 1251, 2019 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-31881855

RESUMO

BACKGROUND: Acute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate. Recent efforts have focused on developing therapies that target specific protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target and thought to be "undrugable" because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A novel technology that circumvents some of these issues is the use of small molecules that stabilize secondary DNA structures present in the promoters of many potential oncogenes and modulate their transcription. METHODS: This study characterizes the in vitro activity of the G-quadruplex-stabilizing small molecule GQC-05 in AML cells. The effect of GQC-05 on three AML cell lines was analyzed using viability and apoptosis assays. GQC-05 has been shown to down-regulate MYC through G-quadruplex stabilization in Burkitt's lymphoma cell lines. MYC expression was evaluated through qPCR and immunoblotting in the three AML cell lines following the treatment of GQC-05. In order to identify other therapeutic agents that potentiate the activity of GQC-05, combination drug screening was performed. The drug combinations were validated using in vitro cytotoxicity assays and compared to other commonly used chemotherapeutic agents. RESULTS: GQC-05 treatment of KG-1a, CMK and TF-1 cells decreased cell viability and resulted in increased DNA damage and apoptosis. Additionally, treatment of KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with a more pronounced effect in KG-1a cells. Combination drug screening identified the Bcl-2/Bcl-XL inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed a synergistic decrease in cell viability of AML cells as determined by Chou-Talalay analysis, and induced more DNA damage, apoptosis, and rapid cytotoxicity. The cytotoxicity induced by GQC-05 and Navitoclax was more potent than that of Navitoclax combined with either cytarabine or doxorubicin. CONCLUSION: These results suggest that the G-quadruplex stabilizing small molecule GQC-05 induces down regulated MYC expression and DNA damage in AML cells. Treatment with both GQC-05 with a Bcl-2/Bcl-XL inhibitor Navitoclax results in increased cytotoxic activity, which is more pronounced than Navitoclax or GQC-05 alone, and more significant than Navitoclax in combination with cytarabine and doxorubicin that are currently being used clinically.


Assuntos
Compostos de Anilina/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Elipticinas/farmacologia , Quadruplex G/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Sulfonamidas/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Dano ao DNA , Elipticinas/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , Resultado do Tratamento
3.
Pediatr Blood Cancer ; 65(9): e27237, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29768711

RESUMO

Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of MAP2K1/MEK1 in 50% and 25% of cases, respectively. We evaluated a patient with treatment-refractory LCH for mutations in the RAS-RAF-MEK-ERK pathway and identified a novel mutation in the MAP2K1 gene resulting in a p.L98_K104 > Q deletion and predicted to be auto-activating. During treatment with the MEK inhibitor trametinib, the patient's disease showed significant progression. In vitro characterization of the MAP2K1 p.L98_K104 > Q deletion confirmed its effect on cellular activation of the ERK pathway and drug resistance.


Assuntos
Resistência a Medicamentos/genética , Histiocitose de Células de Langerhans/tratamento farmacológico , MAP Quinase Quinase 1/genética , Sistema de Sinalização das MAP Quinases/genética , Inibidores de Proteínas Quinases/uso terapêutico , Piridonas/uso terapêutico , Pirimidinonas/uso terapêutico , Adolescente , Corticosteroides/uso terapêutico , Butadienos/farmacologia , Terapia Combinada , Citarabina/uso terapêutico , Progressão da Doença , Quimioterapia Combinada , Ativação Enzimática/genética , Éxons/genética , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Histiocitose de Células de Langerhans/genética , Histiocitose de Células de Langerhans/terapia , Humanos , Masculino , Terapia de Alvo Molecular , Mutação , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Pirazóis/uso terapêutico , Piridonas/farmacologia , Pirimidinonas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Tiofenos/uso terapêutico , Vincristina/uso terapêutico
4.
J Cell Sci ; 125(Pt 18): 4253-63, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22718346

