Metabolic reprogramming and vulnerabilities of prostate cancer stem cells independent or epithelial-mesenchymal transition

Abstract

Metastasis represents the most life-threatening aspect of tumorigenesis and is the leading cause of death by cancer. Intensive research in this field has shed light on some of the molecular strategies employed by the heterogeneous cancer cell populations to leave the primary tumor, disseminate and grow new colonies in distant organs. In any given tumor, one important functional category of cancer cells is represented by cancer stem cells (CSCs), endowed with self-renewal and tumor-initiating potentials. Moreover, the epithelial-mesenchymal transition (EMT) program represents a process of fundamental importance conducive to tumor dissemination and metastatic spread of cancer cells. Some studies have pointed out that the EMT is responsible for the acquisition of the CSC-like state whereas others have shown that both cell entities can exist separately and cooperate to accelerate the process of metastasis.

Here, we propose the combined use of metabolomics and fluxomics strategies to shed light on the metabolic reprogramming and vulnerabilities accompanying specific cancer cell phenotypes that differs in their metastatic and invasive capacities. The main objective of this thesis is focused on the characterization of the metabolic reprogramming and vulnerabilities of uncoupled CSC and EMT phenotypes present in a dual-cell prostate cancer cell model and represented by the highly related cell subpopulations PC-3M and PC-3S cells, respectively.

Our results indicated that epithelial PC-3M cells, displaying CSC features and a high metastatic potential, preferentially rely on aerobic glycolysis (Warburg effect) for bioenergetics. Although these cells show low coupling between glycolysis and oxidative phosphorylation (OXPHOS) because of low pyruvate dehydrogenase activity, they display an increased metabolic flexibility to utilize different carbon sources, such as fatty acids, glutamine and other amino acids, that offset the decreased diversion of glucose-derived carbons into the tricarboxylic acid cycle and OXPHOS.

The characterization of the non-CSC mesenchymal PC-3S cells expressing the EMT program and endowed with a high invasive capacity, showed a strong coupling between aerobic glycolysis and OXPHOS and a strong dependence on the mitochondrial metabolism for bioenergetics, which leads to higher levels of ROS that require increased levels of glutathione to provide an adequate antioxidant defense system.

PC-3M and PC-3S cells differentially reprogram the use of the oxidative and non-oxidative branches of the pentose phosphate pathway to sustain their distinct metabolic needs. Glycolytic intermediates are preferentially directed to ribose synthesis in PC-3M cells to build up nucleotides whereas the generation of NADPH is more crucial for PC-3S cells to counteract their higher oxidative stress and sustain their increased fatty acid synthesis.

Glutamine metabolism substantially contributes to TCA reactions in PC-3. For PC-3S cells, both glucose and glutamine are necessary to display a proper mitochondrial function. PC-3M cells are more dependent than PC-3S cells on the glutaminase reaction for proliferation and survival and this reliance lies mainly on the increased need for PC-3M cells to neutralize the excessive levels of protons (lactic acid) that result from their marked Warburg effect, which is achieved by the ammonia molecules released from glutamine metabolism.

The high metabolic flexibility displayed by the CSC subpopulation including the participation of serine, glycine and one-carbon metabolism, the uptake of ketogenic amino acids, proline metabolism, among others, provide PC-3M cells with an extensive metabolic dynamics to obtain not only precursors but also to balance their redox status (NAD+/NADH and NADP+/NADPH) for metabolic processes to continue (e.g. glycolysis) and protect them from excessive acidity derived from a high glycolytic rate.

Collectively, these results strengthen the notion that specific metabolic signatures are associated to CSC and EMT programs and highlight the importance of studying uncoupled cell phenotypes in order to univocally associate their characteristic metabolic reprogramming.

El proceso de la metástasis es la principal causa de mortalidad en pacientes de cáncer. En los últimos años se ha desvelado la importancia de la cooperación entre distintas subpoblaciones celulares que coexisten en el tumor. Entre estas subpoblaciones, encontramos las células denominadas cancer stem cells (CSCs), con un elevado potencial de autorenovación, pluripotencia y capacidad de iniciar tumores. Por otro lado, ciertas subpoblaciones celulares del tumor son capaces de incrementar sus capacidades migratorias e invasivas, mediante el proceso de epithelial-mesenchymal transition (EMT). Diversos estudios han demostrado que la cooperación entre CSCs y células que han activado el programa EMT facilita la colonización metastásica.

Dado que la reprogramación metabólica es responsable de proveer a las células tumorales aquellos recursos bioenergéticos y de biosíntesis necesarios para el mantenimiento de su fenotipo tumoral, en este trabajo se ha caracterizado el metabolismo y las vulnerabilidades metabólicas de dos subpoblaciones celulares derivadas de la línea celular PC-3, con características diferenciadas de CSCs por un lado (PC-3M) y de EMT por otro (PC-3S). El estudio metabólico de estas subpoblaciones celulares desveló que las células PC-3M presentan una mayor preferencia para el uso de la glucólisis (efecto Warburg más marcado), mientras que las PC-3S son más dependientes del metabolismo energético mitocondrial. Estas subpoblaciones también difieren en el uso de las ramas oxidativa y no oxidativa de la vía de las pentosas fosfato y en las reacciones de biosíntesis y degradación de ácidos grasos, con el fin de satisfacer las distintas necesidades metabólicas que caracterizan estos fenotipos. Por otro lado, las PC-3M muestran una elevada flexibilidad y adaptación metabólica, siendo capaces de metabolizar numerosos substratos, entre ellos diferentes tipos de amino ácidos. Particularmente, el metabolismo de la glutamina en las PC-3M es más esencial que en las PC-3S, no sólo por su papel anaplerótico, si no por su función de tamponamiento de los excesos de ácido. El conjunto de estos resultados han desvelado las particularidades metabólicas y vulnerabilidades asociadas a los fenotipos, no solapados, de CSCs y EMT. El conocimiento adquirido podrá contribuir en el diseño de nuevas estrategias terapéuticas para el tratamiento de la metástasis.