RESUMEN
BACKGROUND: Even though prostate cancer (PCa) patients initially respond to androgen deprivation therapy, some will eventually develop castration resistant prostate cancer (CRPC). Androgen receptor (AR) mediated cell signaling is a major driver in the progression of CRPC while only a fraction of PCa becomes AR negative. This study aimed to understand the regulation of AR levels by N-myristoyltransferase in PCa cells. METHODS: Two enantiomers, (1S,2S)- d-NMAPPD and (1R,2R)- d-NMAPPD (LCL4), were characterized by various methods (1 H and 13 C NMR, UHPLC, high-resolution mass spectra, circular dichroism) and evaluated for the ability to bind to N-myristoyltransferase 1 (NMT1) using computational docking analysis. structure-activity relationship analysis of these compounds led to the synthesis of (1R,2R)-LCL204 and evaluation as a potential NMT1 inhibitor utilizing the purified full length NMT1 enzyme. The NMT inhibitory activity wase determined by Click chemistry and immunoblotting. Regulation of NMT1 on tumor growth was evaluated in a xenograft tumor model. RESULTS: (1R,2R)- d-NMAPPD, but not its enantiomer (1S,2S)- d-NMAPPD, inhibited NMT1 activity and reduced AR protein levels. (1R,2R)-LCL204, a derivative of (1R,2R)- d-NMAPPD, inhibited global protein myristoylation. It also suppressed protein levels, nuclear translocation, and transcriptional activity of AR full-length or variants in PCa cells. This was due to enhanced ubiquitin and proteasome-mediated degradation of AR. Knockdown of NMT1 levels inhibited tumor growth and proliferation of cancer cells. CONCLUSION: Inhibitory efficacy on N-myristoyltransferase activity by d-NMAPPD is stereospecific. (1R,2R)-LCL204 reduced global N-myristoylation and androgen receptor protein levels at low micromolar concentrations in prostate cancer cells. pharmacological inhibition of NMT1 enhances ubiquitin-mediated proteasome degradation of AR. This study illustrates a novel function of N-myristoyltransferase and provides a potential strategy for treatment of CRPC.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Masculino , Humanos , Receptores Androgénicos/metabolismo , Andrógenos , Neoplasias de la Próstata Resistentes a la Castración/patología , Antagonistas de Andrógenos , Complejo de la Endopetidasa Proteasomal , Ubiquitinas , Línea Celular TumoralRESUMEN
Interactions between cells in the stroma and epithelium facilitate prostate stem cell activity and tissue regeneration capacity. Numerous molecular signal transduction pathways, including the induction of sonic hedgehog (Shh) to activate the Gli transcription factors, are known to mediate the cross-talk of these two cellular compartments. However, the details of how these signaling pathways regulate prostate stem and progenitor cell activity remain elusive. Here we demonstrate that, although cell-autonomous epithelial Shh-Gli signaling is essential to determine the expression levels of basal cell markers and the renewal potential of epithelial stem and progenitor cells, stromal Gli signaling regulates prostate stem and progenitor cell activity by increasing the number and size of prostate spheroids in vitro Blockade of stromal Gli signaling also inhibited prostate tissue regeneration in vivo The inhibition of stromal Gli signaling suppressed the differentiation of basal and progenitor cells to luminal cells and limited prostate tubule secretory capability. Additionally, stromal cells were able to compensate for the deficiency of epithelial Shh signaling in prostate tissue regeneration. Mechanistically, suppression of Gli signaling increased the signaling factor transforming growth factor ß (TGFß) in stromal cells. Elevation of exogenous TGFß1 levels inhibited prostate spheroid formation, suggesting that a stromal Gli-TGFß signaling axis regulates the activity of epithelial progenitor cells. Our study illustrates that Gli signaling regulates epithelial stem cell activity and renewal potential in both epithelial and stromal compartments.
