Not to be picky: PEPCK-M ensures metabolic flexibility in cancer cells and neuronal progenitors

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme that catalyses decarboxylation of oxaloacetate to phosphoenolpyruvate and it is part of gluconeogenic/glyceroneogenic pathway. There are two known isoforms of PEPCK, the mitochondrial and the cytosolic isozyme that are catalysing chemically identical reactions, but they differ in regulation and expression pattern. Selective presence of mitochondrial isoform of this enzyme (PEPCK-M, PCK2) in all types of cancer examined and in cycling neuroprogenitors, suggests a functional relationship with the metabolic adaptations of these cells. This thesis has had as its main objectives the characterization of the role of PEPCK-M in tumour cells and in neuronal progenitor cells. Metabolism of cell in CNS is not completely elucidated yet. Here we demonstrate that Tbr2 positive neuronal progenitors are metabolically dependent on lactate, which is favouring maintenance of their undifferentiated state. Lactate as metabolite can feed anabolic pathways and sustain ATP production by its oxidation in the TCA cycle. However, essential pathways like PPP, glycerol synthesis or one carbon metabolism pathways require carbons to feed the glycolytic intermediate pool. PEPCK-M in this setting, with lactate as sole carbon source is the only known pathway to fulfil the above-mentioned anabolic requirements. By using inhibitor of PEPCK-M we were able to prove that Tbr2 positive neuronal progenitors are metabolically dependent on PEPCK-M activity and their number significantly decrease after inhibiting PEPCK-M in vitro and in vivo.

PEPCK-M activity in tumour cells is necessary for survival and growth in 2D and in cultures on semi-solid agar (anchorage-independent growth), which suggests that this enzyme has a fundamental role in the survival program to cell stress. A Kaplan-Meier analysis from datasets available in the GEO database (> 5000 patients) shows that elevated PCK2 expression is significantly associated with a worse prognosis in patients with breast cancer. Despite its potential relevance for metabolic adaptations in cancer, the mechanisms responsible for its pro- survival activity are not known. Therefore, we have proposed to study these mechanisms through metabolomic analysis where we wanted to examine whether PEPCK-M feeds an alternative pathway to glucose using carbons from glutamine in an experimental model with reduced and overexpressed levels of PEPCK-M activity. We demonstrated the functionality of PEPCK-M driven cataplerosis in MCF7 cells grown under glucose deprivation by showing synthesis of serine and glycine from glutamine by observing contribution of 13C-labeled carbons from [U-13C] glutamine into these metabolites. In the absence of nutritional stress (high abundance of glucose and amino acids), the silencing of PEPCK-M induces oxidative stress and the accumulation of succinate, with the consequent induction of p21 and deficiencies in cell growth. Glutamine cataplerosis is not affected by alterations in PEPCK-M activity. However, a higher enrichment of all carbons with 13C in intermediates of the Krebs cycle (TCA cycle) suggests a reduction in flux through this pathway. Together, these data increase our understanding of metabolic adaptations in tumours and the role of PEPCK in providing alternative carbon fluxes to deal with nutritional stress. Finally, these studies allow us to propose PEPCK-M as a new target for the treatment of tumorigenic processes that will need to be validated in the future.

El fosfoenolpiruvato carboxiquinasa mitocondrial (PEPCK-M; PCK2) se regula transcripcionalmente por limitación de aminoácidos y por ER-estrés, de una manera dependiente de ATF4, aumentando así la supervivencia de la célula. La presencia selectiva de esta isoenzima en todos los tipos de cáncer examinado y en células neuroprogenitoras, sugiere una relación funcional con las adaptaciones metabólicas de estas células. Esta tesis ha tenido como objetivos fundamentales la caracterización del rol de la PEPCK-M en célula tumoral y en célula neuroprogenitora En cultivos neuronales, los neuroprogenitores Tbr2 positivos requieren lactato como sustrato metabólico para el mantenimiento de su fenotipo y su metabolismo. La PEPCK-M se expresa a niveles altos en este tipo celular y su actividad es necesaria para mantener la viabilidad de estos progenitores y cumplir con los requerimientos anabólicos a partir de carbonos provenientes del lactato. La actividad PEPCK-M en célula tumoral es necesaria para la supervivencia y crecimiento. A pesar de su potencial relevancia para las adaptaciones metabólicas en cáncer, no se conocen los mecanismos responsables de su actividad pro-supervivencia. Por ello, nos hemos propuesto estudiar estos mecanismos mediante análisis de metabolómica con los que hemos querido examinar si la PEPCK-M alimenta una vía alternativa a la glucosa utilizando carbonos provenientes de glutamina en un modelo experimental con niveles de actividad PEPCK-M reducidos y sobreexpresados. La contribución de carbonos marcados con 13C a partir de [U- 13C] glutamina en los productos de ramificación de glicolisis como serina y glicina, esta correlacionando directamente con los niveles de actividad PEPCK-M en condiciones de estrés nutricional (baja glucosa). La cataplerosis de glutamina no se ve afectada por alteraciones en la actividad de PEPCK-M. Sin embargo, un mayor enriquecimiento de 13C en intermediarios del ciclo de Krebs sugieren una reducción del flujo a través de esta vía. En conjunto, estos datos aumentan nuestra comprensión de las adaptaciones metabólicas en los tumores y el papel de la PEPCK en la provisión de flujos de carbono alternativas para lidiar con el estrés nutricional. Finalmente, estos estudios nos permiten proponer a la PEPCK-M como una nueva diana para el tratamiento de procesos tumorogénicos que necesitará ser validada en el futuro.