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1.
Oncotarget ; 12(21): 2114-2130, 2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34676045

RESUMO

The therapeutic efficacy of temozolomide (TMZ) is hindered by inherent and acquired resistance. Biomarkers such as MGMT expression and MMR proficiency are used as predictors of response. However, not all MGMTlow/-ve/MMRproficient patients benefit from TMZ treatment, indicating a need for additional patient selection criteria. We explored the role of ATR in mediating TMZ resistance and whether ATR inhibitors (ATRi) could reverse this resistance in multiple cancer lines. We observed that only 31% of MGMTlow/-ve/MMRproficient patient-derived and established cancer lines are sensitive to TMZ at clinically relevant concentrations. TMZ treatment resulted in DNA damage signaling in both sensitive and resistant lines, but prolonged G2/M arrest and cell death were exclusive to sensitive models. Inhibition of ATR but not ATM, sensitized the majority of resistant models to TMZ and resulted in measurable DNA damage and persistent growth inhibition. Also, compromised homologous recombination (HR) via RAD51 or BRCA1 loss only conferred sensitivity to TMZ when combined with an ATRi. Furthermore, low REV3L mRNA expression correlated with sensitivity to the TMZ and ATRi combination in vitro and in vivo. This suggests that HR defects and low REV3L levels could be useful selection criteria for enhanced clinical efficacy of an ATRi plus TMZ combination.

2.
BMC Genomics ; 15: 393, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24885658

RESUMO

BACKGROUND: Development of cancer therapeutics partially depends upon selection of appropriate animal models. Therefore, improvements to model selection are beneficial. RESULTS: Forty-nine human tumor xenografts at in vivo passages 1, 4 and 10 were subjected to cDNA microarray analysis yielding a dataset of 823 Affymetrix HG-U133 Plus 2.0 arrays. To illustrate mining strategies supporting therapeutic studies, transcript expression was determined: 1) relative to other models, 2) with successive in vivo passage, and 3) during the in vitro to in vivo transition. Ranking models according to relative transcript expression in vivo has the potential to improve initial model selection. For example, combining p53 tumor expression data with mutational status could guide selection of tumors for therapeutic studies of agents where p53 status purportedly affects efficacy (e.g., MK-1775). The utility of monitoring changes in gene expression with extended in vivo tumor passages was illustrated by focused studies of drug resistance mediators and receptor tyrosine kinases. Noteworthy observations included a significant decline in HCT-15 colon xenograft ABCB1 transporter expression and increased expression of the kinase KIT in A549 with serial passage. These trends predict sensitivity to agents such as paclitaxel (ABCB1 substrate) and imatinib (c-KIT inhibitor) would be altered with extended passage. Given that gene expression results indicated some models undergo profound changes with in vivo passage, a general metric of stability was generated so models could be ranked accordingly. Lastly, changes occurring during transition from in vitro to in vivo growth may have important consequences for therapeutic studies since targets identified in vitro could be over- or under-represented when tumor cells adapt to in vivo growth. A comprehensive list of mouse transcripts capable of cross-hybridizing with human probe sets on the HG-U133 Plus 2.0 array was generated. Removal of the murine artifacts followed by pairwise analysis of in vitro cells with respective passage 1 xenografts and GO analysis illustrates the complex interplay that each model has with the host microenvironment. CONCLUSIONS: This study provides strategies to aid selection of xenograft models for therapeutic studies. These data highlight the dynamic nature of xenograft models and emphasize the importance of maintaining passage consistency throughout experiments.


Assuntos
Perfilação da Expressão Gênica , Neoplasias/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Análise por Conglomerados , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Paclitaxel/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transplante Heterólogo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Clin Cancer Res ; 19(5): 1139-46, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23340293

RESUMO

PURPOSE: High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult because contaminating stromal cells overgrow the malignant cells. EXPERIMENTAL DESIGN: We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. RESULTS: Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified 77 U.S. Food and Drug Administration (FDA)-approved drugs with activity, and two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. CONCLUSION: Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice.


Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Digitoxina/farmacologia , Nogalamicina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Medicina de Precisão , Animais , Antibióticos Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Cardiotônicos/farmacologia , Feminino , Humanos , Hipoxantina Fosforribosiltransferase/genética , Subunidade gama Comum de Receptores de Interleucina , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologia , Células Tumorais Cultivadas
4.
Cancer Med ; 2(5): 687-700, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24403234

RESUMO

Oxyphenisatin (3,3-bis(4-hydroxyphenyl)-1H-indol-2-one) and several structurally related molecules have been shown to have in vitro and in vivo antiproliferative activity. This study aims to confirm and extend mechanistic studies by focusing on oxyphenisatin acetate (OXY, NSC 59687), the pro-drug of oxyphenisatin. Results confirm that OXY inhibits the growth of the breast cancer cell lines MCF7, T47D, HS578T, and MDA-MB-468. This effect is associated with selective inhibition of translation accompanied by rapid phosphorylation of the nutrient sensing eukaryotic translation initiation factor 2α (eIF2α) kinases, GCN2 and PERK. This effect was paralleled by activation of AMP-activated protein kinase (AMPK) combined with reduced phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6K and 4E-BP1. Microarray analysis highlighted activation of pathways involved in apoptosis induction, autophagy, RNA/protein metabolism, starvation responses, and solute transport. Pathway inhibitor combination studies suggested a role for AMPK/mTOR signaling, de novo transcription and translation, reactive oxygen species (ROS)/glutathione metabolism, calcium homeostasis and plasma membrane Na(+) /K(+) /Ca(2+) transport in activity. Further examination confirmed that OXY treatment was associated with autophagy, mitochondrial dysfunction, and ROS generation. Additionally, treatment was associated with activation of both intrinsic and extrinsic apoptotic pathways. In the estrogen receptor (ER) positive MCF7 and T47D cells, OXY induced TNFα expression and TNFR1 degradation, indicating autocrine receptor-mediated apoptosis in these lines. Lastly, in an MCF7 xenograft model, OXY delivered intraperitoneally inhibited tumor growth, accompanied by phosphorylation of eIF2α and degradation of TNFR1. These data suggest that OXY induces a multifaceted cell starvation response, which ultimately induces programmed cell death.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias da Mama/patologia , Mitocôndrias/efeitos dos fármacos , Acetato de Oxifenisatina/farmacologia , Animais , Comunicação Autócrina/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Mitocôndrias/fisiologia , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Análise Serial de Tecidos/métodos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
5.
Cancer Chemother Pharmacol ; 70(1): 207-12, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22526412

RESUMO

PURPOSE: To establish whether NSC80467, a novel fused naphthquinone imidazolium, has a similar spectrum of activity to the well-characterized "survivin suppressant" YM155 and to extend mechanistic studies for this structural class of agent. METHODS: NSC80467 and YM155 were analyzed in parallel using assays measuring viability, survivin suppression, inhibition of DNA/RNA/protein synthesis and the cellular response to DNA damage. RESULTS: GI(50) values generated for both compounds in the NCI-60 screen yielded a correlation coefficient of 0.748, suggesting significant concordance. Both agents were also shown to inhibit protein expression of survivin [BIRC5]. COMPARE analysis identified DNA damaging agents chromomycin A3 and bisantrene HCl and one DNA-directed inhibitor of transcription, actinomycin D, as correlating with the activity of NSC80467 and YM155. Furthermore, both agents were shown to preferentially inhibit DNA, over RNA and protein synthesis. Thus, the ability of NSC80467 and YM155 to induce a DNA damage response was examined further. Treatment of PC3 cells with either agent resulted in dose-dependent induction of γH2AX and pKAP1, two markers of DNA damage. The concentrations of agent required to stimulate γH2AX were considerably lower than those required to inhibit survivin, implicating DNA damage as an initiating event. The DNA damage response was then confirmed in a panel of cell lines treated with NSC80467 or YM155, suggesting that γH2AX and pKAP1 have potential as response biomarkers. CONCLUSIONS: These data provide the first evidence that NSC80467 and YM155 are DNA damaging agents where suppression of survivin is a secondary event, likely a consequence of transcriptional repression.


Assuntos
Antineoplásicos/farmacologia , Dano ao DNA , Imidazóis/farmacologia , Naftoquinonas/farmacologia , Antineoplásicos/química , Western Blotting , Linhagem Celular Tumoral , DNA de Neoplasias/biossíntese , DNA de Neoplasias/genética , Relação Dose-Resposta a Droga , Células HCT116 , Células HT29 , Histonas/metabolismo , Humanos , Imidazóis/química , Proteínas Inibidoras de Apoptose/metabolismo , Células K562 , Estrutura Molecular , Naftoquinonas/química , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteínas Repressoras/metabolismo , Survivina , Fatores de Tempo , Proteína 28 com Motivo Tripartido
6.
Clin Cancer Res ; 15(7): 2291-301, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19258443

