RESUMEN
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.
Asunto(s)
Adenocarcinoma/patología , Carcinoma Ductal Pancreático/patología , Páncreas/patología , Neoplasias Pancreáticas/patología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Humanos , Ganglios Linfáticos/patología , Metástasis Linfática/patología , Masculino , Ratones , Persona de Mediana Edad , Trasplante de Neoplasias , Neoplasias Experimentales/patología , Análisis de Supervivencia , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas de Unión al ARN/metabolismo , Microambiente Tumoral , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Genes myc , Proteínas Hedgehog/metabolismo , Xenoinjertos , Humanos , Ratones , Neoplasias Pancreáticas/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/química , Transducción de Señal , Carga Tumoral , Microambiente Tumoral/genéticaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Isotiocianatos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Tiocianatos/farmacología , Animales , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Isotiocianatos/uso terapéutico , Isotiocianatos/toxicidad , Ratones , Ratones SCID , Mieloma Múltiple/metabolismo , Células del Estroma/efectos de los fármacos , Sulfóxidos , Tiocianatos/uso terapéutico , Tiocianatos/toxicidad , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Antineoplásicos/inmunología , Células de la Médula Ósea/efectos de los fármacos , Factores Inmunológicos/inmunología , Mieloma Múltiple/inmunología , Linfocitos T/efectos de los fármacos , Talidomida/análogos & derivados , Células de la Médula Ósea/inmunología , Línea Celular Tumoral , Citocinas/inmunología , Epigénesis Genética , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Lenalidomida , Mieloma Múltiple/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Células del Estroma/inmunología , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Linfocitos T/inmunología , Talidomida/inmunologíaRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Azepinas/farmacología , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Aurora Quinasa A , Aurora Quinasas , Azepinas/uso terapéutico , Ácidos Borónicos/farmacología , Bortezomib , Ciclo Celular , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Dexametasona/farmacología , Doxorrubicina/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones SCID , Mieloma Múltiple/enzimología , Trasplante de Neoplasias , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/biosíntesis , Pirazinas/farmacología , Pirimidinas/uso terapéutico , Huso Acromático/metabolismo , Factores de Tiempo , Trasplante Heterólogo , Proteínas Supresoras de Tumor/biosíntesis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mieloma Múltiple/patología , Niacinamida/análogos & derivados , Inhibidores de Proteínas Quinasas/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Ratones , Ratones SCID , Mieloma Múltiple/tratamiento farmacológico , Niacinamida/farmacología , Niacinamida/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.