Retinoblastoma Tumor Suppressor Targets dNTP Metabolism to Regulate DNA Replication

Abstract

The retinoblastoma tumor suppressor, RB, is a negative regulator of the cell cycle that is inactivated in the majority of human tumors. Cell cycle inhibition elicited by RB has been attributed to the attenuation of CDK2 activity. Although ectopic cyclins partially overcome RB-mediated S-phase arrest at the replication fork, DNA replication remains inhibited and cells fail to progress to G 2 phase. These data suggest that RB regulates an additional execution point in S phase. We observed that constitutively active RB attenuates the expression of specific dNTP synthetic enzymes: dihydrofolate reductase, ribonucleotide reductase (RNR) subunits R1/R2, and thymidylate synthase (TS). Activation of endogenous RB and related proteins by p16ink4a yielded similar effects on enzyme expression. Conversely, targeted disruption of RB resulted in increased metabolic protein levels (dihydrofolate reductase, TS, RNR-R2) and conferred resistance to the effect of TS or RNR inhibitors that diminish available dNTPs. Analysis of dNTP pools during RB-mediated cell cycle arrest revealed significant depletion, concurrent with the loss of TS and RNR protein. Importantly, the effect of active RB on cell cycle position and available dNTPs was comparable to that observed with specific antimetabolites. Together, these results show that RB-mediated transcriptional repression attenuates available dNTP pools to control Sphase progression. Thus, RB employs both canonical cyclin-dependent kinase/cyclin regulation and metabolic regulation as a means to limit proliferation, underscoring its potency in tumor suppression.The retinoblastoma tumor suppressor (RB) 1 functions as a negative regulator of cell cycle transitions (1-5). Due to its frequent inactivation in tumors (Ͼ60%), it is highly relevant to determine how RB functions to inhibit cellular proliferation and to elucidate its interaction with chemotherapeutic drugs.Biochemically, RB functions as a transcriptional co-repressor that mediates the inhibition of cell cycle progression (1-5). RB interacts with multiple cellular proteins, including the E2F family of transcriptional regulators (6). In addition to binding E2F, RB also interacts with histone deacetylase (HDAC) and SWI/SNF chromatin remodeling proteins to establish a repressor complex on the promoters of E2F-regulated genes (3,7,8). This activity of RB is critical for cell cycle inhibition. In G 0 and early G 1 , RB is hypophosphorylated and forms transcriptional repressor complexes to inhibit cell cycle progression. However, in response to mitogenic signaling, cyclin-dependent kinase (CDK)/cyclin complexes phosphorylate RB (9). Phosphorylation disrupts the association of RB with its interacting proteins, thereby alleviating transcriptional repression of E2F-regulated genes and facilitating cell cycle progression (1-5).Targets of E2F are known to encompass a variety of proteins involved in cell cycle progression (6,10,11). Consistent with the role of RB as a repressor of E2F, in disparate settings the expression/activity of cyclin E, cy...

Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents

“…The loss of RB is accompanied by deregulated expression of a number of known RB/E2F target genes. These targets are similar to those observed to be deregulated in Rb À/À MEFs (Herrera et al, 1996;Hurford et al, 1997;Philips et al, 1998;Angus et al, 2002). Analysis of serum dependence in the presence of these targets revealed that the RB-deficient adult fibroblastic cultures are still serum dependent.…”

“…This suggested that 5-FU might be more efficiently activated to FdUMP in RB-deficient cells than in RB-proficient cells. Consistent with this concept, the promoters of two genes encoding enzymes involved in 5-FU metabolism to FdUMP, thymidine kinase and RNR (Longley et al, 2003), are regulated by the RB/E2F axis (Mudrak et al, 1994;Angus et al, 2002). Indeed, expression of the RNR small subunit (RNR2) was significantly elevated following acute RB loss in primary MAFs (Figure 7e).…”

“…This is attributed to elevated levels of the metabolic enzymes targeted by these compounds (TS, dihydrofolate reductase and ribonucleotide reductase (RNR)) Li et al, 1995;Angus et al, 2002). Therefore, we next probed the consequences of acute RB loss on the response of primary and immortal cultures to antimetabolites.…”

Clinical application of biological markers for treatments of resectable non-small-cell lung cancers

“…Regarding TS, its expression is regulated by a polymorphic tandem repeat sequence in the 5 0 -terminal regulatory region of TS gene (Horie et al, 1995), and it is also regulated by several oncogenes and tumour suppressor genes, including E2F1, retinoblastoma, and p16/INK4 (DeGregori et al, 1995;Omura et al, 2000;Angus et al, 2002). Thymidylate synthase plays a central role in the biosynthesis of thymidylate, an essential precursor for DNA synthesis.…”

RB1: a prototype tumor suppressor and an enigma

“…Based on genetic interactions that were discovered in Drosophila, others have suggested targeting TSC2 to elevate reactive oxygen species (ROS) in RB1 mutant tumors (Li et al 2010;Gordon et al 2013). Potentially, other metabolic features of RB1 mutant cells, such as the changes in mitochondrial activity, depletion of nucleotide pools, or changes in autophagy, might provide alternative therapeutic strategies (Angus et al 2002;Tracy et al 2007;Macleod 2008). Ultimately, there may not be a single vulnerability that is characteristic of all RB1 mutant cells, but different strategies may be necessary for specific types of tumors.…”