RESUMEN
The life expectancy for pancreatic cancer patients has seen no substantial changes in the last 40 years as very few and mostly just palliative treatments are available. As the five years survival rate remains around 5%, the identification of novel pharmacological targets and development of new therapeutic strategies are urgently needed. Here we demonstrate that inhibition of the G protein-coupled receptor GPR55, using genetic and pharmacological approaches, reduces pancreatic cancer cell growth in vitro and in vivo and we propose that this may represent a novel strategy to inhibit pancreatic ductal adenocarcinoma (PDAC) progression. Specifically, we show that genetic ablation of Gpr55 in the KRASWT/G12D/TP53WT/R172H/Pdx1-Cre+/+ (KPC) mouse model of PDAC significantly prolonged survival. Importantly, KPC mice treated with a combination of the GPR55 antagonist Cannabidiol (CBD) and gemcitabine (GEM, one of the most used drugs to treat PDAC), survived nearly three times longer compared to mice treated with vehicle or GEM alone. Mechanistically, knockdown or pharmacologic inhibition of GPR55 reduced anchorage-dependent and independent growth, cell cycle progression, activation of mitogen-activated protein kinase (MAPK) signalling and protein levels of ribonucleotide reductases in PDAC cells. Consistent with this, genetic ablation of Gpr55 reduced proliferation of tumour cells, MAPK signalling and ribonucleotide reductase M1 levels in KPC mice. Combination of CBD and GEM inhibited tumour cell proliferation in KPC mice and it opposed mechanisms involved in development of resistance to GEM in vitro and in vivo. Finally, we demonstrate that the tumour suppressor p53 regulates GPR55 protein expression through modulation of the microRNA miR34b-3p. Our results demonstrate the important role played by GPR55 downstream of p53 in PDAC progression. Moreover our data indicate that combination of CBD and GEM, both currently approved for medical use, might be tested in clinical trials as a novel promising treatment to improve PDAC patients' outcome.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/patología , Receptores de Cannabinoides/metabolismo , Animales , Antineoplásicos/farmacología , Cannabidiol/farmacología , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Ratones , Ratones Noqueados , Neoplasias Pancreáticas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , GemcitabinaRESUMEN
The enzyme mammalian target of rapamycin (mTOR) is a master kinase that regulates several critical intracellular processes. It is now well established that this enzyme has a key role in cancer and its inhibition as therapeutic anti-cancer strategy is well recognised. Several clinical trials using mTOR inhibitors have been and are currently being performed. A huge scientific literature exists not only reporting the results of these trials but also discussing the reasons for the limited efficacy of strategies used so far and the need for new strategies to overcome the problem of resistance. The aim of this review is mainly to reflect on how the complexity of the mTOR-dependent signalling pathway and our difficulty to untangle it can ultimately affect the development of proper strategies to fully exploit the potential benefits of mTOR inhibition as anti-cancer strategy.
Asunto(s)
Antineoplásicos/uso terapéutico , Medicina Clínica/métodos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Medicina Clínica/tendencias , Humanos , Neoplasias/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Recently, the orphan receptor G protein-coupled receptor 55 (GPR55) has been proposed as a potential cannabinoid receptor, although controversy remains on its physiological roles. Current evidence suggests a role for GPR55 as a receptor for the lysophospholipid lysophosphatidylinositol (LPI). In this study, we show that GPR55 is expressed in several prostate and ovarian cancer cell lines, both at the mRNA and at the protein level, and that it has a critical role in regulating proliferation and anchorage-independent growth. We further show that GPR55 mediates the effects of LPI in prostate and ovarian cancer cells. Indeed we demonstrate that LPI is able to induce calcium mobilization and activation of Akt and extracellular signal-regulated kinase (ERK)1/2 in these cells and that both pharmacological blockade of GPR55 and its downregulation using specific small interfering RNA strongly inhibits these processes. We further identify an autocrine loop by which LPI is synthesized by cytosolic phospholipase A2, pumped out of the cell by the ATP-binding cassette transporter ABCC1/MRP1, and is then able to initialize cascades downstream of GPR55. All together, these data demonstrate a role of LPI and its receptor GPR55 in cancer cells in activating an autocrine loop that regulates cell proliferation. These findings may have important implications for LPI as a novel cancer biomarker and for its receptor GPR55 as a potential therapeutic target.