RESUMO

The Forkhead transcription factor, FoxO3a, is a known suppressor of primary tumor growth through transcriptional regulation of key genes regulating cell cycle arrest and apoptosis. In many types of cancer, in response to growth factor signaling, FoxO3a is phosphorylated by Akt, resulting in its exclusion from the nucleus. Here we show that FoxO3a remains nuclear in anaplastic thyroid carcinoma (ATC). This correlates with lack of Akt phosphorylation at serine473 in ATC cell lines and tissues of ATC patients, providing a potential explanation for nuclear FoxO3a. Mechanistically, nuclear FoxO3a promotes cell cycle progression by transcriptional upregulation of cyclin A1, promoting proliferation of human ATC cells. Silencing FoxO3a with a reverse genetics approach leads to downregulation of CCNA1 mRNA and protein. These combined data suggest an entirely novel function for FoxO3a in ATC promotion by enhancing cell cycle progression and tumor growth through transcriptional upregulation of cyclin A1. This is clinically relevant since we detected highly elevated CCNA1 mRNA and protein levels in tumor tissues of ATC patients. Our data indicate therapeutic inactivation of FoxO3a may lead to attenuation of tumor expansion in ATC. This new paradigm also suggests caution in relation to current dogma focused upon reactivation of FoxO3a as a therapeutic strategy against cancers harboring active PI3-K and Akt signaling pathways.


Assuntos
Ciclina A1/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Transcrição Gênica , Sequência de Bases , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Ciclina A1/metabolismo , Proteína Forkhead Box O3 , Inativação Gênica , Células HEK293 , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/terapia
5.
Blood ; 119(12): 2863-72, 2012 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-22267604

RESUMO

To identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Citarabina/farmacologia , Leucemia Mieloide/enzimologia , Proteínas Nucleares/metabolismo , Fosfotransferases/análise , Proteínas Tirosina Quinases/metabolismo , Western Blotting , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala , Humanos , Fosfotransferases/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
Pediatr Blood Cancer ; 60(1): 35-40, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22961763

RESUMO

BACKGROUND: BIRC5 (Survivin), an inhibitor of apoptosis protein (IAP), is over-expressed in several human cancers and increased expression is associated with poor prognosis. The goal of the current study was to evaluate the role of BIRC5 in Ewing sarcoma (ES), the second most common pediatric bone sarcoma. PROCEDURE: BIRC5 protein expression was determined in ES cell lines using Western Blot analysis. Functional role of survivin on growth and viability of ES cells was assessed by siRNA knockdown of BIRC5 and by using a small molecule inhibitor YM155. Immunohistochemical analysis for BIRC5 protein was performed on patient tumor samples using an anti-survivin antibody. The degree of BIRC5 protein expression was correlated with clinical parameters and patient outcome. RESULTS: BIRC5 is over-expressed in a panel of ES cell lines. Gene silencing of BIRC5 in the ES cell line TC-71 decreases cell growth by more than 50% for each BIRC5 siRNA construct compared to non-silencing siRNA control constructs. YM155 also reduces ES cell growth and viability with an EC(50) ranging from 2.8 to 6.2 nM. BIRC5 protein is expressed in majority of the ES tumor samples with minimal expression in normal tissue (P < 0.005). Tumors with more than 50% expression are associated with worse overall survival than tumors with less than 50% expression (Hazard Ratio: 6.05; CI: 1.7-21.4; P = 0.04). CONCLUSION: BIRC5 is over-expressed in ES cell lines and tumor samples. Further, it plays an important role in cell growth and viability in vitro. Higher degree of expression in patients is an independent poor prognostic factor.


Assuntos
Neoplasias Ósseas/patologia , Proteínas Inibidoras de Apoptose/fisiologia , Sarcoma de Ewing/patologia , Adolescente , Adulto , Apoptose , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células , Criança , Pré-Escolar , Feminino , Humanos , Imidazóis/farmacologia , Proteínas Inibidoras de Apoptose/análise , Masculino , Naftoquinonas/farmacologia , Prognóstico , Survivina
7.
Nat Genet ; 36(9): 979-83, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15300251

