RESUMEN
BACKGROUND: Monocarboxylate transporter 1 (MCT1) is a regulator of cell metabolism and a therapeutic target for cancer treatment. Understanding the changes in tumour function accompanying MCT1 inhibition will better characterise the anti-tumour effects of MCT1 inhibitors, potentially enabling the identification of pharmacodynamic biomarkers for the clinical development of these agents. METHODS: We assessed the impact of the MCT1 inhibitor AZD3965 on tumour metabolism and immune cell infiltration as key determinants of tumour biological function in the MCT1-dependent Raji B cell lymphoma model. RESULTS: Treatment of Raji xenograft-bearing severe combined immunodeficiency mice with AZD3965 led to inhibition of tumour growth paralleled with a decrease in tumour choline, as detected by non-invasive in vivo proton nuclear magnetic resonance spectroscopy. This effect was attributed to inhibition of phosphocholine de novo synthesis following decreased choline kinase α protein and messenger RNA expression that correlated with the AZD3965-induced build-up in intracellular lactate. These changes were concomitant with increased tumour immune cell infiltration involving dendritic and natural killer cells. CONCLUSIONS: Our data provide new insights into the metabolic and cellular changes that occur in the tumour microenvironment following MCT1 blockade, which may contribute to the anti-tumour activity of AZD3965 and could have potential as pharmacodynamic biomarkers of MCT1 inhibition.
Asunto(s)
Metabolismo de los Lípidos/efectos de los fármacos , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Transportadores de Ácidos Monocarboxílicos/uso terapéutico , Pirimidinonas/uso terapéutico , Tiofenos/uso terapéutico , Animales , Técnicas de Cultivo de Célula , Línea Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Transportadores de Ácidos Monocarboxílicos/farmacología , Pirimidinonas/farmacología , Tiofenos/farmacologíaRESUMEN
BACKGROUND: BRAF inhibitors, such as vemurafenib, have shown efficacy in BRAF-mutant melanoma treatment but acquired-resistance invariably develops. Unveiling the potential vulnerabilities associated with vemurafenib resistance could provide rational strategies for combinatorial treatment. METHODS: This work investigates the metabolic characteristics and vulnerabilities of acquired resistance to vemurafenib in three generated BRAF-mutant human melanoma cell clones, analysing metabolic profiles, gene and protein expression in baseline and nutrient withdrawal conditions. Preclinical findings are correlated with gene expression analysis from publicly available clinical datasets. RESULTS: Two vemurafenib-resistant clones showed dependency on lipid metabolism and increased prostaglandin E2 synthesis and were more responsive to vemurafenib under EGFR inhibition, potentially implicating inflammatory lipid and EGFR signalling in ERK reactivation and vemurafenib resistance. The third resistant clone showed higher pyruvate-carboxylase (PC) activity indicating increased anaplerotic mitochondrial metabolism, concomitant with reduced GLUT-1, increased PC protein expression and survival advantage under nutrient-depleted conditions. Prostaglandin synthase (PTGES) expression was inversely correlated with melanoma patient survival. Increases in PC and PTGES gene expression were observed in some patients following progression on BRAF inhibitors. CONCLUSIONS: Altogether, our data highlight heterogeneity in metabolic adaptations during acquired resistance to vemurafenib in BRAF-mutant melanoma, potentially uncovering key clinically-relevant mechanisms for combinatorial therapeutic targeting.
Asunto(s)
Antineoplásicos/farmacología , Dinoprostona/biosíntesis , Resistencia a Antineoplásicos/efectos de los fármacos , Melanoma/metabolismo , Mitocondrias/metabolismo , Mutación , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/metabolismo , Vemurafenib/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Gefitinib/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Melanoma/patología , Mitocondrias/efectos de los fármacos , Prostaglandina-E Sintasas/genética , Piruvato Carboxilasa/genética , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/patologíaRESUMEN
Choline metabolites are widely studied in cancer research as biomarkers of malignancy and as indicators of therapeutic response. However, endogenous phosphocholine levels are determined by a number of processes that confound the interpretation of these measurements, including membrane transport rates and a series of enzyme catalysed reactions in the Kennedy pathway. Employing a dynamic (31)P NMR assay that is specific to choline kinase (ChoK) we have measured the rates of this enzyme reaction in cell lysates of MDA-MB-231 breast, PC-3 prostate and HeLa cervical cancer cells and in solutions of purified human ChoK. The rates are sensitive to inhibition by hemicholinium-3 (HC-3), a competitive ChoK inhibitor, and to N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulphonamide (H-89), an agent commercialized as a specific cyclic-AMP-dependent protein kinase A (PKA) inhibitor.
