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1.
Methods Mol Biol ; 2035: 223-231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31444752

RESUMO

Guanine-rich DNA sequences are able to spontaneously fold into G-quadruplex structures in the presence of certain metal cations. In the human genome, the majority of DNA G-quadruplexes form at the telomeres and regulatory regions of cancer-related genes. The formation of these structures is implicated in nuclear processes involving DNA, including transcription, DNA replication, and DNA repair. In the past few decades, small molecules which can stabilize these structures have been shown to suppress the telomere extension and to inhibit oncogene transcription. Therefore, DNA G-quadruplexes are thought to be attractive targets for new anticancer therapies. In this chapter, we describe step by step a DNA polymerase extension method for the characterization of G-quadruplex formation and identification of G-quadruplex-interactive compounds. This method is based on the principle that DNA polymerase is incapable to resolve G-quadruplex structure and pauses at 3'-end of the G-quadruplex forming region when it transverses to the 5'-end of the template. Results from the DNA polymerase stop assay can provide the basis for further studies aimed at elucidating the major G-quadruplexes formed by sequences consisting of more than four runs of contiguous guanines, as well as the specificity of G-quadruplex-interactive molecules in binding different G-quadruplex topologies.

2.
Cancer Treat Res ; 178: 237-252, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31209848

RESUMO

The application of next-generation sequencing in cancer genomics allowed for a better understanding of the genetics and pathogenesis of cancer. Single-cell genomics is a relatively new field that has enhanced our current knowledge of the genetic diversity of cells involved in the complex biological systems of cancer. Single-cell genomics is a rapidly developing field, and current technologies can assay a single cell's gene expression, DNA variation, epigenetic state, and nuclear structure. Statistical and computational methods are central to single-cell genomics and allows for extraction of meaningful information. The translational application of single-cell sequencing in precision cancer therapy has the potential to improve cancer diagnostics, prognostics, targeted therapy, early detection, and noninvasive monitoring. Furthermore, single-cell genomics will transform cancer research as even initial experiments have revolutionized our current understanding of gene regulation and disease.


Assuntos
Genômica , Neoplasias , Medicina de Precisão , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Prognóstico
3.
Trends Pharmacol Sci ; 40(5): 327-341, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30975442

RESUMO

Triptolide, a compound isolated from a Chinese medicinal herb, possesses potent antitumor, immunosuppressive, and anti-inflammatory properties, but is clinically limited due to its poor solubility, bioavailability, and toxicity. Recently, Minnelide, a water-soluble prodrug of triptolide, was shown to have potent antitumor activity in various preclinical cancer models. Minnelide is currently in Phase II clinical trials for treatment of advanced pancreatic cancer, which has fueled increased interest in this promising agent. Here, we review the recent advances in the biological activity of triptolide and its analogs, their mechanisms of actions, and their clinical developments. A special emphasis is given to proteins and pathways within the tumor and stromal compartments that are targeted by triptolide and its analogs as well as the ongoing clinical trials.


Assuntos
Antineoplásicos/farmacologia , Diterpenos/química , Diterpenos/farmacologia , Fenantrenos/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Diterpenos/uso terapêutico , Compostos de Epóxi/química , Compostos de Epóxi/farmacologia , Compostos de Epóxi/uso terapêutico , Humanos , Imunossupressores/química , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Fenantrenos/química , Fenantrenos/uso terapêutico
4.
Cell ; 177(3): 572-586.e22, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30955884