Asunto(s)
Diferenciación Celular , Próstata/citología , Próstata/fisiología , Células Madre/citología , Células Madre/fisiología , Células del Estroma/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Células del Estroma/citología , Factor de Crecimiento Transformador beta/metabolismo , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling facilitates tumor initiation and progression. Although currently approved inhibitors of FGFR kinase have shown therapeutic benefit in clinical trials, overexpression or mutations of FGFRs eventually confer drug resistance and thereby abrogate the desired activity of kinase inhibitors in many cancer types. In this study, we report that loss of myristoylation of fibroblast growth factor receptor substrate 2 (FRS2α), a scaffold protein essential for FGFR signaling, inhibits FGF/FGFR-mediated oncogenic signaling and FGF10-induced tumorigenesis. Moreover, a previously synthesized myristoyl-CoA analog, B13, which targets the activity of N-myristoyltransferases, suppressed FRS2α myristoylation and decreased the phosphorylation with mild alteration of FRS2α localization at the cell membrane. B13 inhibited oncogenic signaling induced by WT FGFRs or their drug-resistant mutants (FGFRsDRM). B13 alone or in combination with an FGFR inhibitor suppressed FGF-induced WT FGFR- or FGFRDRM-initiated phosphoinositide 3-kinase (PI3K) activity or MAPK signaling, inducing cell cycle arrest and thereby inhibiting cell proliferation and migration in several cancer cell types. Finally, B13 significantly inhibited the growth of xenograft tumors without pathological toxicity to the liver, kidney, or lung in vivo In summary, our study suggests a possible therapeutic approach for inhibiting FGF/FGFR-mediated cancer progression and drug-resistant FGF/FGFR mutants.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Amidas/farmacología , Factores de Crecimiento de Fibroblastos/metabolismo , Lipoilación/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/metabolismo , Propanolaminas/farmacología , Neoplasias de la Próstata/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular Tumoral , Factores de Crecimiento de Fibroblastos/genética , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones SCID , Células 3T3 NIH , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/genética , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata/genética , Receptores de Factores de Crecimiento de Fibroblastos/genéticaRESUMEN
Numerous genetic alterations have been identified during prostate cancer progression. The influence of environmental factors, particularly the diet, on the acceleration of tumor progression is largely unknown. Expression levels and/or activity of Src kinase are highly elevated in numerous cancers including advanced stages of prostate cancer. In this study, we demonstrate that high-fat diets (HFDs) promoted pathological transformation mediated by the synergy of Src and androgen receptor in vivo. Additionally, a diet high in saturated fat significantly enhanced proliferation of Src-mediated xenograft tumors in comparison with a diet high in unsaturated fat. The saturated fatty acid palmitate, a major constituent in a HFD, significantly upregulated the biosynthesis of palmitoyl-CoA in cancer cells in vitro and in xenograft tumors in vivo. The exogenous palmitate enhanced Src-dependent mitochondrial ß-oxidation. Additionally, it elevated the amount of C16-ceramide and total saturated ceramides, increased the level of Src kinase localized in the cell membrane, and Src-mediated downstream signaling, such as the activation of mitogen-activated protein kinase and focal adhesion kinase. Our results uncover how the metabolism of dietary palmitate cooperates with elevated Src kinase in the acceleration of prostate tumor progression.
Asunto(s)
Palmitatos/administración & dosificación , Neoplasias de la Próstata/etiología , Familia-src Quinasas/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Dieta Alta en Grasa/efectos adversos , Progresión de la Enfermedad , Células HEK293 , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Células PC-3 , Palmitatos/metabolismo , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patologíaRESUMEN
Exogenous fatty acids provide substrates for energy production and biogenesis of the cytoplasmic membrane, but they also enhance cellular signaling during cancer cell proliferation. However, it remains controversial whether dietary fatty acids are correlated with tumor progression. In this study, we demonstrate that increased Src kinase activity is associated with high-fat diet-accelerated progression of prostate tumors and that Src kinases mediate this pathological process. Moreover, in the in vivo prostate regeneration assay, host SCID mice carrying Src(Y529F)-transduced regeneration tissues were fed a low-fat diet or a high-fat diet and treated with vehicle or dasatinib. The high-fat diet not only accelerated Src-induced prostate tumorigenesis in mice but also compromised the inhibitory effect of the anticancer drug dasatinib on Src kinase oncogenic potential in vivo We further show that myristoylation of Src kinase is essential to facilitate Src-induced and high-fat diet-accelerated tumor progression. Mechanistically, metabolism of exogenous myristic acid increased the biosynthesis of myristoyl CoA and myristoylated Src and promoted Src kinase-mediated oncogenic signaling in human cells. Of the fatty acids tested, only exogenous myristic acid contributed to increased intracellular myristoyl CoA levels. Our results suggest that targeting Src kinase myristoylation, which is required for Src kinase association at the cellular membrane, blocks dietary fat-accelerated tumorigenesis in vivo Our findings uncover the molecular basis of how the metabolism of myristic acid stimulates high-fat diet-mediated prostate tumor progression.