RESUMO

The identification of tumor-suppressor genes in solid tumors by classical cancer genetics methods is difficult and slow. We combined nonsense-mediated RNA decay microarrays and array-based comparative genomic hybridization for the genome-wide identification of genes with biallelic inactivation involving nonsense mutations and loss of the wild-type allele. This approach enabled us to identify previously unknown mutations in the receptor tyrosine kinase gene EPHB2. The DU 145 prostate cancer cell line, originating from a brain metastasis, carries a truncating mutation of EPHB2 and a deletion of the remaining allele. Additional frameshift, splice site, missense and nonsense mutations are present in clinical prostate cancer samples. Transfection of DU 145 cells, which lack functional EphB2, with wild-type EPHB2 suppresses clonogenic growth. Taken together with studies indicating that EphB2 may have an essential role in cell migration and maintenance of normal tissue architecture, our findings suggest that mutational inactivation of EPHB2 may be important in the progression and metastasis of prostate cancer.


Assuntos
Mutação , Neoplasias da Próstata/genética , Receptor EphB2/genética , Linhagem Celular Tumoral , Códon sem Sentido , Emetina/farmacologia , Genes Supressores de Tumor , Humanos , Masculino , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Estabilidade de RNA , Transfecção
8.
J Pathol ; 223(4): 543-52, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21294127

RESUMO

Androgen withdrawal is the standard treatment for advanced prostate cancer. Although this therapy is initially effective, nearly all prostate cancers become refractory to it. Approximately 15% of these castration-resistant prostate cancers harbour a genomic amplification at 10q22. The aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving genes. Application of high-resolution array-CGH using the 244k Agilent microarrays to cell lines with 10q22 amplification allowed us to narrow down the common amplified region to a region of 5.8 megabases. We silenced each of the genes of this region by an RNAi screen in the prostate cancer cell lines PC-3 and 22Rv1. We selected genes with a significant growth reduction in the 10q22 amplified cell line PC-3, but not in the non-amplified 22Rv1 cells, as putative target genes of this amplicon. Immunohistochemical analysis of the protein expression of these candidate genes on a tissue microarray enriched for 10q22 amplified prostate cancers revealed vinculin as the most promising target of this amplicon. We found a strong association between vinculin gene amplification and overexpression (p < 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that vinculin expression was highest in castration-resistant prostate cancers, but negative or very low in benign prostatic hyperplasia (p < 0.0001). Additionally, high tumour cell proliferation measured by Ki67 expression was significantly associated with high vinculin expression in prostate cancer (p < 0.0001). Our data suggest that vinculin is a major driving gene of the 10q22 amplification in prostate cancer and that vinculin overexpression might contribute to prostate cancer progression by enhancing tumour cell proliferation.


Assuntos
Neoplasias da Próstata/metabolismo , Vinculina/biossíntese , Proliferação de Células , Cromossomos Humanos Par 10/genética , Hibridização Genômica Comparativa/métodos , Progressão da Doença , Amplificação de Genes , Estudos de Associação Genética/métodos , Humanos , Masculino , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Hiperplasia Prostática/metabolismo , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , Vinculina/genética
9.
BMC Genomics ; 11: 25, 2010 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-20067632

RESUMO

BACKGROUND: Neurofibrillary tangles (NFT), a cardinal neuropathological feature of Alzheimer's disease (AD) that is highly correlated with synaptic loss and dementia severity, appear to be partly attributable to increased phosphorylation of the microtubule stabilizing protein tau at certain AD-related residues. Identifying the kinases involved in the pathologic phosphorylation of tau may provide targets at which to aim new AD-modifying treatments. RESULTS: We report results from a screen of 572 kinases in the human genome for effects on tau hyperphosphorylation using a loss of function, high-throughput RNAi approach. We confirm effects of three kinases from this screen, the eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), and the A-kinase anchor protein 13 (AKAP13) on tau phosphorylation at the 12E8 epitope (serine 262/serine 356). We provide evidence that EIF2AK2 effects may result from effects on tau protein expression, whereas DYRK1A and AKAP13 are likely more specifically involved in tau phosphorylation pathways. CONCLUSIONS: These findings identify novel kinases that phosphorylate tau protein and provide a valuable reference data set describing the kinases involved in phosphorylating tau at an AD-relevant epitope.