RESUMO

Drug resistance and relapse remain key challenges in pancreatic cancer. Here, we have used RNA sequencing (RNA-seq), chromatin immunoprecipitation (ChIP)-seq, and genome-wide CRISPR analysis to map the molecular dependencies of pancreatic cancer stem cells, highly therapy-resistant cells that preferentially drive tumorigenesis and progression. This integrated genomic approach revealed an unexpected utilization of immuno-regulatory signals by pancreatic cancer epithelial cells. In particular, the nuclear hormone receptor retinoic-acid-receptor-related orphan receptor gamma (RORγ), known to drive inflammation and T cell differentiation, was upregulated during pancreatic cancer progression, and its genetic or pharmacologic inhibition led to a striking defect in pancreatic cancer growth and a marked improvement in survival. Further, a large-scale retrospective analysis in patients revealed that RORγ expression may predict pancreatic cancer aggressiveness, as it positively correlated with advanced disease and metastasis. Collectively, these data identify an orthogonal co-option of immuno-regulatory signals by pancreatic cancer stem cells, suggesting that autoimmune drugs should be evaluated as novel treatment strategies for pancreatic cancer patients.


Assuntos
Adenocarcinoma/patologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/patologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Epigênese Genética , Biblioteca Gênica , Humanos , Camundongos , Camundongos Knockout , Camundongos SCID , Células-Tronco Neoplásicas/citologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Interleucina-10/antagonistas & inibidores , Receptores de Interleucina-10/genética , Receptores de Interleucina-10/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transcriptoma , Células Tumorais Cultivadas
5.
Nucleic Acids Res ; 47(8): e48, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30799483

RESUMO

Characterization of individual cell types is fundamental to the study of multicellular samples. Single-cell RNAseq techniques, which allow high-throughput expression profiling of individual cells, have significantly advanced our ability of this task. Currently, most of the scRNA-seq data analyses are commenced with unsupervised clustering. Clusters are often assigned to different cell types based on the enriched canonical markers. However, this process is inefficient and arbitrary. In this study, we present a technical framework of training the expandable supervised-classifier in order to reveal the single-cell identities as soon as the single-cell expression profile is input. Using multiple scRNA-seq datasets we demonstrate the superior accuracy, robustness, compatibility and expandability of this new solution compared to the traditional methods. We use two examples of the model upgrade to demonstrate how the projected evolution of the cell-type classifier is realized.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/genética , Análise de Célula Única/estatística & dados numéricos , Software , Aprendizado de Máquina Supervisionado , Transcriptoma , Animais , Linhagem da Célula/genética , Análise por Conglomerados , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , RNA Citoplasmático Pequeno/genética , Análise de Sequência de RNA
7.
Cancer Cell ; 34(5): 807-822.e7, 2018 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-30423298

RESUMO

Our recent ERK1/2 inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) indicated ERK1/2-independent mechanisms maintaining MYC protein stability. To identify these mechanisms, we determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, MYC degradation was independent of PI3K-AKT-GSK3ß signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation and performed a kinome-wide proteomics screen. We identified an ERK1/2-inhibition-induced feedforward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing degradation. Concurrent inhibition of ERK1/2 and ERK5 disrupted this mechanism, synergistically causing loss of MYC and suppressing PDAC growth.


Assuntos
Carcinoma Ductal Pancreático/patologia , MAP Quinase Quinase 5/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias Pancreáticas/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinases da Família src/metabolismo
8.
Clin Cancer Res ; 24(19): 4798-4807, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30084839