Asunto(s)
Antineoplásicos/uso terapéutico , Dieta Alta en Grasa/efectos adversos , Próstata/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Acilación/efectos de los fármacos , Sustitución de Aminoácidos , Animales , Antineoplásicos/farmacología , Proteína Tirosina Quinasa CSK , Línea Celular Tumoral , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones SCID , Mutación , Ácido Mirístico/metabolismo , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/etiología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Interferencia de ARN , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Although the differentiation of oncogenically transformed basal progenitor cells is one of the key steps in prostate tumorigenesis, the mechanisms mediating this cellular process are still largely unknown. Here we demonstrate that an expanded p63+ and CK5+ basal/progenitor cell population, induced by the concomitant activation of oncogenic Kras(G12D) and androgen receptor (AR) signaling, underwent cell differentiation in vivo The differentiation process led to suppression of p63-expressing cells with a decreased number of CK5+ basal cells but an increase of CK8+ luminal tumorigenic cells and revealed a hierarchal lineage pattern consisting of p63+/CK5+ progenitor, CK5+/CK8+ transitional progenitor, and CK8+ differentiated luminal cells. Further analysis of the phenotype showed that Kras-AR axis-induced tumorigenesis was mediated by Gli transcription factors. Combined blocking of the activators of this family of proteins (Gli1 and Gli2) inhibited the proliferation of p63+ and CK5+ basal/progenitor cells and development of tumors. Finally, we identified that Gli1 and Gli2 exhibited different functions in the regulation of p63 expression or proliferation of p63+ cells in Kras-AR driven tumors. Gli2, but not Gli1, transcriptionally regulated the expression levels of p63 and prostate sphere formation. Our study provides evidence of a novel mechanism mediating pathological dysregulation of basal/progenitor cells through the differential activation of the Gli transcription factors. Also, these findings define Gli proteins as new downstream mediators of the Kras-AR axis in prostate carcinogenesis and open a potential therapeutic avenue of targeting prostate cancer progression by inhibiting Gli signaling.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Nucleares/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptores Androgénicos/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Transgénicos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores Androgénicos/genética , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Proteína Gli2 con Dedos de ZincRESUMEN
The serine/threonine Pim kinases are overexpressed in solid cancers and hematologic malignancies and promote cell growth and survival. Here, we find that a novel Pim kinase inhibitor, SMI-4a, or Pim-1 siRNA blocked the rapamycin-sensitive mammalian target of rapamycin (mTORC1) activity by stimulating the phosphorylation and thus activating the mTORC1 negative regulator AMP-dependent protein kinase (AMPK). Mouse embryonic fibroblasts (MEFs) deficient for all three Pim kinases [triple knockout (TKO) MEFs] demonstrated activated AMPK driven by elevated ratios of AMPATP relative to wild-type MEFs. Consistent with these findings, TKO MEFs were found to grow slowly in culture and have decreased rates of protein synthesis secondary to a diminished amount of 5'-cap-dependent translation. Pim-3 expression alone in TKO MEFs was sufficient to reverse AMPK activation, increase protein synthesis, and drive MEF growth similar to wild type. Pim-3 expression was found to markedly increase the protein levels of both c-Myc and the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), enzymes capable of regulating glycolysis and mitochondrial biogenesis, which were diminished in TKO MEFs. Overexpression of PGC-1α in TKO MEFs elevated ATP levels and inhibited the activation of AMPK. These results demonstrate the Pim kinase-mediated control of energy metabolism and thus regulation of AMPK activity. We identify an important role for Pim-3 in modulating c-Myc and PGC-1α protein levels and cell growth.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Transactivadores/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Fibroblastos/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Células K562 , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteínas Proto-Oncogénicas c-myc/metabolismo , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismoRESUMEN
Extracellular vesicles (EVs) are heterogeneous biological nanoparticles secreted by all cell types. Identifying the proteins preferentially encapsulated in secreted EVs will help understand their heterogeneity. Src family kinases including Src and Fyn are a group of tyrosine kinases with fatty acylation modifications and/or multiple lysine residues (contributing charge interaction) at their N-terminus. Here, we demonstrate that Src and Fyn kinases were preferentially encapsulated in EVs and fatty acylation including myristoylation and palmitoylation facilitated their encapsulation. Genetic loss or pharmacological inhibition of myristoylation suppressed Src and/or Fyn kinase levels in EVs. Similarly, loss of palmitoylation reduced Fyn levels in EVs. Additionally, mutation of lysine at sites 5, 7, and 9 of Src kinase also inhibited the encapsulation of myristoylated Src into EVs. Knockdown of TSG101, which is a protein involved in the endosomal sorting complexes required for transport (ESCRT) protein complex mediated EVs biogenesis and led to a reduction of Src levels in EVs. In contrast, filipin III treatment, which disturbed the lipid raft structure, reduced Fyn kinase levels, but not Src kinase levels in EVs. Finally, elevated levels of Src protein were detected in the serum EVs of host mice carrying constitutively active Src-mediated prostate tumors in vivo. Collectively, the data suggest that different EVs biogenesis pathways exist and can regulate the encapsulation of specific proteins into EVs. This study provides an understanding of the EVs heterogeneity created by different EVs biogenesis pathways.