Asunto(s)
Colina Quinasa/metabolismo , Neoplasias/enzimología , Línea Celular Tumoral , Colina Quinasa/antagonistas & inhibidores , Humanos , Isoquinolinas/farmacología , Espectroscopía de Resonancia Magnética , Neoplasias/patología , Isótopos de Fósforo , Fosforilación/efectos de los fármacos , Sulfonamidas/farmacología , Factores de TiempoRESUMEN
Several mitogen-activated protein kinase (MAPK) signaling inhibitors are currently undergoing clinical trial as part of novel mechanism-based anticancer treatment strategies. This study was aimed at detecting biomarkers of MAPK signaling inhibition in human breast and colon carcinoma cells using magnetic resonance spectroscopy. We investigated the effect of the prototype MAPK kinase inhibitor U0126 on the (31)P-MR spectra of MDA-MB-231, MCF-7 and Hs578T breast, and HCT116 colon carcinoma cells. Treatment of MDA-MB-231 cells with 50 micromol/L U0126 for 2, 4, 8, 16, 24, 32, and 40 hours caused inhibition of extracellular signal-regulated kinases (ERK1/2) phosphorylation from 2 hours onwards. (31)P-MR spectra of extracted cells indicated that this was associated with a significant drop in phosphocholine levels to 78 +/- 8% at 8 hours, 74 +/- 8% at 16 hours, 66 +/- 7% at 24 hours, 71 +/- 10% at 32 hours, and 65 +/- 10% at 40 hours post-treatment. In contrast, the lower concentration of 10 micromol/L U0126 for 40 hours had no significant effect on either P-ERK1/ 2 or phosphocholine levels in MDA-MB-231 cells. Depletion of P-ERK1/2 in MCF-7 and Hs578T cells with 50 micromol/L U0126 also produced a drop in phosphocholine levels to 51 +/- 17% at 40 hours and 23 +/- 12% at 48 hours, respectively. Similarly, in HCT116 cells, inhibition with 30 micromol/L U0126 caused depletion of P-ERK1/2 and a decrease in phosphocholine levels to 80 +/- 9% at 16 hours and 61 +/- 4% at 24 hours post-treatment. The reduction in phosphocholine in MDA-MB-231 and HCT116 cells correlated positively with the drop in P-ERK1/2 levels. Our results show that MAPK signaling inhibition with U0126 is associated with a time-dependent decrease in cellular phosphocholine levels. Thus, phosphocholine has potential as a noninvasive pharmacodynamic marker for monitoring MAPK signaling blockade.
Asunto(s)
Neoplasias de la Mama/enzimología , Butadienos/farmacología , Neoplasias Colorrectales/enzimología , Inhibidores Enzimáticos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Nitrilos/farmacología , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Células HCT116 , Humanos , Espectroscopía de Resonancia Magnética/métodos , Fosforilcolina/metabolismoRESUMEN
Phosphoinositide 3-kinase (PI3K) is an attractive target for novel mechanism-based anticancer treatment. We used magnetic resonance (MR) spectroscopy (MRS) to detect biomarkers of PI3K signaling inhibition in human breast cancer cells. MDA-MB-231, MCF-7, and Hs578T cells were treated with the prototype PI3K inhibitor LY294002, and the (31)P MR spectra of cell extracts were monitored. In every case, LY294002 treatment was associated with a significant decrease in phosphocholine levels by up to 2-fold (P < 0.05). In addition, a significant increase in glycerophosphocholine levels by up to 5-fold was also observed (P Asunto(s)
Neoplasias de la Mama/metabolismo
, Inhibidores Enzimáticos/farmacología
, Espectroscopía de Resonancia Magnética/métodos
, Inhibidores de las Quinasa Fosfoinosítidos-3
, Androstadienos/farmacología
, Extractos Celulares
, Cromonas/farmacología
, Femenino
, Glicerilfosforilcolina/análisis
, Glicerilfosforilcolina/metabolismo
, Humanos
, Morfolinas/farmacología
, Fosfatidiletanolaminas/análisis
, Fosfatidiletanolaminas/metabolismo
, Fosfatidilinositol 3-Quinasas/metabolismo
, Fosforilcolina/metabolismo
, Transducción de Señal
, Células Tumorales Cultivadas
, Wortmanina
RESUMEN
Targeted therapies specific to the BRAF-MEK-ERK signaling pathway have shown great promise in the treatment of malignant melanoma in the last few years, with these drugs now commonly used in clinic. Melanoma cells treated using these agents are known to exhibit increased levels of melanin pigment and tyrosinase activity. In this study we assessed the potential of non-invasive imaging approaches (photoacoustic imaging (PAI) and magnetic resonance imaging (MRI)) to detect melanin induction in SKMEL28 human melanoma cells, following inhibition of Hsp90 and BRAF signaling using 17-AAG and vemurafenib, respectively. We confirmed, using western blot and spectrophotometry, that Hsp90 or BRAF inhibitor-induced melanoma cell differentiation resulted in an upregulation of tyrosinase and melanin expression levels, in comparison to control cells. This post-treatment increase in cellular pigmentation induced a significant increase in PAI signals that are spectrally identifiable and shortening of the MRI relaxation times T 1 and [Formula: see text]. This proof-of-concept study demonstrates the potential of MRI and PAI for detecting the downstream cellular changes induced by Hsp90 and BRAF-MEK-targeted therapies in melanoma cells with potential significance for in vivo imaging.