RESUMO

Purpose: The tumor microenvironment (TME) evolves to support tumor progression. One marker of more aggressive malignancy is hyaluronan (HA) accumulation. Here, we characterize biological and physical changes associated with HA-accumulating (HA-high) tumors.Experimental Design: We used immunohistochemistry, in vivo imaging of tumor pH, and microdialysis to characterize the TME of HA-high tumors, including tumor vascular structure, hypoxia, tumor perfusion by doxorubicin, pH, content of collagen. and smooth muscle actin (α-SMA). A novel method was developed to measure real-time tumor-associated soluble cytokines and growth factors. We also evaluated biopsies of murine and pancreatic cancer patients to investigate HA and collagen content, important contributors to drug resistance.Results: In immunodeficient and immunocompetent mice, increasing tumor HA content is accompanied by increasing collagen content, vascular collapse, hypoxia, and increased metastatic potential, as reflected by increased α-SMA. In vivo treatment of HA-high tumors with PEGylated recombinant human hyaluronidase (PEGPH20) dramatically reversed these changes and depleted stores of VEGF-A165, suggesting that PEGPH20 may also diminish the angiogenic potential of the TME. Finally, we observed in xenografts and in pancreatic cancer patients a coordinated increase in HA and collagen tumor content.Conclusions: The accumulation of HA in tumors is associated with high tIP, vascular collapse, hypoxia, and drug resistance. These findings may partially explain why more aggressive malignancy is observed in the HA-high phenotype. We have shown that degradation of HA by PEGPH20 partially reverses this phenotype and leads to depletion of tumor-associated VEGF-A165. These results encourage further clinical investigation of PEGPH20. Clin Cancer Res; 24(19); 4798-807. ©2018 AACR.


Assuntos
Carcinogênese/genética , Colágeno/metabolismo , Hialuronoglucosaminidase/administração & dosagem , Neoplasias/terapia , Animais , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Colágeno/genética , Humanos , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/genética , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Pharmaceutics ; 10(3)2018 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-30096910

RESUMO

Multidrug resistance (MDR) is a major hurdle which must be overcome to effectively treat cancer. ATP-binding cassette transporters (ABC transporters) play pivotal roles in drug absorption and disposition, and overexpression of ABC transporters has been shown to attenuate cellular/tissue drug accumulation and thus increase MDR across a variety of cancers. Overcoming MDR is one desired approach to improving the survival rate of patients. To date, a number of modulators have been identified which block the function and/or decrease the expression of ABC transporters, thereby restoring the efficacy of a range of anticancer drugs. However, clinical MDR reversal agents have thus far proven ineffective and/or toxic. The need for new, effective, well-tolerated and nontoxic compounds has led to the development of natural compounds and their derivatives to ameliorate MDR. This review evaluates whether synthetically modifying natural compounds is a viable strategy to generate potent, nontoxic, ABC transporter inhibitors which may potentially reverse MDR.

10.
Expert Rev Hematol ; 11(7): 587-596, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29912583

RESUMO

INTRODUCTION: Pixantrone is a first-in-class aza-anthracenedione approved as monotherapy for treatment of relapsed or refractory aggressive diffuse B-cell non-Hodgkin's lymphoma (NHL), a patient group which is notoriously difficult to treat. It has a unique chemical structure and pharmacologic properties distinguishing it from anthracyclines and anthracenediones. Areas covered: The chemical structure and mode of action of pixantrone versus doxorubicin and mitoxantrone; preclinical evidence for pixantrone's therapeutic effect and cardiac tolerability; efficacy and safety of pixantrone in clinical trials; ongoing and completed trials of pixantrone alone or as combination therapy; and the risk of cardiotoxicity of pixantrone versus doxorubicin and mitoxantrone. Expert commentary: Currently, pixantrone is the only approved therapy for multiply relapsed or refractory NHL, an area with few available effective treatment options. Pixantrone is currently being investigated as combination therapy with other drugs including several targeted therapies, with the ultimate goal of improved survival in heavily pretreated patients. In order for pixantrone to be acknowledged in the treatment of aggressive NHL, the perception of pixantrone as an anthracycline-like agent that has anthracycline-like activity and cardiotoxicity needs to be changed. Further data from ongoing clinical trials will help in confirming pixantrone as an effective and safe option.


