RESUMO
Patient-derived xenograft (PDX) tumors are powerful tools to study cancer biology. However, the ability of PDX tumors to model the biological and histological diversity of pancreatic ductal adenocarcinoma (PDAC) is not well known. In this study, we subcutaneously implanted 133 primary and metastatic PDAC tumors into immunodeficient mice. Fifty-seven tumors were successfully engrafted and even after extensive passaging, the histology of poorly-, moderately-, and well-differentiated tumors was maintained in the PDX models. Moreover, the fibroblast and collagen contents in the stroma of patient tumors were recapitulated in the corresponding PDX models. Analysis of the clinicopathological features of patients revealed xenograft tumor engraftment was associated with lymphovascular invasion (P = 0.001) and worse recurrence-free (median, 7 vs. 16 months, log-rank P = 0.047) and overall survival (median, 13 vs. 21 months, log-rank P = 0.038). Among successful engraftments, median time of growth required for reimplantation into new mice was 151 days. Reflective of the inherent biological diversity between PDX tumors with rapid (<151 days) and slow growth, differences in their growth were maintained during extensive passaging. Rapid growth was additionally associated with lymph node metastasis (P = 0.022). The association of lymphovascular invasion and lymph node metastasis with PDX formation and rapid growth may reflect an underlying biological mechanism that allows these tumors to adapt and grow in a new environment. While the ability of PDX tumors to mimic the cellular and non-cellular features of the parental tumor stroma provides a valuable model to study the interaction of PDAC cells with the tumor microenvironment, the association of successful engraftment with adverse clinicopathological features suggests PDX models over represent more aggressive forms of this disease.
Assuntos
Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Linfonodos/patologia , Metástase Linfática/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Análise de Sobrevida , Transplante HeterólogoRESUMO
PURPOSE: The initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) results from the interplay of genetic and epigenetic events. While the genetic alterations of PDAC have been well characterized, epigenetic pathways regulating PDAC remain, for the most part, elusive. The goal of this study was to identify novel epigenetic regulators contributing to the biology of PDAC. EXPERIMENTAL DESIGN: In vivo pooled shRNA screens targeting 118 epigenetic proteins were performed in two orthotopic PDAC xenograft models. Candidate genes were characterized in 19 human PDAC cell lines, heterotopic xenograft tumor models, and a genetically engineered mouse (GEM) model of PDAC. Gene expression, IHC, and immunoprecipitation experiments were performed to analyze the pathways by which candidate genes contribute to PDAC. RESULTS: In vivo shRNA screens identified BRD2 and BRD3, members of the BET family of chromatin adaptors, as key regulators of PDAC tumor growth. Pharmacologic inhibition of BET bromodomains enhanced survival in a PDAC GEM model and inhibited growth of human-derived xenograft tumors. BET proteins contribute to PDAC cell growth through direct interaction with members of the GLI family of transcription factors and modulating their activity. Within cancer cells, BET bromodomain inhibition results in downregulation of SHH, a key mediator of the tumor microenvironment and canonical activator of GLI. Consistent with this, inhibition of BET bromodomains decreases cancer-associated fibroblast content of tumors in both GEM and xenograft tumor models. CONCLUSIONS: Therapeutic inhibition of BET proteins offers a novel mechanism to target both the neoplastic and stromal components of PDAC. Clin Cancer Res; 22(16); 4259-70. ©2016 AACR.
