RESUMEN
Oncogenic transformation is associated with profound changes in cellular metabolism, but whether tracking these can improve disease stratification or influence therapy decision-making is largely unknown. Using the iKnife to sample the aerosol of cauterized specimens, we demonstrate a new mode of real-time diagnosis, coupling metabolic phenotype to mutant PIK3CA genotype. Oncogenic PIK3CA results in an increase in arachidonic acid and a concomitant overproduction of eicosanoids, acting to promote cell proliferation beyond a cell-autonomous manner. Mechanistically, mutant PIK3CA drives a multimodal signaling network involving mTORC2-PKCζ-mediated activation of the calcium-dependent phospholipase A2 (cPLA2). Notably, inhibiting cPLA2 synergizes with fatty acid-free diet to restore immunogenicity and selectively reduce mutant PIK3CA-induced tumorigenicity. Besides highlighting the potential for metabolic phenotyping in stratified medicine, this study reveals an important role for activated PI3K signaling in regulating arachidonic acid metabolism, uncovering a targetable metabolic vulnerability that largely depends on dietary fat restriction. VIDEO ABSTRACT.
Asunto(s)
Ácido Araquidónico/análisis , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Eicosanoides/metabolismo , Animales , Ácido Araquidónico/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/genética , Citosol/metabolismo , Eicosanoides/fisiología , Activación Enzimática , Femenino , Humanos , Metabolismo de los Lípidos/fisiología , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Redes y Vías Metabólicas/genética , Redes y Vías Metabólicas/fisiología , Ratones Endogámicos BALB C , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasas A2/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) initiates IRE1α, ATF6, and PERK cascades, leading to a transcriptional/translational response known as unfolded protein response (UPR). Here we show that VEGF activates UPR mediators through a PLCγ-mediated crosstalk with the mTORC1 complex without accumulation of unfolded proteins in the ER. Activation of ATF6 and PERK contributes to the survival effect of VEGF on endothelial cells (ECs) by positively regulating mTORC2-mediated phosphorylation of AKT on Ser473, which is required for full activity of AKT. Low levels of CHOP allow ECs to evade the proapoptotic effect of this UPR product. Depletion of PLCγ, ATF6, or eIF2α dramatically inhibited VEGF-induced vascularization in mouse Matrigel plugs, suggesting that the ER and the UPR machinery constitute components of the VEGF signaling circuit that regulates EC survival and angiogenesis, extending their role beyond adaptation to ER stress.
Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Estrés del Retículo Endoplásmico , Células Endoteliales/fisiología , Neovascularización Patológica , Respuesta de Proteína Desplegada , Factor A de Crecimiento Endotelial Vascular/metabolismo , eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 6/genética , Animales , Supervivencia Celular , Estrés del Retículo Endoplásmico/genética , Regulación de la Expresión Génica , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Fosfolipasa C gamma/metabolismo , Fosforilación , Desplegamiento Proteico , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Respuesta de Proteína Desplegada/genética , Factor A de Crecimiento Endotelial Vascular/genética , eIF-2 Quinasa/genéticaRESUMEN
A new tissue sample embedding and processing method is presented that provides downstream compatibility with numerous different histological, molecular biology, and analytical techniques. The methodology is based on the low temperature embedding of fresh frozen specimens into a hydrogel matrix composed of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) and sectioning using a cryomicrotome. The hydrogel was expected not to interfere with standard tissue characterization methods, histologically or analytically. We assessed the compatibility of this protocol with various mass spectrometric imaging methods including matrix-assisted laser desorption ionization (MALDI), desorption electrospray ionization (DESI) and secondary ion mass spectrometry (SIMS). We also demonstrated the suitability of the universal protocol for extraction based molecular biology techniques such as rt-PCR. The integration of multiple analytical modalities through this universal sample preparation protocol offers the ability to study tissues at a systems biology level and directly linking results to tissue morphology and cellular phenotype.
