RESUMEN
The Ras/Raf/MEK pathway represents an important oncogenic signaling pathway in gastrointestinal malignancies, including pancreatic cancer. Although activating B-Raf mutations are infrequent in pancreatic cancer, we hypothesized that targeting Raf could be valuable for therapy of this cancer entity. Moreover, as vascular endothelial growth factor receptor 2 (VEGFR2) is involved in tumor angiogenesis, we sought to investigate the effects of dual inhibition of Raf and VEGFR2 on pancreatic tumor growth, vascularization, and metastasis. Effects of a Raf/VEGFR2 inhibitor (NVP-AAL881) on pancreatic cancer cells, endothelial cells, and vascular smooth muscle cells were determined by Western blotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, and migration assays, respectively. Changes in the expression of VEGF-A or survivin were investigated by ELISA and/or real-time PCR. The growth-inhibitory effects of Raf/VEGFR2 inhibition were additionally evaluated in orthotopic tumor models. Results showed that various Raf isoforms were activated in pancreatic cancer cells and NVP-AAL881 diminished the activation of MEK, Akt, Erk, and also STAT3. Moreover, dual inhibition of Raf/VEGFR2 significantly reduced VEGF expression and impaired cancer cell migration. Importantly, besides blocking VEGF-induced Erk and SAPK phosphorylation in endothelial cells, the Raf inhibitor diminished STAT3 phosphorylation, independent of a VEGFR2 blockade, and reduced the expression of survivin. In addition, cell proliferation and migration of both endothelial cells and vascular smooth muscle cells were significantly reduced. In vivo, blocking Raf/VEGFR2 significantly inhibited orthotopic tumor growth and vascularization and reduced cancer metastasis. In conclusion, blocking Raf exerts growth-inhibitory effects on pancreatic tumor cells, endothelial cells, and pericytes and elicits antiangiogenic properties. Dual targeting of Raf and VEGFR2 appears to be a valid strategy for therapy of pancreatic cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Células Endoteliales/efectos de los fármacos , Isoquinolinas/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Pericitos/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Quinasas raf/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Isoquinolinas/farmacología , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia/patología , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Pericitos/metabolismo , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Quinasas raf/metabolismoRESUMEN
BACKGROUND AND AIMS: Activation of the mitogen-activated protein kinase-extracellular-signal-regulated kinase (ERK) pathways plays an important role in the progression of hepatocellular carcinoma (HCC). Importantly, Raf kinases are principal effectors within this oncogenic signaling cascade. We hypothesized that concomitant inhibition of Raf and vascular endothelial growth factor receptor 2 (VEGFR2) will affect tumor growth and angiogenesis of HCC. MATERIALS AND METHODS: Human HCC cell lines, endothelial cells (EC), and vascular smooth muscle cells (VSMC) were used. For blocking Raf kinase and VEGFR2, the small molecule inhibitor NVP-AAL881 (Novartis, USA) was used. Activation of signaling intermediates was assessed by Western blotting, and changes in cell motility were evaluated in migration assays. Effects of NVP-AAL881 on HCC growth were determined in a subcutaneous tumor model. RESULTS: NVP-AAL881 disrupted activation of ERK and STAT3 in HCC cells and reduced cancer cell motility. In addition, the migration of ECs and VSMC was also significantly impaired. In ECs, HCC-conditioned media-induced activation of STAT3 was diminished by NVP-AAL881 treatment. In vivo, NVP-AAL881 significantly reduced tumor growth, CD31-vessel area, and numbers of BrdU-positive proliferating tumor cells. CONCLUSIONS: Combined inhibition of Raf and VEGFR2 disrupts oncogenic signaling and efficiently reduces tumor growth and vascularization of HCC. Hence, this strategy could prove valuable for therapy of HCC.
Asunto(s)
Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Neoplasias Hepáticas Experimentales/irrigación sanguínea , Neovascularización Patológica/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Quinasas raf/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Humanos , Isoquinolinas/farmacología , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Desnudos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Trasplante de Neoplasias , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
B-Raf is an important mediator of cell proliferation and survival signals transduced via the Ras-Raf-MEK-ERK cascade. BRAF mutations have been detected in several tumors, including papillary thyroid carcinoma, but the precise role of B-Raf as a therapeutic target for thyroid carcinoma is still under investigation. We analyzed a panel of 93 specimens and 14 thyroid carcinoma cell lines for the presence of BRAF mutations and activation of the mitogen-activated protein/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. We also compared the effect of a B-Raf small inhibitory RNA construct and the B-Raf kinase inhibitor AAL881 on both B-Raf wild-type and mutant thyroid carcinoma cell lines. We found a high prevalence of the T1799A (V600E) mutation in papillary and anaplastic carcinoma specimens and cell lines. There was no difference in patient age, B-Raf expression, Ki67 immunostaining, or clinical stage at presentation between wild-type and BRAF(V600E) specimens. Immunodetection of phosphorylated and total forms of MEK and ERK revealed no difference in their phosphorylation between wild-type and BRAF(V600E) patient specimens or cell lines. Furthermore, a small inhibitory RNA construct targeting the expression of both wild-type B-Raf and B-Raf(V600E) induced a comparable reduction of viability in both wild-type and BRAF(V600E) mutant cancer cells. Interestingly, AAL881 inhibited MEK and ERK phosphorylation and induced apoptosis preferentially in BRAF(V600E)-harboring cells than wild-type ones, possibly because of better inhibitory activity against B-Raf(V600E). We conclude that B-Raf is important for the pathophysiology of thyroid carcinomas irrespective of mutational status. Small molecule inhibitors that selectively target B-Raf(V600E) may provide clinical benefit for patients with thyroid cancer.