Assuntos
Doença de Alzheimer/enzimologia , Proteínas Quinases/análise , RNA Interferente Pequeno/análise , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Testes Genéticos , Genoma Humano , Humanos , Fosforilação , Proteínas Quinases/genética , RNA Interferente Pequeno/genética , Regulação para Cima
10.
Mol Cancer ; 9: 218, 2010 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-20718987

RESUMO

BACKGROUND: Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewing's sarcoma cells. RESULTS: Four Ewing's sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewing's sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewing's sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewing's sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis. CONCLUSION: In summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewing's sarcoma.


Assuntos
Interferência de RNA , Sarcoma de Ewing/tratamento farmacológico , Divisão Celular , Linhagem Celular Tumoral , Humanos , Fenótipo , RNA Interferente Pequeno , Sarcoma de Ewing/patologia
11.
Gynecol Oncol ; 118(3): 220-7, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20722101

RESUMO

OBJECTIVE: Ovarian cancer retains a poor prognosis among the female gynaecological malignancies. It constitutes about 3% of all malignancies in women and accounts for 5% of all female cancer related deaths. A standard treatment is cytoreductive surgery followed by adjuvant chemotherapy, and re-treatment with platinum based chemotherapy at the time of relapse. In order to improve cisplatin response in ovarian cancer cells, we utilized a high-throughput RNAi screening to identify kinase modulators. METHODS: A high-throughput RNAi screen was performed using a siRNA library targeting 572 kinases to identify potentiators of cisplatin response in the ovarian cancer cell line SKOV3. RESULTS: RNAi screening identified at least 55 siRNAs that potentiated the growth inhibitory effects of cisplatin in SKOV3 cells. Inhibition of ATR and CHK1 resulted in the greatest modulation of cisplatin response. Drug dose response of cisplatin in the presence of siRNA validated the effects of these target genes. To show that the siRNA data could be successfully translated into potential therapeutic strategies, CHK1 was further targeted with small molecule inhibitor PD 407824 in combination with cisplatin. Results showed that treatment of SKOV3 and OVCAR3 cells with CHK1 inhibitor PD 407824 led to sensitization of ovarian cancer cells to cisplatin. CONCLUSIONS: Our data provides kinase targets that could be exploited to design better therapeutics for ovarian cancer patients. We also demonstrate the effectiveness of high-throughput RNAi screening as a tool for identifying sensitizing targets to known and established chemotherapeutic agents.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Proteínas Quinases/genética , Interferência de RNA , Carbazóis/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Feminino , Humanos , Neoplasias Ovarianas/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética
12.
J Transl Med ; 7: 43, 2009 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-19519883

RESUMO

BACKGROUND: Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement. METHODS: In order to improve gemcitabine response in pancreatic cancer cells, we utilized a synthetic lethal RNAi screen targeting 572 known kinases to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine. RESULTS: Results from the RNAi screens identified several genes that, when silenced, potentiated the growth inhibitory effects of gemcitabine in pancreatic cancer cells. The greatest potentiation was shown by siRNA targeting checkpoint kinase 1 (CHK1). Validation of the screening results was performed in MIA PaCa-2 and BxPC3 pancreatic cancer cells by examining the dose response of gemcitabine treatment in the presence of either CHK1 or CHK2 siRNA. These results showed a three to ten-fold decrease in the EC50 for CHK1 siRNA-treated cells versus control siRNA-treated cells while treatment with CHK2 siRNA resulted in no change compared to controls. CHK1 was further targeted with specific small molecule inhibitors SB 218078 and PD 407824 in combination with gemcitabine. Results showed that treatment of MIA PaCa-2 cells with either of the CHK1 inhibitors SB 218078 or PD 407824 led to sensitization of the pancreatic cancer cells to gemcitabine. CONCLUSION: These findings demonstrate the effectiveness of synthetic lethal RNAi screening as a tool for identifying sensitizing targets to chemotherapeutic agents. These results also indicate that CHK1 could serve as a putative therapeutic target for sensitizing pancreatic cancer cells to gemcitabine.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Carbazóis/farmacologia , Desoxicitidina/análogos & derivados , Inativação Gênica , Neoplasias Pancreáticas/tratamento farmacológico , Interferência de RNA , Idoso , Alcaloides/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Impedância Elétrica , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Transfecção , Gencitabina
13.
Cancer Res ; 67(5): 1943-9, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17332321