RESUMEN
The serine/threonine Pim kinases are up-regulated in specific hematologic neoplasms, and play an important role in key signal transduction pathways, including those regulated by MYC, MYCN, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. We demonstrate that SMI-4a, a novel benzylidene-thiazolidine-2, 4-dione small molecule inhibitor of the Pim kinases, kills a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre-T-LBL/T-ALL) being highly sensitive. Incubation of pre-T-LBL cells with SMI-4a induced G1 phase cell-cycle arrest secondary to a dose-dependent induction of p27(Kip1), apoptosis through the mitochondrial pathway, and inhibition of the mammalian target of rapamycin C1 (mTORC1) pathway based on decreases in phospho-p70 S6K and phospho-4E-BP1, 2 substrates of this enzyme. In addition, treatment of these cells with SMI-4a was found to induce phosphorylation of extracellular signal-related kinase1/2 (ERK1/2), and the combination of SMI-4a and a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor was highly synergistic in killing pre-T-LBL cells. In immunodeficient mice carrying subcutaneous pre-T-LBL tumors, treatment twice daily with SMI-4a caused a significant delay in the tumor growth without any change in the weight, blood counts, or chemistries. Our data suggest that inhibition of the Pim protein kinases may be developed as a therapeutic strategy for the treatment of pre-T-LBL.
Asunto(s)
Compuestos de Bencilideno/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Tiazolidinedionas/farmacología , Animales , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fase G1/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-pim-1/genética , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Serina-Treonina Quinasas TORRESUMEN
While monitoring the reaction of ferric cytochrome P450cam (Cyp101) with substituted peroxybenzoic acids using rapid-scanning, stopped-flow (RSSF) spectroscopy, an intermediate appears en route to formation of the high-valent moiety known as Compound I [Fe(IV)=O/porphyrin radical cation] that is thought to be the key catalytic species for O-atom transfer to substrate. We have previously suggested (Spolitak, T., Dawson, J.H., Ballou, D.P., J. Biol. Chem.2005, 280, 20,300-20,309) that this species is an acylperoxo-ferric heme adduct that subsequently undergoes OO bond cleavage to generate Compound I. Singular value decomposition analysis of the RSSF data for formation of this intermediate shows that the energy of its Soret absorption peak is sensitive to the electron donor properties of the aryl substituents on the peracid. A linear Hammett correlation plot is seen for the energy of the Soret absorption peak vs. the Hammett σ constant. This correlation requires that the aryl substituents remain as part of the ligand bound to the heme iron, providing direct evidence that the adduct is indeed a ferric acylperoxo derivative. Linear Hammett correlation plots are also seen for both the rate of formation of the intermediate as well as for its conversion to Compound I. It is proposed that the electron donating/withdrawing properties of the aryl-bound substituents affect the electrophilic nature for binding substrate, changing the observed rate of formation for the acylperoxo intermediate, as well as the propensity and stability of the substituted benzoic acid to serve as the leaving group during OO bond cleavage yielding Compound I.