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Hidrogeles/química , Derivados de la Hipromelosa/química , Povidona/química , Manejo de Especímenes/métodos , Adhesión del Tejido/métodos , Animales , Masculino , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
In ovarian cancer, the prometastatic RTK AXL promotes motility, invasion and poor prognosis. Here, we show that reduced survival caused by AXL overexpression can be mitigated by the expression of the GPI-anchored tumour suppressor OPCML Further, we demonstrate that AXL directly interacts with OPCML, preferentially so when AXL is activated by its ligand Gas6. As a consequence, AXL accumulates in cholesterol-rich lipid domains, where OPCML resides. Here, phospho-AXL is brought in proximity to the lipid domain-restricted phosphatase PTPRG, which de-phosphorylates the RTK/ligand complex. This prevents AXL-mediated transactivation of other RTKs (cMET and EGFR), thereby inhibiting sustained phospho-ERK signalling, induction of the EMT transcription factor Slug, cell migration and invasion. From a translational perspective, we show that OPCML enhances the effect of the phase II AXL inhibitor R428 in vitro and in vivo We therefore identify a novel mechanism by which two spatially restricted tumour suppressors, OPCML and PTPRG, coordinate to repress AXL-dependent oncogenic signalling.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Benzocicloheptenos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Pollos , Colesterol/metabolismo , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Trompas Uterinas/patología , Femenino , Proteínas Ligadas a GPI/metabolismo , Silenciador del Gen/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Microdominios de Membrana/metabolismo , Invasividad Neoplásica , Neoplasias Ováricas/patología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Resultado del Tratamiento , Triazoles/farmacología , Tirosina Quinasa del Receptor AxlRESUMEN
Tumors are intrinsically heterogeneous and it is well established that this directs their evolution, hinders their classification and frustrates therapy1-3. Consequently, spatially resolved omics-level analyses are gaining traction4-9. Despite considerable therapeutic interest, tumor metabolism has been lagging behind this development and there is a paucity of data regarding its spatial organization. To address this shortcoming, we set out to study the local metabolic effects of the oncogene c-MYC, a pleiotropic transcription factor that accumulates with tumor progression and influences metabolism10,11. Through correlative mass spectrometry imaging, we show that pantothenic acid (vitamin B5) associates with MYC-high areas within both human and murine mammary tumors, where its conversion to coenzyme A fuels Krebs cycle activity. Mechanistically, we show that this is accomplished by MYC-mediated upregulation of its multivitamin transporter SLC5A6. Notably, we show that SLC5A6 over-expression alone can induce increased cell growth and a shift toward biosynthesis, whereas conversely, dietary restriction of pantothenic acid leads to a reversal of many MYC-mediated metabolic changes and results in hampered tumor growth. Our work thus establishes the availability of vitamins and cofactors as a potential bottleneck in tumor progression, which can be exploited therapeutically. Overall, we show that a spatial understanding of local metabolism facilitates the identification of clinically relevant, tractable metabolic targets.
Asunto(s)
Neoplasias de la Mama , Humanos , Ratones , Animales , Femenino , Neoplasias de la Mama/metabolismo , Ácido Pantoténico , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , VitaminasRESUMEN
BACKGROUND: Increased consumption of plant-based diets has been linked to the presence of certain phytochemicals, including polyphenols such as flavonoids. Several of these compounds exert their protective effect via inhibition of tumor angiogenesis. Identification of additional phytochemicals with potential antiangiogenic activity is important not only for understanding the mechanism of the preventive effect, but also for developing novel therapeutic interventions. RESULTS: In an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have screened a set of hitherto untested phytoestrogen metabolites concerning their anti-angiogenic effect, using endothelial cell proliferation as an end point. Here, we show that a novel phytoestrogen, 6-methoxyequol (6-ME), inhibited VEGF-induced proliferation of human umbilical vein endothelial cells (HUVE) cells, whereas VEGF-induced migration and survival of HUVE cells remained unaffected. In addition, 6-ME inhibited FGF-2-induced proliferation of bovine brain capillary endothelial (BBCE) cells. In line with its role in cell proliferation, 6-ME inhibited VEGF-induced phosphorylation of ERK1/2 MAPK, the key cascade responsible for VEGF-induced proliferation of endothelial cells. In this context, 6-ME inhibited in a dose dependent manner the phosphorylation of MEK1/2, the only known upstream activator of ERK1/2. 6-ME did not alter VEGF-induced phosphorylation of p38 MAPK or AKT, compatible with the lack of effect on VEGF-induced migration and survival of endothelial cells. Peri-tumor injection of 6-ME in A-431 xenograft tumors resulted in reduced tumor growth with suppressed neovasularization compared to vehicle controls (P < 0.01). CONCLUSIONS: 6-ME inhibits VEGF- and FGF2-induced proliferation of ECs by targeting the phosphorylation of MEK1/2 and it downstream substrate ERK1/2, both key components of the mitogenic MAPK pathway. Injection of 6-ME in mouse A-431 xenograft tumors results to tumors with decreased neovascularization and reduced tumor volume suggesting that 6-ME may be developed to a novel anti-angiogenic agent in cancer treatment.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Isoflavonas/farmacología , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica , Carga Tumoral/efectos de los fármacos , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/síntesis química , Animales , Bovinos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Equol/análogos & derivados , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Isoflavonas/administración & dosificación , Isoflavonas/síntesis química , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Desnudos , Mitosis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OPCML, a tumor suppressor gene, is frequently silenced epigenetically in ovarian and other cancers. Here we report, by analysis of databases of tumor sequences, the observation of OPCML somatic missense mutations from various tumor types and the impact of these mutations on OPCML function, by solving the X-ray crystal structure of this glycoprotein to 2.65 Å resolution. OPCML consists of an extended arrangement of three immunoglobulin-like domains and homodimerizes via a network of contacts between membrane-distal domains. We report the generation of a panel of OPCML variants with representative clinical mutations and demonstrate clear phenotypic effects in vitro and in vivo including changes to anchorage-independent growth, interaction with activated cognate receptor tyrosine kinases, cellular migration, invasion in vitro and tumor growth in vivo. Our results suggest that clinically occurring somatic missense mutations in OPCML have the potential to contribute to tumorigenesis in a variety of cancers.
