RESUMEN
The repurposing of existing drugs has emerged as an attractive additional strategy to the development of novel compounds in the fight against cancerous diseases. Inhibition of phosphodiesterase 5 (PDE5) has been claimed as a potential approach to target various cancer subtypes in recent years. However, data on the treatment of tumors with PDE5 inhibitors as well as the underlying mechanisms are as yet very scarce. Here, we report that treatment of tumor cells with low concentrations of Sildenafil was associated with decreased cancer cell proliferation and augmented apoptosis in vitro and resulted in impaired tumor growth in vivo. Notably, incubation of cancer cells with Sildenafil was associated with altered expression of HSP90 chaperone followed by degradation of protein kinase D2, a client protein previously reported to be involved in tumor growth. Furthermore, the involvement of low doses of PU-H71, an HSP90 inhibitor currently under clinical evaluation, in combination with low concentrations of Sildenafil, synergistically and negatively impacted on the viability of cancer cells in vivo. Taken together, our study suggests that repurposing of already approved drugs, alone or in combination with oncology-dedicated compounds, may represent a novel cancer therapeutic strategy.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Neoplasias/patología , Inhibidores de Fosfodiesterasa 5/farmacología , Proteolisis , Citrato de Sildenafil/farmacología , Canales Catiónicos TRPP/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Humanos , Neoplasias/metabolismoRESUMEN
Constitutive secretion from the trans-Golgi-network (TGN) is facilitated by a concerted regulation of vesicle biogenesis and fission processes. The protein kinase D family (PKD) has been previously described to enhance vesicle fission by modifying the lipid environment. PKD also phosphorylates the actin regulatory protein cortactin at S298 to impair synergistic actin polymerization. We here report additional functions for PKD2 (also known as PRKD2) and cortactin in the regulation of actin polymerization during the fission of transport carriers from the TGN. Phosphorylation of cortactin at S298 impairs the interaction between WIP (also known as WIPF1) and cortactin. WIP stabilizes the autoinhibited conformation of N-WASP (also known as WASL). This leads to an inhibition of synergistic Arp2/3-complex-dependent actin polymerization at the TGN. PKD2 activity at the TGN is controlled by active CDC42-GTP which directly activates N-WASP, inhibits PKD2 and shifts the balance to non-S298-phosphorylated cortactin, which can in turn sequester WIP from N-WASP. Consequently, synergistic actin polymerization at the TGN and constitutive secretion are enhanced.
Asunto(s)
Cortactina/metabolismo , Canales Catiónicos TRPP/metabolismo , Actinas , Animales , Western Blotting , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Células MCF-7 , Ratones , Células 3T3 NIH , Polimerizacion , Pirazoles/farmacología , Sulfonamidas/farmacología , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/metabolismo , Red trans-Golgi/genéticaRESUMEN
Protein kinase D2 (PKD2) is a serine/threonine kinase that belongs to the PKD family of calcium-calmodulin kinases, which comprises three isoforms: PKD1, PKD2, and PKD3. PKD2 is activated by many stimuli including growth factors, phorbol esters, and G-protein-coupled receptor agonists. PKD2 participation to uncontrolled growth, survival, neovascularization, metastasis, and invasion has been documented in various tumor types including pancreatic, colorectal, gastric, hepatic, lung, prostate, and breast cancer, as well as glioma multiforme and leukemia. This review discusses the versatile functions of PKD2 from the perspective of cancer hallmarks as described by Hanahan and Weinberg. The PKD2 status, signaling pathways affected in different tumor types and the molecular mechanisms that lead to tumorigenesis and tumor progression are presented. The latest developments of small-molecule inhibitors selective for PKD/PKD2, as well as the need for further chemotherapies that prevent, slow down, or eliminate tumors are also discussed in this review.
Asunto(s)
Neoplasias/genética , Neoplasias/patología , Proteínas Quinasas/fisiología , Animales , Proliferación Celular/genética , Humanos , Metástasis de la Neoplasia/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Proteína Quinasa D2 , Proteínas Quinasas/genética , Transducción de Señal/genéticaRESUMEN
We here report a novel function of the armadillo protein p0071 (also known as PKP4) during transport mediated by the KIF3 transport complex. Secretion of chromogranin A and matrix metallopeptidase 9 from pancreatic neuroendocrine tumor cells or pancreatic cancer cells, respectively, was substantially reduced following knockdown of p0071. Vesicle tracking indicated that there was impaired directional persistence of vesicle movement upon p0071 depletion. This suggests a disturbed balance between plus- and minus-end directed microtubule transport in cells lacking p0071. p0071 directly interacts with the KIF3 motor subunit KIF3B. Our data indicate that p0071 also interacts with the kinesin cargo adaptor protein KAP3 (also known as KIFAP3) acting as a stabilizing linker between KIF3B and its KAP3 cargo-binding entity. Thus, p0071 is required for directional vesicle movement and secretion of different KIF3-transported carriers, thereby regulating the transport of intracellular membrane vesicles along microtubules.