Asunto(s)
Alcanfor 5-Monooxigenasa , Porfirinas , Benzoatos , Alcanfor 5-Monooxigenasa/metabolismo , Hemo , Hierro , LigandosRESUMEN
CRISPR/Cas9 genome editing is a very promising avenue for the treatment of a variety of genetic diseases. However, it is still very challenging to encapsulate CRISPR/Cas9 machinery for delivery. Protein N-myristoylation is an irreversible co/post-translational modification that results in the covalent attachment of the myristoyl-group to the N-terminus of a target protein. It serves as an anchor for a protein to associate with the cell membrane and determines its intracellular trafficking and activity. Extracellular vesicles (EVs) are secreted vesicles that mediate cell-cell communication. In this study, we demonstrate that myristoylated proteins were preferentially encapsulated into EVs. The octapeptide derived from the leading sequence of the N-terminus of Src kinase was a favourable substrate for N-myristoyltransferase 1, the enzyme that catalyzes myristoylation. The fusion of the octapeptide onto the N-terminus of Cas9 promoted the myristoylation and encapsulation of Cas9 into EVs. Encapsulation of Cas9 and sgRNA-eGFP inside EVs was confirmed using protease digestion assays. Additionally, to increase the transfection potential, VSV-G was introduced into the EVs. The encapsulated Cas9 in EVs accounted for 0.7% of total EV protein. Importantly, the EVs coated with VSV-G encapsulating Cas9/sgRNA-eGFP showed up to 42% eGFP knock out efficiency with limited off-target effects in recipient cells. Our study provides a novel approach to encapsulate CRISPR/Cas9 protein and sgRNA into EVs. This strategy may open an effective avenue to utilize EVs as vehicles to deliver CRISPR/Cas9 for genome-editing-based gene therapy.
Asunto(s)
Sistemas CRISPR-Cas , Vesículas Extracelulares , Proteína 9 Asociada a CRISPR/genética , Edición Génica , Terapia GenéticaRESUMEN
The Pim-1 protein kinase plays an important role in regulating both cell growth and survival and enhancing transformation by multiple oncogenes. The ability of Pim-1 to regulate cell growth is mediated, in part, by the capacity of this protein kinase to control the levels of the p27, a protein that is a critical regulator of cyclin-dependent kinases that mediate cell cycle progression. To understand how Pim-1 is capable of regulating p27 protein levels, we focused our attention on the SCF(Skp2) ubiquitin ligase complex that controls the rate of degradation of this protein. We found that expression of Pim-1 increases the level of Skp2 through direct binding and phosphorylation of multiple sites on this protein. Along with known Skp2 phosphorylation sites including Ser(64) and Ser(72), we have identified Thr(417) as a unique Pim-1 phosphorylation target. Phosphorylation of Thr(417) controls the stability of Skp2 and its ability to degrade p27. Additionally, we found that Pim-1 regulates the anaphase-promoting complex or cyclosome (APC/C complex) that mediates the ubiquitination of Skp2. Pim-1 phosphorylates Cdh1 and impairs binding of this protein to another APC/C complex member, CDC27. These modifications inhibit Skp2 from degradation. Marked increases in Skp2 caused by these mechanisms lower cellular p27 levels. Consistent with these observations, we show that Pim-1 is able to cooperate with Skp2 to signal S phase entry. Our data reveal a novel Pim-1 kinase-dependent signaling pathway that plays a crucial role in cell cycle regulation.
Asunto(s)
Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Animales , Antígenos CD , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase , Cadherinas/genética , Cadherinas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Células HeLa , Humanos , Immunoblotting , Masculino , Ratones , Ratones Noqueados , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-pim-1/genética , Ratas , Fase S/genética , Fase S/fisiología , Proteínas Quinasas Asociadas a Fase-S/genética , UbiquitinaciónRESUMEN
Fatty acid metabolism is essential for the biogenesis of cellular components and ATP production to sustain proliferation of cancer cells. Long-chain fatty acyl-CoA synthetases (ACSLs), a group of rate-limiting enzymes in fatty acid metabolism, catalyze the bioconversion of exogenous or de novo synthesized fatty acids to their corresponding fatty acyl-CoAs. In this study, systematical analysis of ACSLs levels and the amount of fatty acyl-CoAs illustrated that ACSL1 were significantly associated with the levels of a broad spectrum of fatty acyl-CoAs, and were elevated in human prostate tumors. ACSL1 increased the biosynthesis of fatty acyl-CoAs including C16:0-, C18:0-, C18:1-, and C18:2-CoA, triglycerides and lipid accumulation in cancer cells. Mechanistically, ACSL1 modulated mitochondrial respiration, ß-oxidation, and ATP production through regulation of CPT1 activity. Knockdown of ACSL1 inhibited the cell cycle, and suppressed the proliferation and migration of prostate cancer cells in vitro, and growth of prostate xenograft tumors in vivo. Our study implicates ACSL1 as playing an important role in prostate tumor progression, and provides a therapeutic strategy of targeting fatty acid metabolism for the treatment of prostate cancer.
