The study of RAS-induced metabolic reprogramming and the role of the pentose phosphate pathway in tumor metabolism

Abstract

The present doctoral thesis is focused on the metabolic adaptations induced by oncogene activation as well as the potential role of the metabolic network as antitumor therapy. Over the last years, it has emerged a renewed interest in the field of metabolism, particularly in cancer metabolism. Great efforts have been focused on the association of mutated oncogenes or tumor suppressor genes and tumor metabolic profiles, in the search of metabolic dependencies that offer new potential avenues for cancer treatment. The pursuit of discovering tumor metabolic alterations in which cancer cells rely on has represented the cornerstone of this interesting discipline. Thus, this thesis is part of this recent and promising scientific current and is intended to shed light on the metabolic alterations accompanying oncogene mutation and on potential metabolic pathways that might be of therapeutic interest in the future. Hence, the objectives of this thesis can be divided into two specific aims: i) analysis of the metabolic reprogramming of RAS oncogenic activation using stable transfected cell lines with mutated copies of K-RAS and H-RAS and ii) validation of the pentose phosphate pathway as a potential therapeutic target and exploration of its role within tumor metabolism in colon and breast cancer cell models. Thus, according to the proposed objectives, the main conclusions obtained are as follow: 1. The study of flux distribution in combination with metabolic control analysis performed by analyzing solely the sign of fixed-sign control coefficients, is a reliable approach to identify the key enzymes involved in metabolic reprogramming. The use of this methodology has allowed us to identify an increase in glycolysis and PPP fluxes as metabolic features of KRAS-induced metabolic reprogramming and to propose G6PD, PK and LDH as the key enzymes responsible for this metabolic transition. 2. H-RAS oncogenic activation reprograms glucose and glutamine metabolism by enhancing glycolytic and PPP fluxes as well as mitochondrial metabolism. Glutamine is responsible for sustaining the activated mitochondrial metabolism in BJ-HRasV12, while glucose-derived carbons in the mitochondria are primarily used to fuel lipogenesis. Moreover, lipogenesis is overactivated in BJ-HRasV12 cells, which are more sensitive to FAS inhibition than BJ cells. 3. G6PD enzyme is overactivated in colon cancer cells with oncogenic activation of the RAS signaling pathway. Nevertheless, G6PD seems to be dispensable for proliferation and survival in BRAF-mutated HT29 cell line. Furthermore, a new connection between PPP and glutamine metabolism has been unveiled, as G6PD is overexpressed in HT29 cells under glutamine-deprived conditions by a mechanism involving a concomitantly increase in ROS levels and NRF2 induction. 4. G6PD enzyme is important in proliferation, survival and regulation of ROS levels in breast cancer MCF7 cells. However, it exerts a low regulation over ribose synthesis flux through the oxidative branch of PPP. G6PD inhibition enhances glycolytic flux, promotes lactate secretion and increases glutamine consumption, which is used to maintain energy homeostasis, although it is not essential for cell proliferation. 5. TKT enzyme is dispensable for proliferation of breast cancer MCF7 cells, but it exerts a high control over ribose synthesis flux through the nonoxidative branch of PPP. TKT impairment reduces glycolytic flux and increases the consumption of glutamine, which is intended to maintain energy homeostasis but it is not essential for cell proliferation.

La presente tesis doctoral se centra en las adaptaciones metabólicas inducidas por la activación de oncogenes así como en el potencial del entramado metabólico como diana antitumoral. A lo largo de los últimos años, ha resurgido un renovado interés en el estudio del metabolismo, particularmente en el metabolismo de las células tumorales, dando lugar a una nueva disciplina conocida como metabolismo tumoral. Numerosas investigaciones se han centrado en la asociación entre mutaciones en oncogenes o genes supresores de tumores con perfiles metabólicos característicos, en busca de dependencias metabólicas que ofrezcan nuevas posibilidades para el tratamiento de los tumores. La búsqueda de alteraciones metabólicas que constituyan vulnerabilidades de la célula tumoral representa la piedra angular de esta interesante disciplina. Así, esta tesis doctoral tiene como objetivo general elucidar las alteraciones metabólicas que acompañan a la mutación de oncogenes y explorar el potencial del entramado metabólico como diana antitumoral. Por tanto, los objetivos principales de este trabajo son los siguientes: i) análisis de la reprogramación metabólica inducida por la activación oncogénica de RAS empleando líneas celulares transfectadas de manera estable con copias mutadas de los oncogenes K-RAS y H-RAS y, ii) validación de la vía de las pentosas fosfato como potencial diana antitumoral y estudio de su papel en el metabolismo tumoral de modelos celulares de cáncer de colon y de mama. Así, en este trabajo de tesis doctoral hemos concluido que la activación oncogénica de RAS promueve una profunda reprogramación del metabolismo induciendo cambios significativos en la glucólisis, la vía de las pentosas fosfato, el metabolismo de la glutamina y la lipogénesis. Por otro lado, hemos determinado que la inhibición de la vía de las pentosas fosfato tiene distintos efectos según el tipo de tumor. La inhibición de la G6PD en la línea celular de cáncer colon HT29 no produjo efectos sobre la proliferación mientras que su inhibición en células de cáncer de mama MCF7 indujo una notable reducción de la proliferación y un incremento de la muerte celular. Por otra parte, en la inhibición en MCF7 del otro enzima clave de la vía de las pentosas fosfato, la TKT, no se observaron cambios significativos en términos de proliferación y viabilidad celular. Además, en este trabajo también se ha puesto de manifiesto una conexión funcional entre la vía de las pentosas fosfato y el metabolismo de la glutamina en ambos modelos celulares, sugiriendo un papel complementario de estas dos vías metabólicas.