Asunto(s)
Antineoplásicos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Imagen por Resonancia Magnética , Melanoma/diagnóstico , Melanoma/metabolismo , Técnicas Fotoacústicas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Humanos , Pigmentos Biológicos/biosíntesisRESUMEN
Monocarboxylate transporters (MCT) modulate tumor cell metabolism and offer promising therapeutic targets for cancer treatment. Understanding the impact of MCT blockade on tumor cell metabolism may help develop combination strategies or identify pharmacodynamic biomarkers to support the clinical development of MCT inhibitors now in clinical trials. In this study, we assessed the impact of the MCT1 inhibitor AZD3965 on cancer cell metabolism in vitro and in vivo Exposing human lymphoma and colon carcinoma cells to AZD3965 increased MCT4-dependent accumulation of intracellular lactate, inhibiting monocarboxylate influx and efflux. AZD3965 also increased the levels of TCA cycle-related metabolites and 13C-glucose mitochondrial metabolism, enhancing oxidative pyruvate dehydrogenase and anaplerotic pyruvate carboxylase fluxes. Increased mitochondrial metabolism was necessary to maintain cell survival under drug stress. These effects were counteracted by coadministration of the mitochondrial complex I inhibitor metformin and the mitochondrial pyruvate carrier inhibitor UK5099. Improved bioenergetics were confirmed in vivo after dosing with AZD3965 in mouse xenograft models of human lymphoma. Our results reveal new metabolic consequences of MCT1 inhibition that might be exploited for therapeutic and pharmacodynamic purposes. Cancer Res; 77(21); 5913-24. ©2017 AACR.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfoma/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Pirimidinonas/farmacología , Simportadores/antagonistas & inhibidores , Tiofenos/farmacología , Acrilatos/administración & dosificación , Acrilatos/farmacología , Animales , Western Blotting , Línea Celular Tumoral , Metabolismo Energético/efectos de los fármacos , Femenino , Células HT29 , Humanos , Ácido Láctico/metabolismo , Linfoma/metabolismo , Linfoma/patología , Espectroscopía de Resonancia Magnética , Metformina/administración & dosificación , Metformina/farmacología , Ratones SCID , Mitocondrias/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Pirimidinonas/administración & dosificación , Simportadores/metabolismo , Tiofenos/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acquired resistance (AQR) to drug treatment occurs frequently in cancer patients and remains an impediment to successful therapy. The aim of this study was to gain insight into how AQR arises following the application of PI3K/mTOR inhibitors. H1975 lung cancer cells with EGFR T790M mutations that confer resistance to EGFR inhibitors underwent prolonged treatment with the PI3K/mTOR inhibitor, BEZ235. Monoclonal cells with stable and increased resistance to BEZ235 were obtained after 8 months treatment. These AQR clones showed class-specific resistance to PI3K/mTOR inhibitors, reduced G1 cell cycle arrest and impedance of migration following PI3K/mTOR inhibition, reduced PTEN expression and increased Akt and S6RP phosphorylation. Transcriptome analysis revealed the AQR clones had increased expression of the metabolite transporters SLC16A9 and SLC16A7, suggestive of altered cell metabolism. Subsequent experiments revealed that AQR clones possess features consistent with elevated glycolysis, including increased levels of glucose, lactate, glutamine, glucose dependence, GLUT1 expression, and rates of post-glucose extracellular acidification, and decreased levels of reactive oxygen species and rates of oxygen consumption. Combination treatment of BEZ235 with the glycolysis inhibitor 3-bromopyruvate was synergistic in AQR clones, but only additive in parental cells. DNA sequencing revealed the presence of a mitochondrial DNA (mtDNA) MT-C01 variant in AQR but not parental cells. Depletion of mitochondrial DNA in parental cells induced resistance to BEZ235 and other PI3K/mTOR inhibitors, and was accompanied by increased glycolysis. The results of this study provide the first evidence that a metabolic switch associated with mtDNA mutation can be an underlying mechanism for AQR.