RESUMO

PURPOSE: Aberrant activation of the Notch signaling pathway is commonly observed in human pancreatic cancer, although the mechanism(s) for this activation has not been elucidated. EXPERIMENTAL DESIGN: A panel of 20 human pancreatic cancer cell lines was profiled for the expression of Notch pathway-related ligands, receptors, and target genes. Disruption of intracellular Notch signaling, either genetically by RNA interference targeting NOTCH1 or pharmacologically by means of the gamma-secretase inhibitor GSI-18, was used for assessing requirement of Notch signaling in pancreatic cancer initiation and maintenance. RESULTS: Striking overexpression of Notch ligand transcripts was detectable in the vast majority of pancreatic cancer cell lines, most prominently JAGGED2 (18 of 20 cases, 90%) and DLL4 (10 of 20 cases, 50%). In two cell lines, genomic amplification of the DLL3 locus was observed, mirrored by overexpression of DLL3 transcripts. In contrast, coding region mutations of NOTCH1 or NOTCH2 were not observed. Genetic and pharmacologic inhibition of Notch signaling mitigated anchorage-independent growth in pancreatic cancer cells, confirming that sustained Notch activation is a requirement for pancreatic cancer maintenance. Further, transient pretreatment of pancreatic cancer cells with GSI-18 resulted in depletion in the proportion of tumor-initiating aldehyde dehydrogenase-expressing subpopulation and was associated with inhibition of colony formation in vitro and xenograft engraftment in vivo, underscoring a requirement for the Notch-dependent aldehyde dehydrogenase-expressing cells in pancreatic cancer initiation. CONCLUSIONS: Our studies confirm that Notch activation is almost always ligand dependent in pancreatic cancer, and inhibition of Notch signaling is a promising therapeutic strategy in this malignancy.


Assuntos
Neoplasias Pancreáticas/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Aldeído Desidrogenase/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células , Inibidores Enzimáticos/farmacologia , Amplificação de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Jagged-2 , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/genética , Receptor Notch1/genética , Receptor Notch2/genética
7.
Cancer Biol Ther ; 8(4): 340-6, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19106647

RESUMO

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMNs) are non-invasive precursor lesions of pancreatic cancer. Misexpression of microRNAs (miRNAs) is commonly observed in pancreatic adenocarcinoma. In contrast, miRNA abnormalities in pancreatic cancer precursor lesions have not been documented. EXPERIMENTAL DESIGN: Relative expression levels of a panel of twelve miRNAs upregulated in pancreatic cancers were assessed in 15 non-invasive IPMNs, using quantitative reverse transcription PCR (qRT-PCR). Two significantly overexpressed miRNAs-miR-155 and miR-21-were evaluated by locked nucleic acid in situ hybridization (LNA-ISH) in a panel of 64 archival IPMNs. The expression of miR-155 and miR-21 was also evaluated in pancreatic juice samples obtained from ten patients with surgically resected IPMNs and five patients with non-neoplastic pancreato-biliary disorders ("disease controls"). RESULTS: Significant overexpression by qRT-PCR of ten of the twelve miRNAs was observed in the 15 IPMNs versus matched controls (p < 0.05), with miR-155 (mean 11.6-fold) and miR-21 (mean 12.1-fold) demonstrating highest relative fold-changes in the precursor lesions. LNA-ISH confirmed the expression of miR-155 in 53 of 64 (83%) IPMNs compared to 4 of 54 (7%) normal ducts, and of miR-21 in 52 of 64 (81%) IPMNs compared to 1 of 54 (2%) normal ducts, respectively (p < 0.0001). Upregulation of miR-155 transcripts by qRT-PCR was observed in 6 of 10 (60%) IPMN-associated pancreatic juice samples compared to 0 of 5 (0%) disease controls. CONCLUSIONS: Aberrant miRNA expression is an early event in the multistage progression of pancreatic cancer, and miR-155 warrants further evaluation as a biomarker for IPMNs in clinical samples.


Assuntos
Biomarcadores Tumorais/biossíntese , MicroRNAs/biossíntese , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Biomarcadores Tumorais/genética , Humanos , Hibridização In Situ , MicroRNAs/genética , Suco Pancreático/metabolismo , Neoplasias Pancreáticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
8.
Int J Gastrointest Cancer ; 37(2-3): 57-64, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17827523