Asunto(s)
Cinesinas/metabolismo , Placofilinas/metabolismo , Vesículas Secretoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinesinas/genética , Placofilinas/genética , Transporte de Proteínas/fisiología , Vesículas Secretoras/genéticaRESUMEN
BACKGROUND: Initially identified as a molecule that regulates the final step of glycolysis, the M2 isoform of pyruvate kinase (PKM2) was recently reported to have a central role in the metabolic reprogramming of cancer cells as well as participating in cell cycle progression and gene transcription. Despite intensive efforts, the intricate molecular mechanisms through which PKM2 regulates tumor progression remain elusive. METHODS: The proliferation and apoptosis of various pancreatic cancer cells using lentiviral-mediated PKM2 abrogation were assessed in vitro via Western blot and flow cytometric assay while the in vivo experiments involved tumor xenograft on chicken chorionallantoic membranes and immunohistochemistry on human tissue specimens. In order to decipher the molecular mechanism of HIF-1α and p65/RelA regulation by PKM2 in cancer cells cultivated in hypoxic atmosphere or normoxia we involved various biochemical assays such as Western blotting, immunoprecipitation, reporter gene assay and ELISA. RESULTS: Strong expression of PKM2 was observed in 68 % of human pancreatic adenocarcinoma specimens and almost all analyzed pancreatic cancer cell lines. Abrogation of PKM2 resulted in impaired proliferation and augmented apoptosis in vitro as well as impaired tumor growth and decreased blood vessel formation in vivo. Furthermore, deletion of PKM2 negatively impacted hypoxia-induced HIF-1α accumulation and promoter activity ultimately resulting in impaired secretion of VEGF. CONCLUSIONS: Our study suggests that in hypoxic pancreatic tumors PKM2 interferes both with NF-κB/p65 and HIF-1α activation that ultimately triggers VEGF-A secretion and subsequent blood vessel formation.
Asunto(s)
Proteínas Portadoras/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Neovascularización Patológica/patología , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/metabolismo , Hormonas Tiroideas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Hipoxia de la Célula , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Supervivencia Celular , Pollos , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Persona de Mediana Edad , Modelos Biológicos , Neovascularización Patológica/genética , Neoplasias Pancreáticas/genética , Unión Proteica , Transporte de Proteínas , Transducción de Señal/genética , Factor de Transcripción ReIA/metabolismo , Transcripción Genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Unión a Hormona TiroideRESUMEN
The kinase PRKD2 (protein kinase D) is a crucial regulator of tumor cell-endothelial cell communication in gastrointestinal tumors and glioblastomas, but its mechanistic contributions to malignant development are not understood. Here, we report that the oncogenic chaperone HSP90 binds to and stabilizes PRKD2 in human cancer cells. Pharmacologic inhibition of HSP90 with structurally divergent small molecules currently in clinical development triggered proteasome-dependent degradation of PRKD2, augmenting apoptosis in human cancer cells of various tissue origins. Conversely, ectopic expression of PRKD2 protected cancer cells from the apoptotic effects of HSP90 abrogation, restoring blood vessel formation in two preclinical models of solid tumors. Mechanistic studies revealed that PRKD2 is essential for hypoxia-induced accumulation of hypoxia-inducible factor-1α (HIF1α) and activation of NF-κB in tumor cells. Notably, ectopic expression of PRKD2 was able to partially restore HIF1α and secreted VEGF-A levels in hypoxic cancer cells treated with HSP90 inhibitors. Taken together, our findings indicate that signals from hypoxia and HSP90 pathways are interconnected and funneled by PRKD2 into the NF-κB/VEGF-A signaling axis to promote tumor angiogenesis and tumor growth.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Neovascularización Patológica/metabolismo , Proteína Quinasa C/metabolismo , Animales , Apoptosis/fisiología , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Femenino , Células HCT116 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Desnudos , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In eukaryotes, membrane and soluble proteins of the secretory pathway enter the endoplasmic reticulum (ER) after synthesis in an unfolded state. Directly after entry, most proteins are modified with glycans at suitable glycosylation sites and start to fold. A protein that cannot fold properly will be degraded in a process called ER associated degradation (ERAD). Failures in ERAD, either by loss of function or by premature degradation of proteins, are a cause of severe diseases. Therefore, the search for novel ERAD components to gain better insight in this process is of high importance. Carbohydrate trimming is a relevant process in ER quality control. In this work a novel putative yeast mannosidase encoded by the open reading frame YLR057W was identified and named Mnl2. Deletion of MNL2 diminished the degradation efficiency of misfolded CPY(*) in the absence of the cognate mannosidase Mnl1, indicating a specific role in ERAD.