Niels J. F. van den Broek

FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP

FOXO (Forkhead box O) transcription factors are important regulators of cellular metabolism, cell-cycle progression and cell death. FOXO activity is regulated by multiple post-translational modifications, including phosphorylation, acetylation and polyubiquitination. Here, we show that FOXO becomes monoubiquitinated in response to increased cellular oxidative stress, resulting in its re-localization to the nucleus and an increase in its transcriptional activity. Deubiquitination of FOXO requires the deubiquitinating enzyme USP7/HAUSP (herpesvirus-associated ubiquitin-specific protease), which interacts with and deubiquitinates FOXO in response to oxidative stress. Oxidative stress-induced ubiquitination and deubiquitination by USP7 do not influence FOXO protein half-life. However, USP7 does negatively regulate FOXO transcriptional activity towards endogenous promoters. Our results demonstrate a novel mechanism of FOXO regulation and indicate that USP7 has an important role in regulating FOXO-mediated stress responses.

Mps1 Phosphorylates Borealin to Control Aurora B Activity and Chromosome Alignment

Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.

Acetyl-CoA Synthetase 2 Promotes Acetate Utilization and Maintains Cancer Cell Growth under Metabolic Stress

Summary A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment.

Serine, but Not Glycine, Supports One-Carbon Metabolism and Proliferation of Cancer Cells

Previous work has shown that some cancer cells are highly dependent on serine/glycine uptake for proliferation. Although serine and glycine can be interconverted and either might be used for nucleotide synthesis and one-carbon metabolism, we show that exogenous glycine cannot replace serine to support cancer cell proliferation. Cancer cells selectively consumed exogenous serine, which was converted to intracellular glycine and one-carbon units for building nucleotides. Restriction of exogenous glycine or depletion of the glycine cleavage system did not impede proliferation. In the absence of serine, uptake of exogenous glycine was unable to support nucleotide synthesis. Indeed, higher concentrations of glycine inhibited proliferation. Under these conditions, glycine was converted to serine, a reaction that would deplete the one-carbon pool. Providing one-carbon units by adding formate rescued nucleotide synthesis and growth of glycine-fed cells. We conclude that nucleotide synthesis and cancer cell proliferation are supported by serine--rather than glycine--consumption.

Pyruvate carboxylation enables growth of SDH-deficient cells by supporting aspartate biosynthesis

Succinate dehydrogenase (SDH) is a heterotetrameric nuclear-encoded complex responsible for the oxidation of succinate to fumarate in the tricarboxylic acid cycle. Loss-of-function mutations in any of the SDH genes are associated with cancer formation. However, the impact of SDH loss on cell metabolism and the mechanisms enabling growth of SDH-defective cells are largely unknown. Here, we generated Sdhb-ablated kidney mouse cells and used comparative metabolomics and stable-isotope-labelling approaches to identify nutritional requirements and metabolic adaptations to SDH loss. We found that lack of SDH activity commits cells to consume extracellular pyruvate, which sustains Warburg-like bioenergetic features. We further demonstrated that pyruvate carboxylation diverts glucose-derived carbons into aspartate biosynthesis, thus sustaining cell growth. By identifying pyruvate carboxylase as essential for the proliferation and tumorigenic capacity of SDH-deficient cells, this study revealed a metabolic vulnerability for potential future treatment of SDH-associated malignancies.

Mdm2 induces mono-ubiquitination of FOXO4.

Background The Forkhead box O (FOXO) class of transcription factors are involved in the regulation of several cellular responses including cell cycle progression and apoptosis. Furthermore, in model organisms FOXOs act as tumor suppressors and affect aging. Previously, we noted that FOXOs and p53 are remarkably similar within their spectrum of regulatory proteins [1]. For example, the de-ubiquitinating enzyme USP7 removes ubiquitin from both FOXO and p53. However, Skp2 has been identified as E3 ligase for FOXO1, whereas Mdm2 is the prime E3 ligase for p53. Principal Findings/Methodology Here we provide evidence that Mdm2 acts as an E3 ligase for FOXO as well. In vitro incubation of Mdm2 and FOXO results in ATP-dependent (multi)mono-ubiquitination of FOXO similar to p53. Furthermore, in vivo co-expression of Mdm2 and FOXO induces FOXO mono-ubiquitination and consistent with this result, siRNA-mediated depletion of Mdm2 inhibits mono-ubiquitination of FOXO induced by hydrogen peroxide. Regulation of FOXO ubiquitination by Mdm2 is likely to be direct since Mdm2 and FOXO co-immunoprecipitate. In addition, Mdm2-mediated ubiquitination regulates FOXO transcriptional activity. Conclusions/Significance These data identify Mdm2 as a novel E3 ligase for FOXOs and extend the analogous mode of regulation between FOXO and p53.

