RESUMEN
RalGEFs were recently shown to be critical for Ras-mediated transformed and tumorigenic growth of human cells. We now show that the oncogenic activity of these proteins is propagated by activation of one RalGEF substrate, RalA, but blunted by another closely related substrate, RalB, and that the oncogenic signaling requires binding of the RalBP1 and exocyst subunit effector proteins. Knockdown of RalA expression impeded, if not abolished, the ability of human cancer cells to form tumors. RalA was also commonly activated in a panel of cell lines from pancreatic cancers, a disease characterized by activation of Ras. Activation of RalA signaling thus appears to be a critical step in Ras-induced transformation and tumorigenesis of human cells.
Asunto(s)
Transformación Celular Neoplásica/patología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Proteínas de Unión al GTP ral/fisiología , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Expresión Génica/genética , Guanosina Trifosfato/metabolismo , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias/patología , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Unión Proteica/fisiología , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Interferente Pequeño/genética , Transfección , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismo , Factor de Intercambio de Guanina Nucleótido ral/genética , Factor de Intercambio de Guanina Nucleótido ral/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Pancreatic cancer is one of the leading causes of cancer deaths, with pancreatic ductal adenocarcinoma (PDAC) being the most common subtype. Advanced stage diagnosis of PDAC is common, causing limited treatment opportunities. Gemcitabine is a frequently used chemotherapeutic agent which can be used as a monotherapy or in combination. However, tumors often develop resistance to gemcitabine. Previous studies show that the proto-oncogene PIM kinases (PIM1 and PIM3) are upregulated in PDAC compared to matched normal tissue and are related to chemoresistance and PDAC cell growth. The PIM kinases are also involved in the PI3K/AKT/mTOR pathway to promote cell survival. In this study, we evaluate the effect of the novel multikinase PIM/PI3K/mTOR inhibitor, AUM302, and commercially available PIM inhibitor, TP-3654. Using five human PDAC cell lines, we found AUM302 to be a potent inhibitor of cell proliferation, cell viability, cell cycle progression, and phosphoprotein expression, while TP-3654 was less effective. Significantly, AUM302 had a strong impact on the viability of gemcitabine-resistant PDAC cells. Taken together, these results demonstrate that AUM302 exhibits antitumor activity in human PDAC cells and thus has the potential to be an effective drug for PDAC therapy.
Asunto(s)
Antineoplásicos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Crecimiento/farmacología , Neoplasias Pancreáticas/patología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Ductal Pancreático/patología , Gemcitabina , Serina-Treonina Quinasas TOR , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Proliferación Celular , Línea Celular TumoralRESUMEN
Oncogenic Pim-1 kinase is upregulated in multiple solid cancers, including human pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease with few useful treatment options. Pim-1 is also transcriptionally induced upon oncogenic K-Ras-mediated transformation of the human pancreatic ductal epithelial (HPDE) cell model of PDAC. Given the near ubiquitous presence of mutant K-Ras in PDAC and its critical role in this disease, we wished to study the effects of oncogenic K-Ras signaling on Pim-1 expression, as well as the role of Pim-1 in growth transformation of PDAC cells. Pim-1 protein levels were upregulated in both PDAC cell lines and patient tumor tissues. Furthermore, ectopic oncogenic K-Ras increased Pim-1 expression in human pancreatic nestin-expressing (HPNE) cells, a distinct immortalized cell model of PDAC. Conversely, shRNA-mediated suppression of oncogenic K-Ras decreased Pim-1 protein in PDAC cell lines. These results indicate that oncogenic K-Ras regulates Pim-1 expression. The kinase activity of Pim-1 is constitutively active. Accordingly, shRNA-mediated suppression of Pim-1 in K-Ras-dependent PDAC cell lines decreased Pim-1 activity, as measured by decreased phosphorylation of the pro-apoptotic protein Bad and increased expression of the cyclin-dependent kinase inhibitor p27Kip1. Biological consequences of inhibiting Pim-1 expression included decreases in both anchorage-dependent and -independent cell growth, invasion through Matrigel and radioresistance as measured by standard clonogenic assays. These results indicate that Pim-1 is required for PDAC cell growth, invasion and radioresistance downstream of oncogenic K-Ras. Overall, our studies help to elucidate the role of Pim-1 in PDAC growth transformation and validate Pim-1 kinase as a potential molecular marker for mutated K-Ras activity.
Asunto(s)
Adenocarcinoma/patología , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas c-pim-1/fisiología , Proteínas Proto-Oncogénicas/fisiología , Tolerancia a Radiación , Transducción de Señal/fisiología , Proteínas ras/fisiología , Adenocarcinoma/radioterapia , Carcinoma Ductal Pancreático/radioterapia , Línea Celular Tumoral , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Humanos , Invasividad Neoplásica , Neoplasias Pancreáticas/radioterapia , Fosforilación , Proteínas Proto-Oncogénicas c-pim-1/análisis , Proteínas Proto-Oncogénicas p21(ras) , Proteína Letal Asociada a bcl/metabolismoRESUMEN
Continuous exposure of a pancreatic cancer cell line MIA PaCa-2 (MiaS) to gemcitabine resulted in the formation of a gemcitabine-resistant subline (MiaR). In an effort to discover kinase inhibitors that inhibited MiaR growth, MiaR cells were exposed to kinase inhibitors (PKIS-1 library) in a 384-well screening format. Three compounds (UNC10112721A, UNC10112652A, and UNC10112793A) were identified that inhibited the growth of MiaR cells by more than 50% (at 50 nM). Two compounds (UNC10112721A and UNC10112652A) were classified as cyclin-dependent kinase (CDK) inhibitors, whereas UNC10112793A was reported to be a PLK inhibitor. Dose-response experiments supported the efficacy of these compounds to inhibit growth and increase apoptosis in 2D cultures of these cells. However, only UNC10112721A significantly inhibited the growth of 3D spheroids composed of MiaR cells and GFP-tagged cancer-associated fibroblasts. Multiplexed inhibitor bead (MIB)-mass spectrometry (MS) kinome competition experiments identified CDK9, CLK1-4, DYRK1A, and CSNK1 as major kinase targets for UNC10112721A in MiaR cells. Another CDK9 inhibitor (CDK-IN-2) replicated the growth inhibitory effects of UNC10112721A, whereas inhibitors against the CLK, DYRK, or CSNK1 kinases had no effect. In summary, these studies describe a coordinated approach to discover novel kinase inhibitors, evaluate their efficacy in 3D models, and define their specificity against the kinome.