Assuntos
Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas de Ligação a RNA/metabolismo , Microambiente Tumoral , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Genes myc , Proteínas Hedgehog/metabolismo , Xenoenxertos , Humanos , Camundongos , Neoplasias Pancreáticas/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Transdução de Sinais , Carga Tumoral , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Isothiocyanates, a family of phytochemicals found in cruciferous vegetables, have cytotoxic effects against several types of tumor cells. Multiple myeloma is a fatal disease characterized by clonal proliferation of plasma cells in the bone marrow. The growing body of preclinical information on the anti-cancer activity of isothiocyanates led us to investigate their anti-myeloma properties. DESIGN AND METHODS: We evaluated the anti-myeloma activity of the isothiocyanates, sulforaphane and phenethyl isothiocyanate, on a panel of human myeloma cell lines as well as primary myeloma tumor cells. Cell viability, apoptosis, cell cycle alterations and cell proliferation were then analyzed in vitro and in a xenograft mouse model in vivo. The molecular sequelae of isothiocyanate treatment in multiple myeloma cells were evaluated by multiplex analyses using bead arrays and western blotting. RESULTS: We observed that sulforaphane and phenylethyl isothiocyanate have activity against myeloma cell lines and patients' myeloma cells both in vitro and in vivo using a myeloma xenograft mouse model. Isothiocyanates induced apoptotic death of myeloma cells; depletion of mitochondrial membrane potential; cleavage of PARP and caspases-3 and -9; as well as down-regulation of anti-apoptotic proteins including Mcl-1, X-IAP, c-IAP and survivin. Isothiocyanates induced G(2)/M cell cycle arrest accompanied by mitotic phosphorylation of histone H3. Multiplex analysis of phosphorylation of diverse components of signaling cascades revealed changes in MAPK activation; increased phosphorylation of c-jun and HSP27; as well as changes in the phosphorylation of Akt, and GSK3α/ß and p53. Isothiocyanates suppressed proliferation of myeloma cells alone and when co-cultured with HS-5 stromal cells. Sulforaphane and phenylethyl isothiocyanate enhanced the in vitro anti-myeloma activity of several conventional and novel therapies used in multiple myeloma. CONCLUSIONS: Our study shows that isothiocyanates have potent anti-myeloma activities and may enhance the activity of other anti-multiple myeloma agents. These results indicate that isothiocyanates may have therapeutic potential in multiple myeloma and provide the preclinical framework for future clinical studies of isothiocyanates in multiple myeloma.
Assuntos
Antineoplásicos/farmacologia , Isotiocianatos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Tiocianatos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Isotiocianatos/uso terapêutico , Isotiocianatos/toxicidade , Camundongos , Camundongos SCID , Mieloma Múltiplo/metabolismo , Células Estromais/efeitos dos fármacos , Sulfóxidos , Tiocianatos/uso terapêutico , Tiocianatos/toxicidade , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The bone marrow (BM) microenvironment consists of extracellular-matrix and the cellular compartment including immune cells. Multiple myeloma (MM) cell and BM accessory cell interaction promotes MM survival via both cell-cell contact and cytokines. Immunomodulatory agents (IMiDs) target not only MM cells, but also MM cell-immune cell interactions and cytokine signaling. Here we examined the in vitro effects of IMiDs on cytokine signaling triggered by interaction of effector cells with MM cells and BM stroma cells. IMiDs diminished interleukin-2, interferonγ, and IL-6 regulator suppressor of cytokine signaling (SOCS)1 expression in immune (CD4T, CD8T, natural-killer T, natural-killer) cells from both BM and PB of MM patients. In addition, coculture of MM cells with healthy PBMCs induced SOCS1 expression in effector cells; conversely, treatment with IMiDs down-regulated the SOCS1 expression. SOCS1 negatively regulates IL-6 signaling and is silenced by hypermethylation in MM cells. To define the mechanism of inhibitory-cytokine signaling in effector cells and MM cells, we next analyzed the interaction of immune cells with MM cells that were epigenetically modified to re-express SOCS1; IMiDs induced more potent CTL responses against SOCS1 re-expressing-MM cells than unmodified MM cells. These data therefore demonstrate that modulation of SOCS1 may enhance immune response and efficacy of IMiDs in MM.
