Tumorigenesis requires not only loss of proliferative control, but also a metabolic shift towards increased glucose and glutamine consumption required for increased energy production and to drive the de novo biosynthesis of nucleotides, amino acids, and lipids essential for cell division. These processes are primarily driven through oncogenes or loss of tumor suppressors, which act to circumvent normal regulatory pathways. The retinoblastoma (Rb) protein, the first described tumor suppressor, is extensively involved in cell cycle regulation, and perturbations within the Rb pathway are found in most tumor types. Beyond cell cycle control, Rb has been implicated in multiple additional biochemical pathways known to be involved in tumor progression, such as metastasis and angiogenesis. However, little is known about the role of Rb in regulating the unique changes in metabolism that have been observed in human cancers. Using stable 13C-glucose isotopomer NMR analyses, we found that triple knock-out (TKO) of all three Rb family members in mouse embryonic fibroblasts (MEFs) resulted in increased glucose uptake and flux to lactate, and simultaneously decreased glucose-derived carbon incorporation into TCA cycle intermediates relative to wild-type (WT) MEFs. This metabolic shift was mediated by changes in the expression of multiple glycolytic enzymes including increased Glut-1, HK2, PK-M2 levels and decreased HK1, ALT1, and PCB. To supplement this loss of glucose carbons for anaplerosis within the TCA cycle, we speculated that the Rb TKO MEFs may increase glutamine uptake for both bioenergetic and anabolic precursors. We observed that loss of Rb caused increased 13C- glutamine uptake and flux into glutamate and TCA cycle intermediates via enhanced expression of the glutamine transporter ASCT2 and the activity of glutaminase 1 (GLS1). Further, glutamine carbon is capable of facilitating oxygen consumption, and glutamine withdrawal resulted in significant decrease in ATP levels in the TKO cells. Importantly, this shift towards glutamine utilization was essential fo the survival of Rb TKO MEFs and not for the WT MEFs, and addition of exogenous ?-ketoglutarate was able to rescue both ATP levels and cell viability in the Rb-depleted cells. Mechanistically, E2F-1, -2, &-3 functionally alter both glucose and glutamine uptake, and E2F-1 &-3 were observed to directly associate with the promoters of Glut-1 and ASCT2. Combined, these studies suggest that the Rb/E2F cascade directly regulates two key metabolic pathways that are necessary for neoplastic growth. We hypothesize that inactivation of the Rb protein in human cancers leads to a global metabolic shift towards enhanced glycolysis and glutamine utilization, which in turn is required for neoplastic immortalization and transformation. We will test this hypothesis by conducting the following Specific Aims: 1. To determine the precise metabolic transporters, enzymes and pathways that are modulated by the loss of Rb and the relative requirements of these metabolic targets for the survival and growth of Rb-proficient and Rb-deficient MEFs, human normal epithelial cells and human cancer cells. 2. To determine the effects of Rb1 deletion on glucose/glutamine metabolism and growth of lung adenocarcinomas in vivo. 3. To correlate the loss of Rb function with changes in the 13C-glucose utilization by human lung tumors in vivo.
We anticipate that these studies will further expand our understanding of the role of the retinoblastoma protein (Rb) in tumor initiation and progression by detailing the precise regulatory mechanisms of Rb on tumor cell metabolism. These findings also may enable the identification of new molecular targets for development of anti-cancer agents as well as potential biomarkers for tumors that may be more responsive to anti-metabolic therapies.
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|Reynolds, Miriam R; Clem, Brian F (2015) Troglitazone suppresses glutamine metabolism through a PPAR-independent mechanism. Biol Chem 396:937-47|
|Reynolds, M R; Lane, A N; Robertson, B et al. (2014) Control of glutamine metabolism by the tumor suppressor Rb. Oncogene 33:556-66|
|Clem, Brian (2014) RB in glutamine metabolism. Oncoscience 1:304-5|