Assuntos
Isoquinolinas/uso terapêutico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Ensaios Clínicos como Assunto , Intervalo Livre de Doença , Doxorrubicina/efeitos adversos , Doxorrubicina/uso terapêutico , Humanos , Isoquinolinas/efeitos adversos , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/mortalidade , Linfoma Difuso de Grandes Células B/patologia , Mitoxantrona/efeitos adversos , Mitoxantrona/uso terapêutico , Taxa de Sobrevida
11.
Methods Mol Biol ; 1706: 293-302, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29423805

RESUMO

RNA interference (RNAi) is a biological process by which double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. As a tool for knocking down the expression of individual genes posttranscriptionally, RNAi has been widely used to study the cellular function of genes. In this chapter, I describe procedures for using gene-specific, synthetic, short interfering RNA (siRNA) to induce gene silencing in mammalian cells. Protocols for using lipid-based transfection reagents and electroporation techniques are provided. Potential challenges and problems associated with the siRNA technology are also discussed.


Assuntos
Eletroporação/métodos , Técnicas de Silenciamento de Genes/métodos , Interferência de RNA , Estabilidade de RNA , Transfecção/métodos , Animais , Humanos
12.
Oncotarget ; 9(4): 4485-4495, 2018 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-29435118

RESUMO

A number of folate receptor (FR) targeted small molecular drugs and monoclonal antibodies have been introduced into clinical trials to treat FR positive cancers. Because the therapeutic efficacy of these drugs depends prominently on the level of FR-α expression on the cancer cells, patients have been commonly selected for FR-targeted therapies based on the intensity of a folate-targeted radioimaging agent. Unfortunately, uptake of such imaging agents can be mediated by both major isoforms of the folate receptor, FR-α and FR-ß. Logically, if the FR positive cell population in a tumor mass is dominated by FR-ß positive macrophages, patients could be selected for therapy that have few FR-expressing cancer cells. Although several IHC studies have examined expression of either FR-α or FR-ß, no study to date has investigated expression of both FR-α and FR-ß in the same tumor mass. Herein, we utilize monoclonal antibodies specific for FR-α (mAb343) and FR-ß (m909) to query each isoform's expression in a range of cancers. We show that lung and pancreatic adenocarcinomas express the full spectrum of FR-α and FR-ß combinations with ~76% of lung adenocarcinomas expressing both FR-α and FR-ß while pancreatic cancers express primarily FR-ß. Thus, while folate-targeted imaging of lung cancer patients might accurately reflect the expression of FR-α on lung cancer cells, imaging of pancreatic cancer patients could mislead a physician into treating a nonresponding patient. Overall, these data suggest that an independent analysis of both FR-α and FR-ß should be obtained to predict the potential efficacy of a folate-targeted drug.

13.
Theranostics ; 8(21): 5986-5994, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30613276

RESUMO

Rationale: Exosomes are small extracellular vesicles secreted by most cells that are found in blood and other bodily fluids, and which contain cytoplasmic material and membrane factors corresponding to their cell type of origin. Exosome membrane factors and contents have been reported to alter adjacent and distant cell behavior in multiple studies, but the impact of cancer-derived exosomes on chemoresistance is less clear. Methods: Exosomes isolated from three pancreatic cancer (PC) cell lines displaying variable gemcitabine (GEM) resistance (PANC-1, MIA PaCa-2, and BxPC-3) were tested for their capacity to transmit chemoresistance among these cell lines. Comparative proteomics was performed to identify key exosomal proteins that conferred chemoresistance. Cell survival was assessed in GEM responsive PC cell lines treated with recombinant Ephrin type-A receptor 2 (EphA2), a candidate chemoresistance transfer factor, or exosomes from a chemoresistant PC cell line treated with or without EphA2 shRNA. Results: Exosomes from chemoresistant PANC-1 cells increased the GEM resistance of MIA PaCa-2 and BxPC-3 cell cultures. Comparative proteomics determined that PANC-1 exosomes overexpressed Ephrin type-A receptor 2 (EphA2) versus exosomes of less chemoresistant PC cell lines MIA PaCa-2 and BxPC-3. EphA2-knockdown in PANC-1 cells inhibited their ability to transmit exosome-mediated chemoresistance to MIA PaCa-2 and BxPC-3, while treatment of MIA PaCa-2 and BxPC-3 cells with soluble EphA2 did not promote chemoresistance, indicating that membrane carried EphA2 was important for the EphA2 chemoresistance effect. Conclusion: Exosomal EphA2 expression could transmit chemoresistance and may potentially serve as a minimally-invasive predictive biomarker for PC treatment response. Further work should address whether additional exosomal factors regulate resistance to other cancer therapeutic agents for PC or other cancer types.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Exossomos/metabolismo , Neoplasias Pancreáticas/patologia , Receptor EphA2/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Humanos , Proteoma/análise
14.
World J Gastroenterol ; 23(45): 7945-7951, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29259370

