Regulation of Metabolism by RB

Clinical Cancer Molecular Pathways Research

Molecular Pathways: Regulation of Metabolism by RB

Brian F. Clem and Jason Chesney

Abstract The discovery of the retinoblastoma (RB-1) gene as a tumor suppressor that is disrupted in a majority of human cancers either via direct or indirect genetic alterations has resulted in increased interest in its functions and downstream effectors. Although the canonical pathway that links this tumor suppressor to human cancers details its interaction with the E2F transcription factors and cell-cycle progression, recent studies have shown an essential role for RB-1 in the suppression of glycolytic and glutaminolytic metabolism. Characterization of the precise metabolic transporters and enzymes suppressed by the RB- E2F axis should enable the identiﬁcation of small molecule antagonists that have selective and potent antitumor properties. Clin Cancer Res; 18(22); 6096–100. 2012 AACR.

Background anomas, and hepatocellular carcinomas, causes the activa- In the early 1970s, Alfred Knudson described a statistical tion of cyclin-dependent kinases (CDKs) that phosphory- study of 48 cases of a rare childhood malignancy, retino- late and suppress the activity of RB-1 (5, 6). Given that RB-1 blastoma, which indicated that only 2 mutational events introduction of wild-type RB-1 into negative cancer were required for its development (1). Subsequent pedi- cells suppresses their anchorage-independent growth in soft gree analyses of retinoblastoma patients identified a linked agar and as tumors in mice (7), RB-1 became known as the locus on chromosome 13 (2) and, within this locus, the first gene to fulfill the equivalent of Koch’s postulates for a retinoblastoma target gene RB-1 was sequenced by Lee and tumor suppressor. On the basis of these prior genetic and colleagues in the 1980s (3). Multiple RB-1 deletions and biologic studies, it is now anticipated that understanding mutations that prevent the expression and function of the precise biochemical functions of RB-1 will identify the normal retinoblastoma protein product (RB-1) were proteins that become activated from the loss of RB-1 and then identified in retinoblastomas (4) and the two-hit that have utility as molecular targets for the development of hypothesis by Knudson was supported by the observations novel chemotherapeutic approaches. that one mutated allele and one wild-type allele were Canonical Function of RB in Cell-Cycle present in normal, somatic cells of hereditary retinoblas- Regulation toma patients whereas their retinoblastomas contained 2 mutated alleles (3). In normal cells, activation of signaling pathways down- Apart from retinoblastoma, genetic alterations of RB-1 stream of growth factor receptor tyrosine kinases causes also have been found in in osteosarcomas, renal cell carci- increased expression of D-type cyclins that form the regu- nomas, soft-tissue sarcomas, and adenocarcinomas of latory domains of CDK-4 and CDK-6 to enhance catalytic breast, lung (small cell), prostate, cervix, and the bladder, activity and nuclear translocation (8). Activated CDKs suggesting an extensive role for RB dysfunction in the phosphorylate RB-1, which causes its dissociation from the development of human cancers (4). In addition, mutations E2F transcription factors that, depending on their RB-bind- of gene products that lead to suppression of RB function ing state, modulate the expression of genes such as CDC25A have been found to be a ubiquitous feature of malignancies. that are essential for the G1/S cell-cycle progression (9). INK4A For example, genetic loss of p16 , which is common in Perhaps not surprisingly, overexpression of wild-type RB-1 RB-1 non–small cell lung and pancreatic adenocarcinomas, mel- protein in negative cells in the early G1 phase of the cell-cycle causes a G1 arrest and cytostasis (10). The E2F family consists of nine distinct members, which are differ- entiated by their putative activator (E2F-1, 2, 3a) or repres- Authors' Affiliation: Department of Medicine, Molecular Targets Group, James Graham Brown Cancer Center, University of Louisville, Louisville, sor functions (E2F-3b, 4, 5, 6, 7, 8). RB-1 associates specif- Kentucky ically with the E2F activator transcription factors and, when Corresponding Authors: Jason Chesney, Department of Medicine, bound, recruits selective corepressor complexes, such as Molecular Targets Group, James Graham Brown Cancer Center, University histone deacetylases (HDAC), to silence gene transcription of Louisville, 529 S. Jackson Street, Louisville, KY 40202. Phone: 502-852- 3679; E-mail: [email protected] and Brian F. Clem, 505 S. (11–15). Although the precise mechanisms for RB-E2F- Hancock Street, CTRB, Rm. 422, Louisville, KY 40202. Phone: 502-852- mediated modulation of gene expression remain contro- 8427; E-mail: [email protected] versial, several developmental mouse models in which E2F doi: 10.1158/1078-0432.CCR-11-3164 activators have been genetically deleted support this canon- 2012 American Association for Cancer Research. ical pathway (9).