Asunto(s)
Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor , Línea Celular Tumoral , Supervivencia Celular , Desoxicitidina/química , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Conformación Molecular , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Relación Estructura-Actividad , Flujo de Trabajo , GemcitabinaRESUMEN
This study investigated student learning outcomes using a case-based approach focused on cellular respiration. Students who used the case study, relative to students who did not use the case study, exhibited a significantly greater learning gain, and demonstrated use of higher-order thinking skills. Preliminary data indicate that after engaging with the case study, students were more likely to answer a question addressing misconceptions about cellular respiration correctly when compared with students who did not use the case study. More rigorous testing is needed to fully elucidate whether case-based learning can effectively clarify student misconceptions related to biological processes.
RESUMEN
Cancer is a multistep genetic process that includes mutational activation of oncogenes and inactivation of tumor suppressor genes. The Ras oncogenes are the most frequently mutated oncogenes in human cancers (30%), with a high frequency associated with cancers of the lung, colon, and pancreas. Mutational activation of Ras is commonly an early event in the development of these cancers. Thus, whether mutated Ras is required for tumor maintenance and what aspects of the complex malignant phenotype might be promoted by mutated Ras are issues that remain unresolved for these and other human cancers. The recent development of interfering RNA to selectively impair expression of mutated Ras provides a powerful approach to begin to resolve these issues. In this chapter, we describe the use of retrovirus-based RNA interference approaches to study the functions of Ras and Ras effectors (Raf, RalA, RalB, and Tiam1) in the growth of pancreatic carcinoma and other human tumor cell lines. Finally, we also compare the use of constitutive and inducible shRNA expression vectors for analyses of mutant Ras function.
Asunto(s)
Genes ras/fisiología , Neoplasias/fisiopatología , Retroviridae/fisiología , Proteínas ras/fisiología , Secuencia de Bases , Línea Celular Tumoral , Genes ras/efectos de los fármacos , Humanos , Neoplasias/genética , Neoplasias Pancreáticas/patología , Interferencia de ARN , ARN Interferente Pequeño/fisiología , Transfección/métodos , Quinasas raf/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a 5-year survival rate of only 6%. Although the cytosine analog gemcitabine is the drug commonly used to treat PDAC, chemoresistance unfortunately renders the drug ineffective. Thus, strategies that can decrease this resistance will be essential for improving the dismal outcome of patients suffering from this disease. We previously observed that oncogenic Pim-1 kinase was aberrantly expressed in PDAC tissues and cell lines and was responsible for radioresistance. Furthermore, members of the Pim family have been shown to reduce the efficacy of chemotherapeutic drugs in cancer. Therefore, we attempted to evaluate the role of Pim-3 in chemoresistance of PDAC cells. We were able to confirm upregulation of the Pim-3 oncogene in PDAC tissues and cell lines versus normal samples. Biological consequences of inhibiting Pim-3 expression with shRNA-mediated suppression included decreases in anchorage-dependent growth, invasion through Matrigel and chemoresistance to gemcitabine as measured by caspase-3 activity. Additionally, we were able to demonstrate that Pim-1 and Pim-3 play overlapping but non-identical roles as it relates to gemcitabine sensitivity of pancreatic cancer cells. To further support the role of Pim-3 suppression in sensitizing PDAC cells to gemcitabine, we used the pharmacological Pim kinase inhibitor SGI-1776. Treatment of PDAC cells with SGI-1776 resulted in decreased phosphorylation of the proapoptotic protein Bad and cell cycle changes. When SGI-1776 was combined with gemcitabine, there was a greater decrease in cell viability in the PDAC cells versus cells treated with either of the drugs separately. These results suggest combining drug therapies that inhibit Pim kinases, such as Pim-3, with chemotherapeutic agents, to aid in decreasing chemoresistance in pancreatic cancer.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/enzimología , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular , Desoxicitidina/farmacología , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Imidazoles/farmacología , Invasividad Neoplásica , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Piridazinas/farmacología , ARN Interferente Pequeño , Proteína Letal Asociada a bcl/metabolismo , GemcitabinaRESUMEN
The RAS oncogenes (HRAS, NRAS and KRAS) comprise the most frequently mutated class of oncogenes in human cancers (33%), thus stimulating intensive effort in developing anti-Ras inhibitors for cancer treatment. Despite intensive effort, to date, no effective anti-Ras strategies have successfully made it to the clinic. We present an overview of past and ongoing strategies to inhibit oncogenic Ras in cancer. Since approaches to directly target mutant Ras have not been successful, most efforts have focused on indirect approaches to block Ras membrane association or downstream effector signaling. While inhibitors of effector signaling are currently under clinical evaluation, genome-wide unbiased genetic screens have identified novel directions for future anti-Ras drug discovery.