RESUMO

BACKGROUND: Somatic mutations of mitochondrial DNA (mtDNA) are increasingly being recognized in many human cancers, but automated sequencing of 16.5 kb of DNA poses an onerous task. We have recently described an oligonucleotide microarray (MitoChip) for rapid and accurate sequencing of the entire mitochondrial genome (Zhou et al., J Mol Diagnostics, 8: 9_14, 2006), greatly facilitating the analysis of mtDNA mutations in cancer. In this report, we perform a comprehensive cataloging of somatic mutations in the mitochondrial genome of human pancreatic cancers using our novel array-based approach. MATERIALS AND METHODS: MitoChip analysis was performed on DNA isolated from 15 histologically confirmed resection specimens of pancreatic ductal adenocarcinomas. In all cases, matched nonneoplastic pancreatic tissue was obtained as germline control for mtDNA sequence. DNA was extracted from snap-frozen cryostat-embedded specimens and hybridized to the sequencing microarray after appropriate polymerase chain reaction amplification and labeling steps. The vast majority of somatic mutational analyses of mtDNA in human cancers utilize lymphocyte DNA as germline control, without excluding the potential for organ-specific polymorphisms. Therefore, we also examined a series of 15 paired samples of DNA obtained from nonneoplastic pancreata and corresponding EBV-immortalized lymphoblastoid cell lines to determine whether lymphocyte DNA provides an accurate surrogate for the mtDNA sequence of pancreatic tissue. RESULTS: We sequenced 497,070 base pairs of mtDNA in the 15 matched samples of pancreatic cancer and nonneoplastic pancreatic tissue, and 467,269 base pairs (94.0%) were assigned by the automated genotyping software. All 15 pancreatic cancers demonstrated at least one somatic mtDNA mutation compared to the control germline DNA with a range of 1-14 alterations. Of the 71 somatic mutations observed in our series, 18 were nonsynonymous coding region alterations (i.e., resulting in an amino acid change), 22 were synonymous coding region alterations, and 31 involved noncoding mtDNA segments (including ribosomal and transfer RNAs). Overall, somatic mutations in the coding region most commonly involved the ND4, COI, and CYTB genes; of note, an A-G transition at nucleotide position 841 in the 12sRNA was observed in three independent samples. In the paired analysis of nonneoplastic pancreata and lymphoblastoid cell line DNA, 14 nucleotide discrepancies were observed out of 226,876 nucleotide sequences (a concordance rate of 99.99%), with 9 samples demonstrating a perfect match across all bases assigned. CONCLUSIONS: Our findings confirm that somatic mtDNA mutations are common in pancreatic cancers, and therefore, have the potential to be a clinically useful biomarker for early detection. Further, our studies confirm that lymphocyte DNA is an excellent, albeit not perfect, surrogate for nonneoplastic pancreatic tissues in terms of being utilized as a germline control. Finally, our report confirms the utility of a high-throughput array-based platform for mtDNA mutational analyses of human cancers.


Assuntos
DNA Mitocondrial/genética , DNA de Neoplasias/genética , Genoma Mitocondrial , Mutação/genética , Neoplasias Pancreáticas/genética , Análise Mutacional de DNA , Genótipo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Pâncreas/metabolismo , Reação em Cadeia da Polimerase , Estudos Retrospectivos
9.
Ann N Y Acad Sci ; 1014: 189-98, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15153434

RESUMO

Multiple endocrine neoplasia type 1 (MEN1), among all syndromes, causes tumors in the highest number of tissue types. Most of the tumors are hormone producing (e.g., parathyroid, enteropancreatic endocrine, anterior pituitary) but some are not (e.g., angiofibroma). MEN1 tumors are multiple for organ type, for regions of a discontinuous organ, and for subregions of a continuous organ. Cancer contributes to late mortality; there is no effective prevention or cure for MEN1 cancers. Morbidities are more frequent from benign than malignant tumor, and both are indicators for screening. Onset age is usually earlier in a tumor type of MEN1 than of nonhereditary cases. Broad trends contrast with those in nonneoplastic excess of hormones (e.g., persistent hyperinsulinemic hypoglycemia of infancy). Most germline or somatic mutations in the MEN1 gene predict truncation or absence of encoded menin. Similarly, 11q13 loss of heterozygosity in tumors predicts inactivation of the other MEN1 copy. MEN1 somatic mutation is prevalent in nonhereditary, MEN1-like tumor types. Compiled germline and somatic mutations show almost no genotype/phenotype relation. Normal menin is 67 kDa, widespread, and mainly nuclear. It may partner with junD, NF-kB, PEM, SMAD3, RPA2, FANCD2, NM23beta, nonmuscle myosin heavy chain II-A, GFAP, and/or vimentin. These partners have not clarified menin's pathways in normal or tumor tissues. Animal models have opened approaches to menin pathways. Local overexpression of menin in Drosophila reveals its interaction with the jun-kinase pathway. The Men1+/- mouse has robust MEN1; its most important difference from human MEN1 is marked hyperplasia of pancreatic islets, a tumor precursor stage.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasia Endócrina Múltipla/genética , Neoplasia Endócrina Múltipla/fisiopatologia , Proteínas Proto-Oncogênicas/genética , Animais , Humanos , Neoplasia Endócrina Múltipla/patologia
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