Asunto(s)
Adenocarcinoma Folicular/genética , Carcinoma Papilar/genética , Isoquinolinas/farmacología , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Tiroides/genética , Adenocarcinoma Folicular/patología , Adenocarcinoma Folicular/prevención & control , Adulto , Anciano , Apoptosis/efectos de los fármacos , Carcinoma/genética , Carcinoma/patología , Carcinoma/prevención & control , Carcinoma Papilar/patología , Carcinoma Papilar/prevención & control , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/prevención & control , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/prevención & control , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Monotherapies have proven largely ineffective for the treatment of glioblastomas, suggesting that increased patient benefit may be achieved by combining therapies. Two protumorigenic pathways known to be active in glioblastoma include RAS/RAF/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT/target of rapamycin (TOR). We investigated the efficacy of a combination of novel low molecular weight inhibitors LBT613 and RAD001 (everolimus), which were designed to target RAF and TOR, respectively. LBT613 decreased phosphorylation of extracellular signal-regulated kinase 1 and 2, downstream effectors of RAF, in a human glioma cell line. RAD001 resulted in decreased phosphorylation of the TOR effector S6. To determine if targeting RAF and TOR activities could result in decreased protumorigenic glioma cellular behaviors, we evaluated the abilities of LBT613 and RAD001 to affect the proliferation, migration, and invasion of human glioma cells. Treatment with either LBT613 or RAD001 alone significantly decreased the proliferation of multiple human glioma cell lines. Furthermore, LBT613 and RAD001 in combination synergized to decrease glioma cell proliferation in association with G(1) cell cycle arrest. Glioma invasion is a critical contributor to tumor malignancy. The combination of LBT613 and RAD001 inhibited the invasion of human glioma cells through Matrigel to a greater degree than treatment with either drug alone. These data suggest that the combination of LBT613 and RAD001 reduces glioma cell proliferation and invasion and support examination of the combination of RAF and TOR inhibitors for the treatment of human glioblastoma patients.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Glioma/patología , Inmunosupresores/uso terapéutico , Isoquinolinas/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Sirolimus/análogos & derivados , Quinasas raf/antagonistas & inhibidores , Anexina A5/metabolismo , Western Blotting , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/patología , Movimiento Celular/efectos de los fármacos , Colágeno/metabolismo , Replicación del ADN/efectos de los fármacos , Combinación de Medicamentos , Quimioterapia Combinada , Everolimus , Citometría de Flujo , Glioma/tratamiento farmacológico , Humanos , Laminina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Proteoglicanos/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR , Quinasas raf/metabolismoRESUMEN
The Raf-MEK-ERK signaling pathway is critical for cell survival, growth, proliferation and tumorigenesis. Among the three isoforms of Raf protein kinases, in vitro and in vivo studies have shown that B-Raf functions as the primary MEK activator. B-Raf is one of the most frequently mutated genes in human cancers with a high prevalence in melanoma, and many of the B-Raf mutations activate the kinase activity of B-Raf. B-Raf kinase represents an excellent target for anticancer therapy based on preclinical target validation, epidemiology and drugability. Several small-molecule inhibitors of B-Raf kinase are currently undergoing clinical evaluation, with others due to enter clinical development in the near future.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Bencenosulfonatos/farmacología , Bencenosulfonatos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Piridinas/farmacología , Piridinas/uso terapéutico , SorafenibRESUMEN
PURPOSE: Papillary thyroid carcinomas are associated with nonoverlapping activating mutations of RET, NTRK, RAS and BRAF, which altogether are present in approximately 70% of cases. We postulated that compounds that inhibit a distal effector in the mitogen-activated protein kinase (MAPK) pathway would inhibit growth and tumorigenicity of human thyroid cancer cell lines with mutations of RET or BRAF. EXPERIMENTAL DESIGN AND RESULTS: We first examined the effects of AAL-881 and LBT-613, two inhibitors of RAF kinase activity, on RAF-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK activation in thyroid PCCL3 cells after conditional induction of expression of H-RAS(G12V) or BRAF(V600E). Both compounds blocked RAS and RAF-dependent MEK and ERK phosphorylation. They also potently blocked MEK phosphorylation in human thyroid cancer