Asunto(s)
Coenzima A Ligasas/genética , Ácidos Grasos/metabolismo , Lipogénesis/genética , Neoplasias de la Próstata/genética , Adenosina Trifosfato/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Progresión de la Enfermedad , Ácidos Grasos/genética , Xenoinjertos , Humanos , Masculino , Ratones , Oxidación-Reducción , Neoplasias de la Próstata/patologíaRESUMEN
Androgen deprivation therapy has led to elevated cases of androgen receptor (AR) pathway-independent prostate cancer with dysregulated fatty acid metabolism. However, it is unclear how prostate cancer cells sustain dysregulated fatty acid metabolism to drive AR-independent prostate cancer. Long-chain acyl-CoA synthetases (ACSL) catalyze the conversion of fatty acids into fatty acyl-CoAs that are required for fatty acid metabolism. In this study, we demonstrate that expression levels of ACSL3 and 4 were oppositely regulated by androgen-AR signaling in prostate cancer cells. AR served as a transcription suppressor to bind at the ACSL4 promoter region and inhibited its transcription. Inhibition of androgen-AR signaling significantly downregulated ACSL3 and PSA, but elevated ACSL4 levels. ACSL4 regulated a broad spectrum of fatty acyl-CoA levels, and its catalytic efficiency in fatty acyl-CoAs biosynthesis was about 1.9- to 4.3-fold higher than ACSL3. In addition, in contrast to ACSL3, ACSL4 significantly regulated global protein myristoylation or myristoylation of Src kinase in prostate cancer cells. Knockdown of ACSL4 inhibited the proliferation, migration, invasion, and xenograft growth of AR-independent prostate cancer cells. Our results suggest that the surge of ACSL4 levels by targeting AR signaling increases fatty acyl-CoAs biosynthesis and protein myristoylation, indicating the opposite, yet complementary or Yin-Yang regulation of ACSL3 and 4 levels in sustaining fatty acid metabolism when targeting androgen-AR signaling. This study reveals a mechanistic understanding of ACSL4 as a potential therapeutic target for treatment of AR-independent prostate cancer. IMPLICATIONS: AR coordinately regulates the expression of ACSL3 and ACSL4, such that AR pathway-independent prostate tumors become dependent on ACSL4-mediated fatty acid metabolism.
Asunto(s)
Coenzima A Ligasas/metabolismo , Ácidos Grasos/metabolismo , Neoplasias de la Próstata/genética , Receptores Androgénicos/metabolismo , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones SCID , Neoplasias de la Próstata/patología , Transducción de SeñalRESUMEN
Cross talk of stromal-epithelial cells plays an essential role in both normal development and tumor initiation and progression. Fibroblast growth factor (FGF)-FGF receptor (FGFR)-Src kinase axis is one of the major signal transduction pathways to mediate this cross talk. Numerous genomic studies have demonstrated that expression levels of FGFR/Src are deregulated in a variety of cancers including prostate cancer; however, the role that paracrine FGF (from stromal cells) plays in dysregulated expression of epithelial FGFRs/Src and tumor progression in vivo is not well evaluated. In this study, we demonstrate that ectopic expression of wild-type FGFR1/2 or Src kinase in epithelial cells was not sufficient to initiate prostate tumorigenesis under a normal stromal microenvironment in vivo. However, paracrine FGF10 synergized with ectopic expression of epithelial FGFR1 or FGFR2 to induce epithelial-mesenchymal transition. Additionally, paracrine FGF10 sensitized FGFR2-transformed epithelial cells to initiate prostate tumorigenesis. Next, paracrine FGF10 also synergized with overexpression of epithelial Src kinase to high-grade tumors. But loss of the myristoylation site in Src kinase inhibited paracrine FGF10-induced prostate tumorigenesis. Loss of myristoylation alters Src levels in the cell membrane and inhibited FGF-mediated signaling including inhibition of the phosphotyrosine pattern and FAK phosphorylation. Our study demonstrates the potential tumor progression by simultaneous deregulation of proteins in the FGF/FGFRs/Src signal axis and provides a therapeutic strategy of targeting myristoylation of Src kinase to interfere with the tumorigenic process.