RESUMO

Pancreatic carcinomas with acinar differentiation are rare, accounting for 1%-2% of adult pancreatic tumors; they include pancreatic acinar cell carcinoma (PACC), pancreatoblastoma, and carcinomas of mixed differentiation. Patients with PACC have a prognosis better than pancreatic ductal adenocarcinomas but worse than pancreatic neuroendocrine tumors. Reports of overall survival range from 18 to 47 mo. A literature review on PACCs included comprehensive genomic profiling and whole exome sequencing on a series of more than 70 patients as well as other diagnostic studies including immunohistochemistry. Surgical resection of PACC is the preferred treatment for localized and resectable tumors. The efficacy of adjuvant treatment is unclear. Metastatic PACCs are generally not curable and treated with systemic chemotherapy. They are moderately responsive to chemotherapy with different regimens showing various degrees of response in case reports/series. Most of these regimens were developed to treat patients with pancreatic ductal adenocarcinomas or colorectal adenocarcinomas. Review of PACC's molecular profiling showed a number of gene alterations such as: SMAD4, BRAF, BRCA2, TP53, RB1, MEN1, JAK-1, BRCA-1, BRCA-2, and DNA mismatch repair abnormalities. PACCs had multiple somatic mutations with some targetable with available drugs. Therefore, molecular profiling of PACC should be an option for patients with refractory PACC.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Carcinoma de Células Acinares/genética , Neoplasias Pancreáticas/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Acinares/mortalidade , Carcinoma de Células Acinares/patologia , Carcinoma de Células Acinares/terapia , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Mutação , Pâncreas/patologia , Pâncreas/cirurgia , Pancreatectomia , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Prognóstico , Transdução de Sinais/genética , Taxa de Sobrevida , Sequenciamento Completo do Exoma
15.
Carcinogenesis ; 38(10): 1036-1046, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29048549

RESUMO

Development of resistance to chemotherapeutic drugs is a major challenge in the care of patients with pancreatic ductal adenocarcinoma (PDAC). Acquired resistance to chemotherapeutic agents in PDAC has been linked to a subset of cancer cells termed 'cancer stem cells' (CSCs). Therefore, an improved understanding of the molecular events underlying the development of pancreatic CSCs is required to identify new therapeutic targets to overcome chemoresistance. Accumulating evidence indicates that curcumin, a phenolic compound extracted from turmeric, can overcome de novo chemoresistance and re-sensitize tumors to various chemotherapeutic agents. However, the underlying mechanisms for curcumin-mediated chemosensitization remain unclear. The Enhancer of Zeste Homolog-2 (EZH2) subunit of Polycomb Repressive Complex 2 (PRC2) was recently identified as a key player regulating drug resistance. EZH2 mediates interaction with several long non-coding RNAs (lncRNAs) to modulate epithelial-mesenchymal transition and cancer stemness, phenomena commonly associated with drug resistance. Here, we report the re-sensitization of chemoresistant PDAC cells by curcumin through the inhibition of the PRC2-PVT1-c-Myc axis. Using gemcitabine-resistant PDAC cell lines, we found that curcumin sensitized chemoresistant cancer cells by inhibiting the expression of the PRC2 subunit EZH2 and its related lncRNA PVT1. Curcumin was also found to prevent the formation of spheroids, a hallmark of CSCs, and to down-regulate several self-renewal driving genes. In addition, we confirmed our in vitro findings in a xenograft mouse model where curcumin inhibited gemcitabine-resistant tumor growth. Overall, this study indicates clinical relevance for combining curcumin with chemotherapy to overcome chemoresistance in PDAC.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Curcumina/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Curcumina/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , RNA Longo não Codificante/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
J Vis Exp ; (126)2017 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-28872142

RESUMO

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 µm within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.