Asunto(s)
Comunicación Autocrina , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Lisofosfolípidos/metabolismo , Neoplasias Ováricas/patología , Neoplasias de la Próstata/patología , Receptores Acoplados a Proteínas G/metabolismo , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/análisis , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/análisis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Fosfolipasas A2/análisis , Fosfolipasas A2/metabolismo , Piperidinas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazoles/farmacología , ARN Interferente Pequeño/genética , Receptores de Cannabinoides , Receptores Acoplados a Proteínas G/análisis , Receptores Acoplados a Proteínas G/genética , RimonabantRESUMEN
BACKGROUND: Owing to its role in cancer, the phosphoinositide 3-kinase (PI3K)/Akt pathway is an attractive target for therapeutic intervention. We previously reported that the inhibition of Akt by inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) results in anti-tumour properties. To further develop this compound we modified its structure to obtain more potent inhibitors of the PI3K/Akt pathway. METHODS: Cell proliferation/survival was determined by cell counting, sulphorhodamine or acridine orange/ethidium bromide assay; Akt activation was determined by western blot analysis. In vivo effect of compounds was tested on PC3 xenografts, whereas in vitro activity on kinases was determined by SelectScreen Kinase Profiling Service. RESULTS: The derivative 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP(5)) is active towards cancer types resistant to InsP(5) in vitro and in vivo. 2-O-Bn-InsP(5) possesses higher pro-apoptotic activity than InsP(5) in sensitive cells and enhances the effect of anti-cancer compounds. 2-O-Bn-InsP(5) specifically inhibits 3-phosphoinositide-dependent protein kinase 1 (PDK1) in vitro (IC(50) in the low nanomolar range) and the PDK1-dependent phosphorylation of Akt in cell lines and excised tumours. It is interesting to note that 2-O-Bn-InsP(5) also inhibits the mammalian target of rapamycin (mTOR) in vitro. CONCLUSIONS: InsP(5) and 2-O-Bn-InsP(5) may represent lead compounds to develop novel inhibitors of the PI3K/Akt pathway (including potential dual PDK1/mTOR inhibitors) and novel potential anti-cancer drugs.
Asunto(s)
Antineoplásicos/farmacología , Fosfatos de Inositol/química , Fosfatos de Inositol/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Línea Celular Tumoral/trasplante , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Fosfatos de Inositol/síntesis química , Fosfatos de Inositol/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Masculino , Ratones , Ratones Desnudos , Estructura Molecular , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although it is now well established that PI3K (phosphoinositide 3-kinase) is a key enzyme in several intracellular processes, there are still relatively few reports that precisely identify the specific isoforms of PI3K actually involved in such events. The lack of specific inhibitors has made it particularly difficult to address the physiological roles of some isoforms, such as the members of class II. As a consequence, there is still relatively little understanding of the role of these enzymes and the question about the intracellular role of these isoforms still waits for more answers.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Androstadienos/farmacología , Animales , Fenómenos Fisiológicos Celulares , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/metabolismo , Microsomas/enzimología , Fosfatidilinositol 3-Quinasas/clasificación , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , WortmaninaRESUMEN
Activation of the enzyme PLC (phospholipase C) leads to the formation of second messengers Ins(1,4,5)P(3) and diacylglycerol. RTKs (receptor tyrosine kinases) activate this reaction through PLCgamma isoenzymes. It has been shown that PI3K (phosphoinositide 3-kinase) may regulate PLCgamma activity through the interaction of PI3K product PtdIns(3,4,5)P(3) and the PLCgamma PH domain (pleckstrin homology domain). Here, we analyse the potential functional roles of the PI3K/PLC pathway.
Asunto(s)
Fosfatidilinositol 3-Quinasas/fisiología , Fosfolipasa C gamma/metabolismo , Animales , Vasos Sanguíneos/fisiología , Activación Enzimática , Cinética , Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Sistemas de Mensajero Secundario , VenasRESUMEN
Pleckstrin homology (PH) domains are protein modules found in proteins involved in many cellular processes. The majority of PH domain-containing proteins require membrane association for their function. It has been shown that most PH domains interact directly with the cell membrane by binding to phosphoinositides with a broad range of specificity and affinity. While a highly specific binding of the PH domain to a phosphoinositide can be necessary and sufficient for the correct recruitment of the host protein to the membrane, a weaker and less specific interaction may be necessary but not sufficient, thus probably requiring alternative, co-operative mechanisms.
Asunto(s)
Proteínas Sanguíneas/química , Fosfatidilinositoles/metabolismo , Fosfoproteínas/química , Proteínas/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Fosfolipasa C delta , Unión Proteica , Fosfolipasas de Tipo C/química , Fosfolipasas de Tipo C/metabolismoRESUMEN
Integrating the pleomorphic membranes of the intermediate compartment (IC) into the array of Golgi cisternae is a crucial step in membrane transport, but it is poorly understood. To gain insight into this step, we investigated the dynamics by which cis-Golgi matrix proteins such as GM130 and GRASP65 associate with, and incorporate, incoming IC elements. We found that GM130 and GRASP65 cycle via membranous tubules between the Golgi complex and a constellation of mobile structures that we call late IC stations. These stations are intermediate between the IC and the cis-Golgi in terms of composition, and they receive cargo from earlier IC elements and deliver it to the Golgi complex. Late IC elements are transient in nature and sensitive to fixatives; they are seen in only a fraction of fixed cells, whereas they are always visible in living cells. Finally, late IC stations undergo homotypic fusion and establish tubular connections between themselves and the Golgi. Overall, these features indicate that late IC stations mediate the transition between IC elements and the cis-Golgi face.