Asunto(s)
Moléculas de Adhesión Celular/genética , Epigénesis Genética , Neoplasias Ováricas/genética , Moléculas de Adhesión Celular/química , Transformación Celular Neoplásica , Cristalografía por Rayos X , Femenino , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Glicosilación , Humanos , Mutación Missense , Invasividad Neoplásica , Agregación Patológica de Proteínas/genética , Estructura Terciaria de ProteínaRESUMEN
Opioid-binding protein/cell adhesion molecule-like (OPCML) is a tumor-suppressor gene that is frequently inactivated in ovarian cancer and many other cancers by somatic methylation. We have previously shown that OPCML exerts its suppressor function by negatively regulating a spectrum of receptor tyrosine kinases (RTK), such as ErbB2/HER2, FGFR1, and EphA2, thus attenuating their related downstream signaling. The physical interaction of OPCML with this defined group of RTKs is a prerequisite for their downregulation. Overexpression/gene amplification of EGFR and HER2 is a frequent event in multiple cancers, including ovarian and breast cancers. Molecular therapeutics against EGFR/HER2 or EGFR only, such as lapatinib and erlotinib, respectively, were developed to target these receptors, but resistance often occurs in relapsing cancers. Here we show that, though OPCML interacts only with HER2 and not with EGFR, the interaction of OPCML with HER2 disrupts the formation of the HER2-EGFR heterodimer, and this translates into a better response to both lapatinib and erlotinib in HER2-expressing ovarian and breast cancer cell lines. Also, we show that high OPCML expression is associated with better response to lapatinib therapy in breast cancer patients and better survival in HER2-overexpressing ovarian cancer patients, suggesting that OPCML co-therapy could be a valuable sensitizing approach to RTK inhibitors. Mol Cancer Ther; 16(10); 2246-56. ©2017 AACR.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Moléculas de Adhesión Celular/genética , Receptores ErbB/genética , Neoplasias Ováricas/tratamiento farmacológico , Receptor ErbB-2/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib/administración & dosificación , Femenino , Proteínas Ligadas a GPI/genética , Amplificación de Genes/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lapatinib , Terapia Molecular Dirigida , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Quinazolinas/administración & dosificación , Receptor ErbB-2/antagonistas & inhibidores , Trastuzumab/administración & dosificaciónRESUMEN
Vascular endothelial growth factor (VEGF) activates unfolded protein response sensors in the endoplasmic reticulum through phospholipase C gamma (PLCγ)-mediated crosstalk with mammalian target of rapamycin complex 1 (mTORC1). Activation of transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) activate mTORC2, ensuring maximal endothelial cell survival and angiogenic activity through phosphorylation of AKT on Ser473. As mTORC1 is a metabolic sensor, metabolic signals may be integrated with signals from VEGF in the regulation of angiogenesis.
RESUMEN
Bcl-2 family proteins are important regulators of apoptosis and its antiapoptotic members, which are overexpressed in many types of cancer, are of high prognostic significance, establishing them as attractive therapeutic targets. Quercetin, a natural flavonoid, has drawn much attention because it exerts anticancer effects, while sparing normal cells. A multidisciplinary approach has been employed herein, in an effort to reveal its mode of action including dose-response antiproliferative activity and induced apoptosis effect, biochemical and physicochemical assays, and computational calculations. It may be concluded that, quercetin binds directly to the BH3 domain of Bcl-2 and Bcl-xL proteins, thereby inhibiting their activity and promoting cancer cell apoptosis.