RESUMEN
Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long-term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity, and potential as noninvasive imaging response biomarkers. 1H NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF-mutant (WM266.4 and SKMEL28) but not BRAFWT (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine, and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. 13C NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of hexokinase 2, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase, and monocarboxylate transporters (MCT) 1 and 4 in BRAF-mutant but not BRAFWT cells and, interestingly, decreased BRAF-mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized 13C-pyruvate-lactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as noninvasive imaging of response. Mol Cancer Ther; 15(12); 2987-99. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Indoles/farmacología , Lactatos/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Ácido Pirúvico/metabolismo , Sulfonamidas/farmacología , Línea Celular Tumoral , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Metabolómica/métodos , Modelos Biológicos , Piruvato Carboxilasa/metabolismo , VemurafenibRESUMEN
The RAS/BRAF/MEK/ERK signaling pathway is a central driver in cancer with many BRAF and MEK inhibitors being evaluated in clinical trials. Identifying noninvasive biomarkers of early pharmacodynamic responses is important for development of these targeted drugs. As increased aerobic glycolysis is often observed in cancer, we hypothesized that MEK1/2 (MAP2K1/MAP2K2) inhibitors may reduce lactate levels as detected by magnetic resonance spectroscopy (MRS), as a metabolic biomarker for the pharmacodynamic response. MRS was used to monitor intracellular and extracellular levels of lactate in human cancer cells in vitro and in melanoma tumors ex vivo. In addition, we used (1)H MRS and a fluorescent glucose analog to evaluate the effect of MEK inhibition on glucose uptake. MEK1/2 signaling inhibition reduced extracellular lactate levels in BRAF-dependent cells but not BRAF-independent cells. The reduction in extracellular lactate in BRAF-driven melanoma cells was time-dependent and associated with reduced expression of hexokinase-II driven by c-Myc depletion. Taken together, these results reveal how MEK1/2 inhibition affects cancer cell metabolism in the context of BRAF oncogene addiction. Furthermore, they offer a preclinical proof-of-concept for the use of MRS to measure lactate as a noninvasive metabolic biomarker for pharmacodynamic response to MEK1/2 inhibition in BRAF-driven cancers.