Asunto(s)
Transformación Celular Neoplásica/patología , Factores de Crecimiento de Fibroblastos/genética , Genes src/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Oncogenes/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular , Transformación Celular Neoplásica/genética , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Tirosina Quinasas Receptoras/genética , Transducción de Señal/genética , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
Protein N-myristoylation enables localization to membranes and helps maintain protein conformation and function. N-myristoyltransferases (NMT) catalyze co- or posttranslational myristoylation of Src family kinases and other oncogenic proteins, thereby regulating their function. In this study, we provide genetic and pharmacologic evidence that inhibiting the N-myristoyltransferase NMT1 suppresses cell-cycle progression, proliferation, and malignant growth of prostate cancer cells. Loss of myristoylation abolished the tumorigenic potential of Src and its synergy with androgen receptor in mediating tumor invasion. We identified the myristoyl-CoA analogue B13 as a small-molecule inhibitor of NMT1 enzymatic activity. B13 exposure blocked Src myristoylation and Src localization to the cytoplasmic membrane, attenuating Src-mediated oncogenic signaling. B13 exerted its anti-invasive and antitumor effects against prostate cancer cells, with minimal toxic side-effects in vivo Structural optimization based on structure-activity relationships enabled the chemical synthesis of LCL204, with enhanced inhibitory potency against NMT1. Collectively, our results offer a preclinical proof of concept for the use of protein myristoylation inhibitors as a strategy to block prostate cancer progression. Cancer Res; 77(24); 6950-62. ©2017 AACR.
Asunto(s)
Aciltransferasas/fisiología , Ácido Mirístico/metabolismo , Fosfotransferasas/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/genética , Aciltransferasas/metabolismo , Sustitución de Aminoácidos , Animales , Células Cultivadas , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Mutación Missense , Fosforilación/efectos de los fármacos , Fosforilación/genética , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Procesamiento Proteico-Postraduccional/genética , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Relación Estructura-Actividad , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The metallo-beta-lactamase IMP-1 catalyzes the hydrolysis of a broad range of beta-lactam antibiotics to provide bacterial resistance to these compounds. In this study, 29 amino acid residue positions in and near the active-site pocket of the IMP-1 enzyme were randomized individually by site-directed mutagenesis of the corresponding codons in the bla(IMP-1) gene. The 29 random libraries were used to identify positions that are critical for the catalytic and substrate-specific properties of the IMP-1 enzyme. Mutants from each of the random libraries were selected for the ability to confer to Escherichia coli resistance to ampicillin, cefotaxime, imipenem or cephaloridine. The DNA sequence of several functional mutants was determined for each of the substrates. Comparison of the sequences of mutants obtained from the different antibiotic selections indicates the sequence requirements for each position in the context of each substrate. The zinc-chelating residues in the active site were found to be essential for hydrolysis of all antibiotics tested. Several positions, however, displayed context-dependent sequence requirements, in that they were essential for one substrate(s) but not others. The most striking examples included Lys69, Asp84, Lys224, Pro225, Gly232, Asn233, Asp236 and Ser262. In addition, comparison of the results for all 29 positions indicates that hydrolysis of imipenem, cephaloridine and ampicillin has stringent sequence requirements, while the requirements for hydrolysis of cefotaxime are more relaxed. This suggests that more information is required to specify active-site pockets that carry out imipenem, cephaloridine or ampicillin hydrolysis than one that catalyzes cefotaxime hydrolysis.