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Control of Glucose Metabolism by RB function causes an increase in glycolysis, a hallmark of Until recently, the central dogma related to mutations cancer. that facilitate neoplastic transformation by disabling the Entry of glucose carbons into the TCA is largely mediated RB-E2F suppressive activity supported a scenario whereby by conversion of pyruvate to acetyl-CoA through the activity loss of RB function resulted in transcriptional upregulation of pyruvate dehydrogenase (PDH). PDH activity is inhib- of cell-cycle mediators which in turn caused a shift in the ited through phosphorylation by members of the pyruvate diffusion of nutrients and biosynthetic enzyme equilibria. dehydrogenase kinase (PDK) family, and several oncogenes However, an early indication that RB-E2F might directly and protumorigenic factors regulate the activity of PDKs regulate metabolic enzyme expression came from the obser- (37–39). For example, whereas c-Myc activity under normal vation that two enzymes required for nucleotide synthesis, oxygen tension increases mitochondrial glucose oxidation, thymidine kinase (TK1) and dihydrofolate reductase c-Myc coordinates with hypoxia-stabilized HIF-1a to stim- (DHFR), are direct transcriptional targets of E2F family ulate the expression of PDK1, thereby suppressing PDH members (16–23). Because RB suppresses the transcription function and glycolytic flux into the TCA (40). RB-E2F-1 of these nucleotide biosynthetic enzyme mRNAs via phys- also is able to block glucose oxidation under normoxia ical interaction with E2Fs, investigators postulated that RB through another PDK family member, PDK4, which has might also suppress the transcription of mRNAs required to been identified as a direct transcriptional target of Rb-E2F-1, translate the multitude of regulators, transporters, and and loss of E2F-1 or RB function causes increased PDK4 enzymes required for energetic and anabolic metabolism. expression and reduction of glucose oxidation (41, 42). Glycolysis provides carbons required for the synthesis of Taken together, these studies suggest that glycolytic flux into ribose, amino acids, lipids, and ATP necessary for biomass the TCA cycle may be determined not only by oxygen expansion and cytokinesis. Early studies identified elevated availability, but also by distinct combinations of tumor- glycolytic enzyme expression and activity levels in retino- promoting mutations. blastomas. For example, the expression of the first enzyme of the glycolytic pathway, hexokinase (isozyme II), is signifi- Control of Mitochondrial Glutaminolysis and cantly elevated in retinoblastomas relative to the normal Biogenesis by RB retina (24) and the glycolytic hub enzyme, lactate dehydro- Reduced glycolytic flux into the TCA as a result of RB-1 genase (LDH), has been found to be activated in the aqueous deletion may result in the preferential use of glutamine- humor surrounding retinoblastomas compared with non- derived carbons to replenish TCA intermediates for reduc- malignant ocular disorders (25). The precise mechanisms ing equivalents (e.g., NADH) required for oxidative phos- for increased expression and activity of glycolytic enzymes in phorylation and for the synthesis of TCA-derived macro- retinoblastomas as well as other RB-1 deleted cancers have molecules (43–46). In addition, glutamate, a product of not been completely defined. However, derepressed E2F-1 the glutaminases, functions as an essential precursor for activity leads to increased expression of c-Myc through direct the production of glutathione, which serves as a reductive transcriptional activation of the MYC promoter, and the role sink for reactive oxygen species and thiol-containing of c-Myc in stimulating glycolytic flux to lactate is well proteins (47). Over the last 10 years, the activation of established via its control of glycolytic mRNAs including several oncogenes and deletion of tumor suppressors GLUT1, HK2, PKM2, and LDH-A (26–29). In addition, loss have been found to regulate glutaminolysis via direct of RB function in 3T3 fibroblasts causes a 30-fold increase in modulation of glutamine transporters and glutaminase. activated Ras whereas restoration of wild-type RB in these Importantly, overexpression of c-Myc increases expres- cells reverses this activation (30, 31). Ras induces the sion of glutamine transporters and glutaminase 1 (GLS1) expression of 6-phosphofructo-2-kinase 3 (PFKFB3), whose along with concomitant and obligatory reliance on glu- product, fructose-2,6-bisphosphate, allosterically activates tamine for survival (48–50). Given the symbiotic inter- the first committed and irreversible step of glycolysis, 6- action between oncogenes and tumor suppressors that phosphofructo-1-kinase, which in turn results in increased drives neoplastic transformation (51), it is not surprising glycolytic flux to lactate (32, 33). Furthermore, the down- that genetic deletion of the RB family in mouse embry- stream Ras effector kinase AKT can directly phosphorylate onic fibroblasts also causes increased glutamine uptake and stimulate PFKFB3 activity (34), which, combined with and usage via elevated expression of the glutamine trans- c-Myc-mediated induction of glucose transporters and gly- porter ASCT2 and the activity of GLS1 (52). Furthermore, colytic enzyme mRNAs, may synergistically increase glyco- exposure to a physiologic concentration of glutamine has lytic flux to lactate. Importantly, cells lacking a functional RB been found to markedly increase oxygen consumption, pathway also have been found to express elevated phos- ATPproduction,andglutathione formation in RB-null phorylated-AKT and mTOR, which, through the action of cells relative to RB wild-type cells, indicating high reliance mTORC2, can suppress the nuclear translocation of Foxo3a on glutaminolysis as a result of an inactive RB pathway. (35), a negative regulator of the proglycolytic transcription Importantly, the effects of RB deletion are mediated in factor HIF-1a (36). Taken together, there is substantial, part by the RB-E2F cascade as ChIP analysis has revealed albeit indirect, evidence that activation of the RB-E2F path- specific E2F family members associated with promoter way suppresses glucose metabolism and that loss of RB regions of glutaminolytic enzymes (52). Together, these

