Fulvio Chiacchiera

Tet Proteins Connect the O-Linked N-acetylglucosamine Transferase Ogt to Chromatin in Embryonic Stem Cells

O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) activity is essential for embryonic stem cell (ESC) viability and mouse development. Ogt is present both in the cytoplasm and the nucleus of different cell types and catalyzes serine and threonine glycosylation. We have characterized the biochemical features of nuclear Ogt and identified the ten-eleven translocation (TET) proteins Tet1 and Tet2 as stable partners of Ogt in the nucleus of ESCs. We show at a genome-wide level that Ogt preferentially associates with Tet1 to genes promoters in close proximity of CpG-rich transcription start sites. These regions are characterized by low levels of DNA modification, suggesting a link between Tet1 and Ogt activities in regulating CpG island methylation. Finally, we show that Tet1 is required for binding of Ogt to chromatin affecting Tet1 activity. Taken together, our data characterize how O-GlcNAcylation is recruited to chromatin and interacts with the activity of 5-methylcytosine hydroxylases.

The AMPK-FoxO3A axis as a target for cancer treatment.

FoxO proteins are an evolutionarily conserved subfamily of transcription factors involved in tumor suppression, regulation of energy metabolism and development in several tissues, and are mainly regulated by phosphorylation-dependent nuclear/cytoplasmic shuttling. The transcriptional activity of FoxO3A, one of the four members of the family, is further modulated by AMPK, one of the key regulators of cellular metabolism, which basically shifts cell machinery from energy-consuming to energy-producing pathways. We recently demonstrated that the AMPK/FoxO3A energy sensor pathway is still inducible in human cancer cells in response to metabolic stress, as it becomes activated in colorectal and ovarian cancer cells in response to the inhibition of p38α. Activation of the FoxO3A transcriptional program initially induces autophagy as an attempt to retain energy to survive, whereas under persistent stress conditions it triggers autophagic cell death. In this review, we focus on the connections between AMPK and FoxO3A, describing their central role as modulators of fundamental processes such as stress resistance, cell metabolism, autophagy and cell death, and highlighting the therapeutic potential of pharmacological modulation of the AMPK-FoxO3A axis.

Inhibition of p38α unveils an AMPK-FoxO3A axis linking autophagy to cancer-specific metabolism

Autophagy is an essential process for the maintenance of cellular and metabolic homeostasis. Indeed, it is required for the recovery of ATP-generating substrates in cells subjected to different types of stress insults. Thus, the activity of the autophagic machinery strongly depends on the metabolic status of the cell.1 It has been proposed that this principle applies not only to normal, but also to cancer cells,2 despite the profound differences in their metabolism. Cancer cells predominantly produce ATP through the constitutive activation of aerobic glycolysis, a process that generally relies on the stabilization and activation of the transcription factor HIF1α, which regulates the expression of glycolytic genes.3 We recently showed that p38α is required to sustain the expression of HIF1α target genes, and that its inhibition causes a rapid drop in ATP levels in colorectal cancer cells (CRCs). This acute energy need triggers AMPK-dependent nuclear accumulation of FoxO3A and subsequent activation of its transcriptional program, leading to sequential induction of autophagy, cell cycle arrest and cell death. In vivo, pharmacological blockade of p38α has both a cytostatic and cytotoxic effect on colorectal neoplasms, associated with nuclear enrichment of FoxO3A and expression of its target genes p21 and PTEN.4 Our data suggest that CRCs impaired in their glycolytic metabolism trigger autophagy as a reversible recovery mechanism and undergo cell cycle arrest; however, the persistence of the stress insults inevitably leads to cell death.

A novel AMPK-dependent FoxO3A-SIRT3 intramitochondrial complex sensing glucose levels

Reduction of nutrient intake without malnutrition positively influences lifespan and healthspan from yeast to mice and exerts some beneficial effects also in humans. The AMPK-FoxO axis is one of the evolutionarily conserved nutrient-sensing pathways, and the FOXO3A locus is associated with human longevity. Interestingly, FoxO3A has been reported to be also a mitochondrial protein in mammalian cells and tissues. Here we report that glucose restriction triggers FoxO3A accumulation into mitochondria of fibroblasts and skeletal myotubes in an AMPK-dependent manner. A low-glucose regimen induces the formation of a protein complex containing FoxO3A, SIRT3, and mitochondrial RNA polymerase (mtRNAPol) at mitochondrial DNA-regulatory regions causing activation of the mitochondrial genome and a subsequent increase in mitochondrial respiration. Consistently, mitochondrial transcription increases in skeletal muscle of fasted mice, with a mitochondrial DNA-bound FoxO3A/SIRT3/mtRNAPol complex detectable also in vivo. Our results unveil a mitochondrial arm of the AMPK-FoxO3A axis acting as a recovery mechanism to sustain energy metabolism upon nutrient restriction.