Assuntos
Bioensaio/métodos , Técnicas de Cocultura/métodos , Fibroblastos/metabolismo , Neoplasias Pancreáticas/metabolismo , Esferoides Celulares/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Pancreáticas/patologia
17.
PLoS One ; 12(8): e0183871, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28841710

RESUMO

ROCK, or Rho-associated coiled coil-containing protein kinase, is a member of the AGC kinase family and has been shown to play a role in cell migration, ECM synthesis, stress-fiber assembly, and cell contraction. Increased ROCK expression has been reported in multiple pathological conditions, including cancer. Here, we report increased expression of ROCK 1 in pancreatic tumor epithelial cells as well as in cancer associated fibroblasts (CAF). In our analysis, 62% of tumor samples exhibited ≥2+ in staining intensity by IHC analysis, versus 40% of adjacent normal tissue samples (P<0.0001). Thus, we hypothesized that ROCKs may play a significant role in pancreatic cancer progression, and may serve as a suitable target for treatment. We report a low frequency (4/34) amplification of the ROCK1 gene locus at chromosome 18q11.1 in pancreatic ductal adenocarcinoma (PDAC) patient tissue samples by aCGH analysis. Inhibition of ROCK kinase activity by a small molecule inhibitor (fasudil) resulted in moderate (IC50s of 6-71 µM) inhibition of PDAC cell proliferation, migration, and activation of co-cultured stellate cells. In the KPC mouse model for pancreatic cancer, fasudil decreased tumor collagen deposition. This translated to an enhanced overall survival of the mice and an increase in gemcitabine uptake. Though fasudil may target both the tumor epithelial cells and the CAFs, our findings are consistent with the hypothesis that inhibition of tumor stroma enhances drug penetration and efficacy in PDAC. Overall, our data suggests that ROCK1 may serve as a potential therapeutic target to enhance current treatment regimens for pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Células Estromais/patologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/genética , Hibridização Genômica Comparativa , Inativação Gênica , Humanos , Camundongos , Metástase Neoplásica , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , RNA Interferente Pequeno/genética , Quinases Associadas a rho/metabolismo
18.
Br J Cancer ; 117(4): 572-582, 2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-28720843

RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer with complex genomes and dense fibrotic stroma. This study was designed to identify clinically relevant somatic aberrations in pancreatic cancer genomes of patients with primary and metastatic disease enrolled and treated in two clinical trials. METHODS: Tumour nuclei were flow sorted prior to whole genome copy number variant (CNV) analysis. Targeted or whole exome sequencing was performed on most samples. We profiled biopsies from 68 patients enrolled in two Stand Up to Cancer (SU2C)-sponsored clinical trials. These included 38 resected chemoradiation naïve tumours (SU2C 20206-003) and metastases from 30 patients who progressed on prior therapies (SU2C 20206-001). Patient outcomes including progression-free survival (PFS) and overall survival (OS) were observed. RESULTS: We defined: (a) CDKN2A homozygous deletions that included the adjacent MTAP gene, only its' 3' region, or excluded MTAP; (b) SMAD4 homozygous deletions that included ME2; (c) a pancreas-specific MYC super-enhancer region; (d) DNA repair-deficient genomes; and (e) copy number aberrations present in PDA patients with long-term (⩾ 40 months) and short-term (⩽ 12 months) survival after surgical resection. CONCLUSIONS: We provide a clinically relevant framework for genomic drivers of PDA and for advancing novel treatments.