Asunto(s)
Antineoplásicos/farmacología , Bencimidazoles/farmacología , Ácido Láctico/metabolismo , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Animales , Benzamidas/farmacología , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Desoxiglucosa/farmacología , Difenilamina/análogos & derivados , Difenilamina/farmacología , Femenino , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Hexoquinasa/antagonistas & inhibidores , Hexoquinasa/metabolismo , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Ratones , Mutación Missense , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/genética , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Real-time detection of the rates of metabolic flux, or exchange rates of endogenous enzymatic reactions, is now feasible in biological systems using Dynamic Nuclear Polarization Magnetic Resonance. Derivation of reaction rate kinetics from this technique typically requires multi-compartmental modeling of dynamic data, and results are therefore model-dependent and prone to misinterpretation. We present a model-free formulism based on the ratio of total areas under the curve (AUC) of the injected and product metabolite, for example pyruvate and lactate. A theoretical framework to support this novel analysis approach is described, and demonstrates that the AUC ratio is proportional to the forward rate constant k. We show that the model-free approach strongly correlates with k for whole cell in vitro experiments across a range of cancer cell lines, and detects response in cells treated with the pan-class I PI3K inhibitor GDC-0941 with comparable or greater sensitivity. The same result is seen in vivo with tumor xenograft-bearing mice, in control tumors and following drug treatment with dichloroacetate. An important finding is that the area under the curve is independent of both the input function and of any other metabolic pathways arising from the injected metabolite. This model-free approach provides a robust and clinically relevant alternative to kinetic model-based rate measurements in the clinical translation of hyperpolarized (13)C metabolic imaging in humans, where measurement of the input function can be problematic.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Animales , Línea Celular Tumoral , Humanos , Indazoles , Cinética , Ácido Láctico/química , Redes y Vías Metabólicas/efectos de los fármacos , Ratones , Modelos Teóricos , Piruvatos/química , Sulfonamidas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Histone deacetylase (HDAC) inhibitors are currently approved for cutaneous T-cell lymphoma and are in mid-late stage trials for other cancers. The HDAC inhibitors LAQ824 and SAHA increase phosphocholine (PC) levels in human colon cancer cells and tumor xenografts as observed by magnetic resonance spectroscopy (MRS). In this study, we show that belinostat, an HDAC inhibitor with an alternative chemical scaffold, also caused a rise in cellular PC content that was detectable by (1)H and (31)P MRS in prostate and colon carcinoma cells. In addition, (1)H MRS showed an increase in branched chain amino acid and alanine concentrations. (13)C-choline labeling indicated that the rise in PC resulted from increased de novo synthesis and correlated with an induction of choline kinase α expression. Furthermore, metabolic labeling experiments with (13)C-glucose showed that differential glucose routing favored alanine formation at the expense of lactate production. Additional analysis revealed increases in the choline/water and phosphomonoester (including PC)/total phosphate ratios in vivo. Together, our findings provide mechanistic insights into the impact of HDAC inhibition on cancer cell metabolism and highlight PC as a candidate noninvasive imaging biomarker for monitoring the action of HDAC inhibitors.
Asunto(s)
Biomarcadores de Tumor/análisis , Colina Quinasa/metabolismo , Neoplasias del Colon/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Fosforilcolina/análisis , Fosforilcolina/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Humanos , Espectroscopía de Resonancia Magnética/métodos , Masculino , Ratones , Ratones Desnudos , Sulfonamidas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Rational drug discovery and development requires biomarkers to inform on target modulation and treatment efficacy. Many aspects of metabolism are altered in cancer, compared to normal tissues, and are often regulated by oncogene activation. Non-invasive imaging of spatio-temporal effects of molecularly targeted anticancer agents on tumor metabolism has considerable potential in the development and use of personalized molecular medicine approaches to cancer treatment. Here we describe how non-invasive monitoring of metabolism, using primarily magnetic resonance spectroscopy (MRS), can be used to follow treatment with novel molecularly targeted anticancer agents. We discuss how the regulation of metabolic pathways by oncogenic signaling can affect MRS-detectable metabolic signals together with the physiological factors that can influence the measured changes. Finally, the translation of these metabolic measurements from pre-clinical models to patients will be discussed.
Asunto(s)
Antineoplásicos/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Terapia Molecular Dirigida/métodos , Neoplasias/metabolismo , Antineoplásicos/uso terapéutico , Biomarcadores , Colina/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/uso terapéutico , Glucosa/metabolismo , Humanos , Redes y Vías Metabólicas , Imagen Molecular/métodos , Neoplasias/patología , Neoplasias/terapia , Tomografía de Emisión de Positrones , Transducción de SeñalRESUMEN
Molecular chaperone heat shock protein 90 (Hsp90) inhibitors are promising targeted cancer therapeutic drugs, with the advantage that they deplete multiple oncogenic client proteins and modulate all the classical hallmarks of cancer. They are now in clinical trial and show potential for activity in melanoma and other malignancies. Here we explore the metabolic response to Hsp90 inhibition in human melanoma cells using magnetic resonance spectroscopy. We show that, concomitant with growth inhibition and re-differentiation, Hsp90 inhibition in human melanoma cells is associated with increased glycerophosphocholine content. This was seen with both the clinical geldanamycin-based Hsp90 drug 17-AAG and the structurally dissimilar Hsp90 inhibitor CCT018159. The effect was noted in both BRAF mutant SKMEL28 and BRAF wildtype CHL-1 melanoma cells. Elevated content of the -CH2+CH3 fatty acyl chains and cytoplasmic mobile lipid droplets was also observed in 17-AAG-treated SKMEL28 cells. Importantly, the phospholipase A2 inhibitor bromoenol lactone prevented the rise in glycerophosphocholine seen with 17-AAG, suggesting a role for phospholipase A2 activation in the Hsp90 inhibitor-induced metabolic response. Our findings provide a basis for using metabolic changes as non-invasive indicators of Hsp90 inhibition and potentially as biomarkers of anticancer activity with Hsp90 drugs in malignant melanoma and possibly in other cancers.