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studies extend the tumor suppressor role of the RB-E2F genes involved in electron transport chain activity and oxi- pathway to a metabolic pathway, glutaminolysis that is dative phosphorylation have been found to be directly essential for the promotion of the growth and survival of suppressedbyRB-E2F-1,includingsubunitsofATPsynthase, neoplastic cells and identiﬁes several metabolic enzymes cytochrome c oxidase, ubiquinol-cytochrome c reductase, that may be useful for the development of novel anti- and the succinate dehydrogenase complex (41). Combined, neoplastic strategies. these reports suggest that dysfunction of RB should facilitate Recent studies also have showed a direct role for the RB- the usage of glutamine for oxidative phosphorylation. E2F interaction in mitochondrial biogenesis and electron transport chain activity, both of which are involved in – glutaminolysis. Adipocyte-speciﬁc deletion of RB causes Clinical Translational Advances increased expression of peroxisome proliferator-activated Targeting the loss of RB-E2F function in cancer receptor g co-activator 1-a (PGC-1a) and estrogen receptor Several metabolic transporters and enzymes that are related a (ERRa), which directly promote mitochondrial transcriptionally repressed by the RB-E2F pathway may transcription, biogenesis, and oxygen consumption (53). prove useful as targets for the development of antineoplastic This has been similarly observed in gastrocnemius myocytes agents (Fig. 1). For example, gossypol and its derivatives are À À from E2F-1 / knockout animals (41). In addition, several competitive inhibitors of the NADH binding site of the RB-

Glucose Glutamine

GLUT1 ASCT2 Ribose-5P Glucose Glutamine HK2 GLS1

Ras G6P Glutamate X5P c-Myc ؉؉؉ F6P PFK1 Mitochondria F26BP H+ Rb HIF-1α F16P IV PFKFB3 III ATP II E2Fs – + DHA G3P I 2e 2H + Rb NAD ½O2 → H2O E2Fs NADH c-Myc