Asunto(s)
Aminoácidos/metabolismo , beta-Lactamasas/metabolismo , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Modelos Moleculares , Especificidad por Sustrato , beta-Lactamasas/químicaRESUMEN
One of the major processes for aerobic biodegradation of aromatic compounds is initiated by Rieske dioxygenases. Benzoate dioxygenase contains a reductase component, BenC, that is responsible for the two-electron transfer from NADH via FAD and an iron-sulfur cluster to the terminal oxygenase component. Here, we present the structure of BenC from Acinetobacter sp. strain ADP1 at 1.5 A resolution. BenC contains three domains, each binding a redox cofactor: iron-sulfur, FAD and NADH, respectively. The [2Fe-2S] domain is similar to that of plant ferredoxins, and the FAD and NADH domains are similar to members of the ferredoxin:NADPH reductase superfamily. In phthalate dioxygenase reductase, the only other Rieske dioxygenase reductase for which a crystal structure is available, the ferredoxin-like and flavin binding domains are sequentially reversed compared to BenC. The BenC structure shows significant differences in the location of the ferredoxin domain relative to the other domains, compared to phthalate dioxygenase reductase and other known systems containing these three domains. In BenC, the ferredoxin domain interacts with both the flavin and NAD(P)H domains. The iron-sulfur center and the flavin are about 9 A apart, which allows a fast electron transfer. The BenC structure is the first determined for a reductase from the class IB Rieske dioxygenases, whose reductases transfer electrons directly to their oxygenase components. Based on sequence similarities, a very similar structure was modeled for the class III naphthalene dioxygenase reductase, which transfers electrons to an intermediary ferredoxin, rather than the oxygenase component.
Asunto(s)
Acinetobacter/enzimología , Oxigenasas/química , Acinetobacter/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , Transporte de Electrón , Ferredoxina-NADP Reductasa/química , Ferredoxinas/química , Flavinas/metabolismo , Hierro/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , NAD/metabolismo , Oxigenasas/genética , Oxigenasas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Azufre/metabolismoRESUMEN
Elevation of the chromatin repression factor enhancer of zeste homolog (EZH2) is associated with progression and poor prognosis in several human cancers including prostate cancer. However, the mechanisms driving EZH2 expression are not fully understood. In this study, we investigated the functional synergy in prostate cancers in mice resulting from activation of the androgen receptor, Kras, and Akt, which drives three of the most frequently activated oncogenic signaling pathways in prostate cancer. Although, any two of these three events were sufficient to promote the formation and progression of prostate cancer, only the synergy of androgen receptor and Kras signaling could elevate EZH2 expression and expand prostate cancer progenitor cells in vivo. Our findings have revealed a genetic mechanism resulting in enhanced EZH2 expression during the progression of aggressive prostate cancer, with important implications for understanding how to target advanced disease where cancer progenitor cells may be critical.
Asunto(s)
Células Madre Neoplásicas/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal/fisiología , Animales , Proteína Potenciadora del Homólogo Zeste 2 , Regulación Neoplásica de la Expresión Génica/fisiología , Masculino , Ratones , Células Madre Neoplásicas/patología , Neoplasias de la Próstata/patologíaRESUMEN
PIM1 is a serine/threonine kinase that has diverse biological roles in cell survival, proliferation and differentiation. PIM1 has been implicated in early transformation and tumor progression in haematopoietic malignancies and prostate carcinomas. The ability of PIM1 to regulate these processes is thought to be in part secondary to its activity in stimulating 4EBP1 phosphorylation and enhancement of protein synthesis. Because 4EBP1 is an mTOR substrate, we have investigated how PIM1 might regulate this latter enzyme. We have examined the ability of PIM1 to modulate PRAS40, a protein known to negatively regulate mTOR activity in FDCP1 cells. Upon phosphorylation, PRAS40 dissociates from the mTOR complex and increases mTOR kinase activity. We find that enforced overexpression of PIM1 increases PRAS40 phosphorylation at Thr(246), an AKT phosphorylation site, whether grown in complete media or deprived of IL-3 and serum. The increase in PRAS40 phosphorylation was independent of AKT activation and not inhibited by wortmannin. In vitro kinase assays indicate that the PIM1 protein kinase is capable of directly phosphorylating Thr(246) in PRAS40. PIM1 protein kinase overexpression reduced the association of PRAS40 with mTOR, and increased the mTOR directed phosphorylation of 4EBP1 and p70S6Kinase. Treatment of FDCP1 cells transfected with PIM1 (FD/mpim44) with small molecule inhibitors of PIM1 kinase activity reduced both PRAS40 and 4EBP1 phosphorylation. These results suggest that PIM1 regulates mTOR activity through phosphorylation of PRAS40. Thus, increases in mTOR activity mediated by the PIM protein kinase may have the potential to control cell growth.