Assuntos
Antineoplásicos/imunologia , Células da Medula Óssea/efeitos dos fármacos , Fatores Imunológicos/imunologia , Mieloma Múltiplo/imunologia , Linfócitos T/efeitos dos fármacos , Talidomida/análogos & derivados , Células da Medula Óssea/imunologia , Linhagem Celular Tumoral , Citocinas/imunologia , Epigênese Genética , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Lenalidomida , Mieloma Múltiplo/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/imunologia , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/imunologia , Linfócitos T/imunologia , Talidomida/imunologiaRESUMO
Aurora-A is a mitotic kinase that regulates mitotic spindle formation and segregation. In multiple myeloma (MM), high Aurora-A gene expression has been correlated with centrosome amplification and proliferation; thus, inhibition of Aurora-A in MM may prove to be therapeutically beneficial. Here we assess the in vitro and in vivo anti-MM activity of MLN8237, a small-molecule Aurora-A kinase inhibitor. Treatment of cultured MM cells with MLN8237 results in mitotic spindle abnormalities, mitotic accumulation, as well as inhibition of cell proliferation through apoptosis and senescence. In addition, MLN8237 up-regulates p53 and tumor suppressor genes p21 and p27. Combining MLN8237 with dexamethasone, doxorubicin, or bortezomib induces synergistic/additive anti-MM activity in vitro. In vivo anti-MM activity of MLN8237 was confirmed using a xenograft-murine model of human-MM. Tumor burden was significantly reduced (P = .007) and overall survival was significantly increased (P < .005) in animals treated with 30 mg/kg MLN8237 for 21 days. Induction of apoptosis and cell death by MLN8237 were confirmed in tumor cells excised from treated animals by TdT-mediated dUTP nick end labeling assay. MLN8237 is currently in phase 1 and phase 2 clinical trials in patients with advanced malignancies, and our preclinical results suggest that MLN8237 may be a promising novel targeted therapy in MM.
Assuntos
Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Aurora Quinase A , Aurora Quinases , Azepinas/uso terapêutico , Ácidos Borônicos/farmacologia , Bortezomib , Ciclo Celular , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Dexametasona/farmacologia , Doxorrubicina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos SCID , Mieloma Múltiplo/enzimologia , Transplante de Neoplasias , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/biossíntese , Pirazinas/farmacologia , Pirimidinas/uso terapêutico , Fuso Acromático/metabolismo , Fatores de Tempo , Transplante Heterólogo , Proteínas Supressoras de Tumor/biossíntese , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This study investigated the cytotoxicity and mechanism of action of AS703026, a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026 inhibited growth and survival of MM cells and cytokine-induced osteoclast differentiation more potently (9- to 10-fold) than AZD6244. Inhibition of proliferation induced by AS703026 was mediated by G0-G1 cell cycle arrest and was accompanied by reduction of MAF oncogene expression. AS703026 further induced apoptosis via caspase 3 and Poly ADP ribose polymerase (PARP) cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti-MM therapies. Significant tumour growth reduction in AS703026- vs. vehicle-treated mice bearing H929 MM xenograft tumours correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nmol/l) was cytotoxic against the majority of tumour cells tested from patients with relapsed and refractory MM (84%), regardless of mutational status of RAS and BRAF genes. Importantly, BMSC-induced viability of MM patient cells was similarly blocked within the same dose range. Our results therefore support clinical evaluation of AS703026, alone or in combination with other anti-MM agents, to improve patient outcome.
Assuntos
Antineoplásicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mieloma Múltiplo/patologia , Niacinamida/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Camundongos , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CS1 is highly expressed on tumor cells from the majority of multiple myeloma (MM) patients regardless of cytogenetic abnormalities or response to current treatments. Furthermore, CS1 is detected in MM patient sera and correlates with active disease. However, its contribution to MM pathophysiology is undefined. We here show that CS1 knockdown using lentiviral short-interfering RNA decreased phosphorylation of ERK1/2, AKT, and STAT3, suggesting that CS1 induces central growth and survival signaling pathways in MM cells. Serum deprivation markedly blocked survival at earlier time points in CS1 knockdown compared with control MM cells, associated with earlier activation of caspases, poly(ADP-ribose) polymerase, and proapoptotic proteins BNIP3 and BIK. CS1 knockdown further delayed development of MM tumor and prolonged survival in mice. Conversely, CS1 overexpression promoted myeloma cell growth and survival by significantly increasing myeloma adhesion to bone marrow stromal cells (BMSCs) and enhancing myeloma colony formation in semisolid culture. Moreover, CS1 increased c-maf-targeted cyclin D2-dependent proliferation, -integrin beta7/alphaE-mediated myeloma adhesion to BMSCs, and -vascular endothelial growth factor-induced bone marrow angiogenesis in vivo. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.