PEP Acetyl CoA TCA Cycle PK-M2 PDH PDKs Pyruvate LDH-A

Lactate

Figure 1. The prototypical tumor suppressor RB-1 has pleiotropic inhibitory effects on metabolism. Deletions of RB-1 or certain E2Fs that result in the loss of RB-1–mediated transcriptional control have been found to directly and/or indirectly increase the expression of several key mediators of glycolysis, glutaminolysis, electron transport chain activity, and mitochondrial biogenesis including Ras, HIF-1a, and c-Myc. These signaling proteins and transcription factors increase the expression of metabolic transporters and enzymes causing a reprogramming of metabolic usage that supports the enhanced energetic and anabolic requirements of immortalized and transformed cells. In addition, the RB-1–E2F axis can directly regulate the expression of PDK4 and the glutamine transporter ASCT2, which can stimulate glycolytic ﬂux to lactate and glutaminolysis, respectively. Black lines indicate suppression and red lines indicate stimulation of activity and/or expression of the ﬁnal enzyme or transporter. Key regulators, transporters, and enzymes are highlighted in red.

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E2F-c-Myc target LDH that inhibits xenograft growth in Summary mice (54) and another target, Glut1, confers sensitivity to The discovery of mutations that disrupt the activities of the hexokinase inhibitor, 3-bromopyruvate (3BP; ref. 55). the RB-E2F pathway in a majority of human cancers has In addition, a competitive antagonist of the PFKFB family of highlighted the clinical importance of downstream effector glycolytic regulators was recently discovered, 3-(3-pyridi- enzymes that regulate cell-cycle progression. More recent nyl)-1-(4-pyridinyl)-2-propen-1-one (termed 3PO), that studies have showed that this pathway also regulates the reduces the glucose uptake, intracellular F2,6BP, lactate expression and activities of several metabolic transporters in vitro secretion, steady-state concentration of ATP, and and enzymes that are required for the production of ATP and in vivo growth of multiple transformed cells (56). V12 and anabolic precursors. Given that these metabolic effector Importantly, H-Ras –transformed bronchial epithelial proteins are directly suppressed by the RB-E2F axis and are cells that have low RB function are markedly more sensitive required for the immortalization and subsequent transfor- to the cytotoxic effects of 3PO than are normal bronchial mation of normal cells into neoplastic cells, small molecule epithelial cells (56). Finally, the induction of glutaminolysis antagonists that target their substrate-binding domains may RB in -null cells indicates that this metabolic pathway may have selective antitumor properties. be selectively required for their survival and growth. Indeed, RB glutamine withdrawal is cytotoxic to -null cells and the Disclosure of Potential Conflicts of Interest cells can be rescued by the addition of the TCA cycle No potential conflicts of interest were disclosed. intermediate, a-ketoglutarate (52). These studies suggest that suppression of glutamine metabolism may be cata- Authors' Contributions strophic to RB-deficient tumors. Unfortunately, glutamine Conception and design: B.F. Clem, J. Chesney Writing, review, and/or revision of the manuscript: B.F. Clem, J. Chesney antagonists have not been proven to be effective in clinical trials (for example, ref. 57) thereby highlighting the need for Acknowledgments a more targeted therapeutic approach against enzymes We thank Drs. Abdullah Yalcin, John Eaton, Alden Klarer, Yoannis whose activity may be altered in neoplastic cells lacking RB Imbert-Fernandez, and Sucheta Telang for their review and comments. function. For example, several small molecular inhibitors Because of space limitations, the authors apologize for the exclusion of primary references and the lack of detailed discussion on the RB-E2F of glutaminase, including BPTES and compound 968, have regulatory pathway. been found to have potent antitumor activities (58, 59). To validate these approaches and justify clinical trials, Grant Support analyses of the relative sensitivities of RB-null human cancer This work was supported by NIH/NCRR 3P20RR018733-09 (B.F. Clem) cells versus RB wild-type normal cells to pharmacologic and NIH/NCI 1RO1CA149438-01 (J. Chesney). inhibition of the RB-E2F-regulated metabolic enzymes are Received August 20, 2012; accepted August 23, 2012; published online necessary. November 15, 2012.

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