RESUMO

Pancreatic cancer is a highly aggressive disease characterized by poor prognosis and vast genetic instability. Recent microarray-based, genome-wide surveys have identified multiple recurrent copy number aberrations in pancreatic cancer; however, the target genes are, for the most part, unknown. Here, we characterized the 19q13 amplicon in pancreatic cancer to identify putative new drug targets. Copy number increases at 19q13 were quantitated in 16 pancreatic cancer cell lines and 31 primary tumors by fluorescence in situ hybridization. Cell line copy number data delineated a 1.1 Mb amplicon, the presence of which was also validated in 10% of primary pancreatic tumors. Comprehensive expression analysis by quantitative real-time reverse transcription-PCR indicated that seven transcripts within this region had consistently elevated expression levels in the amplified versus nonamplified cell lines. High-throughput loss-of-function screen by RNA interference was applied across the amplicon to identify genes whose down-regulation affected cell viability. This screen revealed five genes whose down-regulation led to significantly decreased cell viability in the amplified PANC-1 cells but not in the nonamplified MiaPaca-2 cells, suggesting the presence of multiple biologically interesting genes in this region. Of these, the transcriptional regulator intersex-like (IXL) was consistently overexpressed in amplified cells and had the most dramatic effect on cell viability. IXL silencing also resulted in G(0)-G(1) cell cycle arrest and increased apoptosis in PANC-1 cells. These findings implicate IXL as a novel amplification target gene in pancreatic cancer and suggest that IXL is required for cancer cell survival in 19q13-amplified tumors.


Assuntos
Apoptose/genética , Cromossomos Humanos Par 19 , Amplificação de Genes , Neoplasias Pancreáticas/patologia , Fatores de Transcrição/fisiologia , Sobrevivência Celular , Cromossomos Artificiais Bacterianos , Dosagem de Genes , Humanos , Complexo Mediador , Neoplasias Pancreáticas/genética , Interferência de RNA , Análise Serial de Tecidos , Células Tumorais Cultivadas
14.
Int J Cancer ; 123(2): 330-339, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18452169

RESUMO

S100P protein regulates calcium signal transduction and mediates cytoskeletal interaction, protein phosphorylation and transcriptional control. We have previously shown how elevated S100P levels in prostate cancer strongly correlate with progression to metastatic disease. In our study, we evaluated the functional significance of S100P expression on prostate tumor growth in vitro and in vivo. S100P levels were modulated by overexpressing S100P in PC3 prostate cancer cells and by silencing S100P levels in 22Rv1 prostate cancer cells. Overexpression of S100P in PC3 cells promoted cell growth, increased the percentage of S-phase cells, decreased basal apoptosis rate and promoted anchorage independent growth in soft agar. Furthermore, prostate cancer cells overexpressing S100P were protected against camptothecin-induced apoptosis. Conversely, silencing of S100P in 22Rv1 cells using siRNA resulted in a prominent cytostatic effect. The influence of S100P on tumor growth and metastases were assessed in vivo. S100P-overexpressing PC3 cells had a dramatically increased tumor formation compared to controls. Microarray analysis showed the involvement of growth pathways including increased androgen receptor expression in S100P-overexpressing cells. These results provide the first functional proof that S100P overexpression can upregulate androgen receptor expression and thereby promote prostate cancer progression by increasing cell growth. Moreover, the results confirm the oncogenic nature of S100P in prostate cancer and suggest that the protein may directly confer resistance to chemotherapy. Hence, S100P could be considered a potential drug target or a chemosensitization target, and could also serve as a biomarker for aggressive, hormone-refractory and metastatic prostate cancer.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Apoptose , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Progressão da Doença , Regulação para Baixo , Impedância Elétrica , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Regulação para Cima
15.
Mol Cell Biol ; 23(1): 140-9, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12482968