The Peptidyl-Isomerase Pin1 Regulates p27kip1 Expression through Inhibition of Forkhead Box O Tumor Suppressors

The Forkhead box O (FOXO) protein family is an evolutionarily conserved subclass of transcription factors recently identified as bona fide tumor suppressors. Preventing the accumulation of cellular damage due to oxidative stress is thought to underlie its tumor-suppressive role. Oxidative stress, in turn, also feedback controls FOXO4 function. Regulation of this process, however, is poorly understood but may be relevant to the ability of FOXO to control tumor suppression. Here, we characterize novel FOXO4 phosphorylation sites after increased cellular oxidative stress and identify the isomerase Pin1, a protein frequently found to be overexpressed in cancer, as a critical regulator of p27(kip1) through FOXO4 inhibition. We show that Pin1 requires these phosphorylation events to act negatively on FOXO4 transcriptional activity. Consistent with this, oxidative stress induces binding of Pin1 to FOXO, thereby attenuating its monoubiquitination, a yet uncharacterized mode of substrate modulation by Pin1. We have previously shown that monoubiquitination is involved in controlling nuclear translocation in response to cellular stress, and indeed, Pin1 prevents nuclear FOXO4 accumulation. Interestingly, Pin1 acts on FOXO through stimulation of the activity of the deubiquitinating enzyme HAUSP/USP7. Ultimately, this results in decreased transcriptional activity towards target genes, including the cell cycle arrest gene p27(kip1). Notably, in a primary human breast cancer panel, low p27(kip1) levels inversely correlated with Pin1 expression. Thus, Pin1 is identified as a novel negative FOXO regulator, interconnecting FOXO phosphorylation and monoubiquitination in response to cellular stress to regulate p27(kip1).

Regulation of histone H3K4 tri-methylation and PAF complex recruitment by the Ccr4-Not complex

Efficient transcription is linked to modification of chromatin. For instance, tri-methylation of lysine 4 on histone H3 (H3K4) strongly correlates with transcriptional activity and is regulated by the Bur1/2 kinase complex. We found that the evolutionarily conserved Ccr4-Not complex is involved in establishing H3K4 tri-methylation in Saccharomyces cerevisiae. We observed synthetic lethal interactions of Ccr4-Not components with BUR1 and BUR2. Further analysis indicated that the genes encoding the Not-proteins are essential for efficient regulation of H3K4me3, but not H3K4me1/2, H3K36me2 or H3K79me2/3 levels. Moreover, regulation of H3K4me3 levels by NOT4 is independent of defects in RNA polymerase II loading. We found NOT4 to be important for ubiquitylation of histone H2B via recruitment of the PAF complex, but not for recruitment or activation of the Bur1/2 complex. These results suggest a mechanism in which the Ccr4-Not complex functions parallel to or downstream of the Bur1/2 kinase to facilitate H3K4me3 via PAF complex recruitment.

Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence.

Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAF(V600E) oncogene, which arises commonly in melanoma. BRAF(V600E) signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH(2) terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr(223), Ser(226), Thr(447), and Thr(451). BRAF(V600E)-induced FOXO4 phosphorylation resulted in p21(cip1)-mediated cell senescence independent of p16(ink4a) or p27(kip1). Importantly, melanocyte-specific activation of BRAF(V600E) in vivo resulted in the formation of skin nevi expressing Thr(223)/Ser(226)-phosphorylated FOXO4 and elevated p21(cip1). Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging.

Insulin/IGF-1-mediated longevity is marked by reduced protein metabolism.

Mutations in the daf‐2 gene of the conserved Insulin/Insulin‐like Growth Factor (IGF‐1) pathway double the lifespan of the nematode Caenorhabditis elegans. This phenotype is completely suppressed by deletion of Forkhead transcription factor daf‐16. To uncover regulatory mechanisms coordinating this extension of life, we employed a quantitative proteomics strategy with daf‐2 mutants in comparison with N2 and daf‐16; daf‐2 double mutants. This revealed a remarkable longevity‐specific decrease in proteins involved in mRNA processing and transport, the translational machinery, and protein metabolism. Correspondingly, the daf‐2 mutants display lower amounts of mRNA and 20S proteasome activity, despite maintaining total protein levels equal to that observed in wild types. Polyribosome profiling in the daf‐2 and daf‐16;daf‐2 double mutants confirmed a daf‐16‐dependent reduction in overall translation, a phenotype reminiscent of Dietary Restriction‐mediated longevity, which was independent of germline activity. RNA interference (RNAi)‐mediated knockdown of proteins identified by our approach resulted in modified C. elegans lifespan confirming the importance of these processes in Insulin/IGF‐1‐mediated longevity. Together, the results demonstrate a role for the metabolism of proteins in the Insulin/IGF‐1‐mediated extension of life.