Assuntos
Sequência de Bases , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Deleção de Sequência , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/secundário , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p18/genética , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Reparo do DNA/genética , Intervalo Livre de Doença , Elementos Facilitadores Genéticos , Exoma , Feminino , Genes myc , Homozigoto , Humanos , Malato Desidrogenase/genética , Masculino , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Pâncreas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Purina-Núcleosídeo Fosforilase/genética , Proteína Smad4/genética , Taxa de Sobrevida , Proteína Supressora de Tumor p53/genética
19.
PLoS One ; 11(10): e0165586, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27792755

RESUMO

Perineural invasion (PNI) is thought to be one of the factors responsible for the high rate of tumor recurrence after surgery and the pain generation associated with pancreatic cancer. Signaling via the nerve growth factor (NGF) pathway between pancreatic cancer cells and the surrounding nerves has been implicated in PNI, and increased levels of these proteins have been correlated to poor prognosis. In this study, we examine the molecular mechanism of the NGF signaling pathway in PNI in pancreatic cancer. We show that knocking down NGF or its receptors, TRKA and p75NTR, or treatment with GW441756, a TRKA kinase inhibitor, reduces the proliferation and migration of pancreatic cancer cells in vitro. Furthermore, pancreatic cancer cells migrate towards dorsal root ganglia (DRG) in a co-culture assay, indicating a paracrine NGF signaling between the DRGs and pancreatic cancer cells. Knocking down the expression of NGF pathway proteins or inhibiting the activity of TRKA by GW441756 reduced the migratory ability of Mia PaCa2 towards the DRGs. Finally, blocking NGF signaling by NGF neutralizing antibodies or GW441756 inhibited the neurite formation in PC-12 cells in response to conditioned media from pancreatic cancer cells, indicating a reciprocal signaling pathway between the pancreatic cancer cells and nerves. Our results indicate that NGF signaling pathway provides a potential target for developing molecularly targeted therapies to decrease PNI and reduce pain generation. Since there are several TRKA antagonists currently in early clinical trials they could now be tested in the clinical situation of pancreatic cancer induced pain.


Assuntos
Fator de Crescimento Neural/metabolismo , Sistema Nervoso/patologia , Neoplasias Pancreáticas/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Gânglios Espinais/patologia , Técnicas de Inativação de Genes , Humanos , Indóis/farmacologia , Invasividade Neoplásica , Fator de Crescimento Neural/deficiência , Fator de Crescimento Neural/genética , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Receptor trkA/antagonistas & inibidores , Receptor trkA/deficiência , Receptor trkA/genética , Receptores de Fator de Crescimento Neural/deficiência , Receptores de Fator de Crescimento Neural/genética
20.
World J Gastrointest Oncol ; 7(9): 132-40, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26380056

RESUMO

Adenosquamous carcinoma of the pancreas (ASCP) is a rare entity. Like adenocarcinoma of the pancreas, overall survival is poor. Characteristics of ASCP include central tumor necrosis, along with osteoclasts and hypercalcemia. Various theories exist as to why this histological subtype exists, as normal pancreas tissue has no benign squamous epithelium. Due to the rarity of this disease, limited molecular analysis has been performed, and those reports indicate unique molecular features of ASCP. In this paper, we characterize 23 patients diagnosed with ASCP through molecular profiling using immunohistochemistry staining, fluorescent in situ hybridization, chromogenic in situ hybridization, and gene sequencing, Additionally, we provide a comprehensive literature review of what is known to date of ASCP. Molecular characterization revealed overexpression in MRP1 (80%), MGMT (79%), TOP2A (75), RRM1 (42%), TOPO1 (42%), PTEN (45%), CMET (40%), and C-KIT (10%) among others. One hundred percent of samples tested were positive for KRAS mutations. This analysis shows heretofore unsuspected leads to be considered for treatments of this rare type of exocrine pancreas cancer. Molecular profiling may be appropriate to provide maximum information regarding the patient's tumor. Further work should be pursued to better characterize this disease.

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