Asunto(s)
Antineoplásicos/farmacología , Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Antineoplásicos/uso terapéutico , Benzoquinonas/química , Benzoquinonas/uso terapéutico , Biomarcadores Farmacológicos , Glicerilfosforilcolina/metabolismo , Compuestos Heterocíclicos con 2 Anillos/química , Compuestos Heterocíclicos con 2 Anillos/farmacología , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/uso terapéutico , Metabolismo de los Lípidos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Melanoma/patología , Mutación/genética , Naftalenos/farmacología , Inhibidores de Fosfolipasa A2 , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pirazoles/química , Pirazoles/farmacología , Pironas/farmacología , Neoplasias Cutáneas/patologíaRESUMEN
Phosphoinositide-specific phospholipase Cγ1 (PLCγ1) is activated downstream of many receptor tyrosine kinases to promote cell motility. Inhibition of this protein is being explored as a therapeutic strategy for blocking cancer cell invasion and metastasis. The clinical development of such cytostatic therapies requires the implementation of pharmacodynamic biomarkers of target modulation. In this study, we use magnetic resonance spectroscopy to explore metabolic biomarkers of PLCγ1 down-regulation in PC3LN3 prostate cancer cells. We show that inhibition of PLCγ1 via an inducible short hairpin RNA system causes a reduction in phosphocholine levels by up to 50% relative to the control as detected by (1)H and (31)P magnetic resonance spectroscopy analyses. This correlated with a rounded-up morphology and reduced cell migration. Interestingly, the fall in phosphocholine levels was not recorded in cells with constitutive PLCγ1 knockdown where the rounded-up phenotype was no longer apparent. This study reveals alterations in metabolism that accompany the cellular effects of PLCγ1 knockdown and highlights phosphocholine as a potential pharmacodynamic biomarker for monitoring the action of inhibitors targeting PLCγ1 signaling.
Asunto(s)
Biomarcadores de Tumor , Regulación Neoplásica de la Expresión Génica , Fosfolipasa C gamma/metabolismo , Fosforilcolina/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular , Movimiento Celular/genética , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Fosfolipasa C gamma/genética , Neoplasias de la Próstata/patología , Interferencia de ARNRESUMEN
The aim of this work was to use phosphorus magnetic resonance spectroscopy ((31)P MRS) to investigate the pharmacodynamic effects of LAQ824, a histone deacetylase (HDAC) inhibitor. Human HT29 colon carcinoma cells were examined by (31)P MRS after treatment with LAQ824 and another HDAC inhibitor, suberoylanilide hydroxamic acid. HT29 xenografts and tumor extracts were also examined using (31)P MRS, pre- and post-LAQ824 treatment. Histone H3 acetylation was determined using Western blot analysis, and tumor microvessel density by immunohistochemical staining of CD31. Phosphocholine showed a significant increase in HT29 cells after treatment with LAQ824 and suberoylanilide hydroxamic acid. In vivo, the ratio of phosphomonoester/total phosphorus (TotP) signal was significantly increased in LAQ824-treated HT29 xenografts, and this ratio was inversely correlated with changes in tumor volume. Statistically significant decreases in intracellular pH, beta-nucleoside triphosphate (beta-NTP)/TotP, and beta-NTP/inorganic phosphate (Pi) and an increase in Pi/TotP were also seen in LAQ824-treated tumors. Tumor extracts showed many significant metabolic changes after LAQ824 treatment, in parallel with increased histone acetylation and decreased microvessel density. Treatment with LAQ824 resulted in altered phospholipid metabolism and compromised tumor bioenergetics. The phosphocholine and phosphomonoester increases may have the potential to act as pharmacodynamic markers for noninvasively monitoring tumor response after treatment with LAQ824 or other HDAC inhibitors.