cell lines with either RET/PTC1 (TPC1) or BRAF(V600E) (NPA, ARO, and FRO) mutations. Inhibition of ERK phosphorylation was transient in TPC1 and ARO cells, with recovery of ERK phosphorylation associated with concomitant down-regulation of the MAPK phosphatases MKP-3 and DUSP5. Both compounds inhibited growth of all cell lines, with LBT-613 being approximately 10-fold more potent than AAL-881. TPC1 cells were more sensitive to growth inhibition (IC50 0.1-0.25 and approximately 0.05 micromol/L for AAL-881 and LBT-613, respectively) than BRAF + lines (IC50 2.5-5 and 0.1-0.5 micromol/L, respectively). Growth inhibition was associated with G1 arrest, and induction of cell death. Growth of ARO and NPA tumor xenografts was inhibited by LBT-613 or AAL-881. MEK and ERK phosphorylation was inhibited by both compounds in ARO but not in NPA cell xenografts. CONCLUSIONS: Compounds that inhibit kinase activity are effective growth inhibitors for poorly differentiated thyroid cancer cell lines with either RET or RAF mutations, and hold promise for treatment of most forms of papillary thyroid carcinoma.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Isoquinolinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-ret/genética , Neoplasias de la Tiroides/prevención & control , Quinasas raf/antagonistas & inhibidores , Animales , Western Blotting , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Fosfatasa 6 de Especificidad Dual , Fosfatasas de Especificidad Dual , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Desnudos , Mutación/genética , Fosforilación/efectos de los fármacos , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas raf/metabolismoRESUMEN
Ovarian cancer is a leading cause of death from gynecologic malignancies. Treatment for advanced-stage disease remains limited and, to date, targeted therapies have been incompletely explored. By systematically suppressing each human tyrosine kinase in ovarian cancer cell lines by RNAi, we found that an autocrine signal-transducing loop involving NRG1 and activated ErbB3 operates in a subset of primary ovarian cancers and ovarian cancer cell lines. Perturbation of this circuit with ErbB3-directed RNAi decreased cell growth in three-dimensional culture and resulted in decreased disease progression and prolonged survival in a xenograft mouse model of ovarian cancer. Furthermore, a monoclonal ErbB3-directed antibody (MM-121) also significantly inhibited tumor growth in vivo. These findings identify ErbB3 as a potential therapeutic target in ovarian cancer.
Asunto(s)
Proliferación Celular , Neurregulina-1/fisiología , Neoplasias Ováricas/patología , Receptor ErbB-3/fisiología , Animales , Comunicación Autocrina , Células Cultivadas , Femenino , Humanos , Ratones , Ratones Endogámicos , Neurregulina-1/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Fosforilación , Interferencia de ARN , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Transducción de Señal , Trasplante HeterólogoRESUMEN
Malignant gliomas are highly proliferative and angiogenic cancers resistant to conventional therapies. Although RAS and RAF mutations are uncommon in gliomas, RAS activity is increased in gliomas. Additionally, vascular endothelial growth factor and its cognate receptors are highly expressed in gliomas. We now report that AAL881, a novel low-molecular weight inhibitor of the kinase activities associated with B-RAF, C-RAF (RAF-1), and VEGF receptor-2 (VEGFR2), showed activity against glioma cell lines and xenografts. In culture, AAL881 inhibited the downstream effectors of RAF in a concentration-dependent manner, with inhibition of proliferation associated with a G(1) cell cycle arrest, induction of apoptosis, and decreased colony formation. AAL881 decreased the proliferation of bovine aortic endothelial cells as well as the tumor cell secretion of vascular endothelial growth factor and inhibited the invasion of glioma cells through an artificial extracellular matrix. Orally administered AAL881 was well tolerated with minimal weight loss in non-tumor-bearing mice. Established s.c. human malignant glioma xenografts grown in immunocompromised mice treated with a 10-day course of oral AAL881 exhibited growth delays relative to control tumors, frequently resulting in long-term complete regressions. AAL881 treatment extended the survival of immunocompromised mice bearing orthotopic glioma xenografts compared with placebo controls. The intraparenchymal portions of orthotopic AAL881-treated tumors underwent widespread necrosis consistent with vascular disruption compared with the subarachnoid elements. These effects are distinct from our prior experience with VEGFR2 inhibitors, suggesting that targeting RAF itself or in combination with VEGFR2 induces profound tumor responses in gliomas and may serve as a novel therapeutic approach in patients with malignant gliomas.