RESUMO

Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, alpha/beta-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte de Cátions , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Lisina/metabolismo , Substâncias Macromoleculares , Metilação , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coativadores de Receptor Nuclear , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Tubulina (Proteína)/metabolismo
16.
J Exp Clin Cancer Res ; 36(1): 22, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28143565

RESUMO

BACKGROUND: Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL. METHODS: Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu2+) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo®, ALDH activity by ALDELUORTM, and proteasome inhibition by western blot of ubiquitinated proteins and the Proteasome-Glo™ Chymotrypsin-Like (CT-like) assay, apoptosis by Annexin V Fluos/Propidium iodide staining, and mutations were detected using PCR, cloning and sequencing. RESULTS: Ara-C-resistant AML cell lines were sensitive to BTZ and DSF/Cu2+. The Ara-C-resistant DS-AMKL CMY cells had a high percentage of ALDHbright "stem-like" populations that may underlie Ara-C resistance. One percent of these cells were still resistant to BTZ but sensitive to DSF/Cu2+. To understand the mechanism of BTZ resistance, BTZ resistant (CMY-BR) and (CMK-BR) were generated. A novel mutation PSMB5 Q62P underlied BTZ resistance, and was associated with an overexpression of the ß5 proteasome subunit. BTZ-resistance conferred increased resistance to Ara-C due to G1 arrest in the CMY-BR cells, which protected the cells from S-phase damage by Ara-C. CMY-BR and CMK-BR cells were cross-resistant to CFZ and MG-132 but sensitive to DSF/Cu2+. In this setting, DSF/Cu2+ induced apoptosis and proteasome inhibition independent of CT-like activity inhibition. CONCLUSIONS: We provide evidence that DSF/Cu2+ overcomes Ara-C and BTZ resistance in cell lines from DS-AMKL patients. A novel mutation underlying BTZ resistance was detected that may identify BTZ-resistant patients, who may not benefit from treatment with CFZ or Ara-C, but may be responsive to DSF/Cu2+. Our findings support the clinical development of DSF/Cu2+ as a less toxic efficacious treatment approach in patients with relapsed/refractory DS-AMKL.


Assuntos
Dissulfiram/farmacologia , Síndrome de Down/complicações , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda/genética , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Adolescente , Adulto , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Criança , Pré-Escolar , Citarabina/administração & dosagem , Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Lactente , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Adulto Jovem
17.
Methods Mol Biol ; 1470: 15-24, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27581281

RESUMO

RNAi screening of mammalian cells is often performed using siRNAs and cationic lipids as transfection reagents. Efficiency of transfection depends on growth characteristics of the cells and the cationic lipid used. With a large selection of cationic lipids available, it can often be difficult to select the optimal lipid and lipid:siRNA (vol:wt) ratio. Here, we describe the process of optimizing siRNA transfection conditions for efficient reverse transfection of mammalian cells using specific positive and negative siRNA controls.


Assuntos
RNA Interferente Pequeno , Transfecção/métodos , Animais , Cátions/química , Humanos , Indicadores e Reagentes , Lipídeos/química
18.
Methods Mol Biol ; 1470: 247-60, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27581298

RESUMO

High-throughput RNA interference (HT-RNAi) screening is an effective technology to help identify important genes and pathways involved in a biological process. Analysis of high-throughput RNAi screening data is a critical part of this technology, and many analysis methods have been described. Here, we summarize the workflow and types of analyses commonly used in high-throughput RNAi screening.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Interferência de RNA , Controle de Qualidade , RNA Interferente Pequeno , Fluxo de Trabalho
19.
Drug Discov Today Technol ; 2(2): 141-7, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-24981841

RESUMO

The combination of RNAi-mediated knockdown of gene expression and high content screening (HCS) allows the determination of the contribution of single genes to a variety of cellular effects varying between growth and survival to subtle alterations in cellular morphology and phenotype. This review examines the current status of research in combining these tools.:

20.
Oncotarget ; 6(34): 35247-62, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26497213

RESUMO

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials.


Assuntos
Aurora Quinase B/antagonistas & inibidores , Neuroblastoma/enzimologia , Neuroblastoma/terapia , Animais , Apoptose/fisiologia , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Neuroblastoma/genética , Neuroblastoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
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