Signal-dependent regulation of gene expression as a target for cancer treatment: Inhibiting p38α in colorectal tumors

In the last year, several evidences indicated that pharmacological manipulation of relevant signaling pathways could selectively affect gene expression to influence cell fate. These findings render of extreme importance the elucidation of how external stimuli are transduced to mediate chromatin modifications, resulting in a permissive or repressive environment for gene expression. These signaling cascades activate or repress the function of chromatin binding proteins that represent attractive pharmacological targets for human diseases. Actually, the closer the target is to chromatin, the more the transcriptional effect will be selective. Recent studies suggest that pharmacological manipulation of signaling pathways to modulate cell fate is indeed possible and that chromatin-associated kinases could represent an optimal target. The p38 MAPK is the prototype of this class of enzymes and its central role in the transcription process is evolutionary conserved. In this review we will focus on the possibility to inhibit p38alpha in colorectal cancer to arrest tumor progression and induce autophagic cell death.

p38alpha is required for ovarian cancer cell metabolism and survival

Introduction: Ovarian cancer is highly sensitive to chemotherapy but also shows a high rate of recurrence and drug resistance. These negative outcomes mostly depend on altered apoptotic pathways, making the design of new therapeutic strategies based on the induction of other types of cell death highly desirable. Several lines of research are now addressing cancer-specific features to specifically target tumor cells, thus reducing adverse effects. In this light, a great deal of attention has been devoted to the metabolic reprogramming occurring in cancer cells, which display increased levels of glycolysis compared with their normal counterparts. We recently showed that inhibition of p38&agr; impairs key metabolic functions of colorectal cancer cells, inducing growth arrest, autophagy, and cell death both in vivo and in vitro. These effects are mediated by a switch from hypoxia-inducible factor 1&agr; (HIF1&agr;) to forkhead transcription factor O (FoxO)-dependent transcription. Methods: We first characterized p38 expression in OVCAR-3, A2780, and SKOV-3 ovarian cancer cell lines. Then, we treated these cells with the p38&agr;/p38&bgr;-specific inhibitor SB202190 and performed a morphological, proliferation, and survival analyses. Finally, we studied HIF1&agr; and FoxO3A expressions and signaling pathways to evaluate their role in SB202190-induced effects. Results: p38&agr; blockade induces the formation of intracellular autophagic vacuoles and reduces growth and viability of ovarian cancer cells. As in colorectal cancer, the underlying molecular mechanism seems to rely on a shift from HIF1&agr;- to FoxO3A-dependent transcription, which is promoted by the activation of the adenosine monophosphate-activated protein kinase pathway. Conclusions: These data corroborate the hypothesis that pharmacological modulation of genes involved in cancer-specific homeostasis, such as p38&agr;, might be exploited to design new therapeutic approaches to cancer treatment.

Polycomb Complex PRC1 Preserves Intestinal Stem Cell Identity by Sustaining Wnt/β-Catenin Transcriptional Activity

Polycomb repressive complexes (PRCs) are among the most important gatekeepers of establishing and maintaining cell identity in metazoans. PRC1, which plays a dominant role in this context, executes its functions via multiple subcomplexes, which all contribute to H2AK119 mono-ubiquitination (H2Aubq). Despite our comprehensive knowledge of PRC1-dependent H2Aubq in embryonic stem cells and during early development, its role in adult stem cells still remains poorly characterized. Here we show that PRC1 activity is required for the integrity of the intestinal epithelium, regulating stem cell self-renewal via a cell-autonomous mechanism that is independent from Cdkn2a expression. By dissecting the PRC1-dependent transcription program in intestinal stem cells, we demonstrate that PRC1 represses a large number of non-lineage-specific transcription factors that directly affect β-catenin/Tcf transcriptional activity. Our data reveal that PRC1 preserves Wnt/β-catenin activity in adult stem cells to maintain intestinal homeostasis and supports tumor formation induced by the constitutive activation of this pathway.

Blocking p38/ERK crosstalk affects colorectal cancer growth by inducing apoptosis in vitro and in preclinical mouse models

We recently demonstrated that p38α is required to maintain colorectal cancer (CRC) metabolism, as its inhibition leads to FoxO3A activation, autophagy, cell death, and tumor growth reduction both in vitro and in vivo. Here we show that inhibition of p38α is followed by TRAIL-mediated activation of caspase-8 and FoxO3A-dependent HER3 upregulation with consequent overactivation of the MEK-ERK1/2 survival pathway. p38α and MEK combined inhibition specifically induces apoptosis by enabling TRAIL signaling propagation through t-Bid and caspase-3, and fosters cell death in CRC cells and preclinical mouse models. Current MEK1-directed pharmacological strategies could thus be exploited, in combination with p38α inhibition, to develop new approaches for CRC treatment.

Epigenetic methylations and their connections with metabolism.

Metabolic pathways play fundamental roles in several processes that regulate cell physiology and adaptation to environmental changes. Altered metabolic pathways predispose to several different pathologies ranging from diabetes to cancer. Specific transcriptional programs tightly regulate the enzymes involved in cell metabolism and dictate cell fate regulating the differentiation into specialized cell types that contribute to metabolic adaptation in higher organisms. For these reasons, it is of extreme importance to identify signaling pathways and transcription factors that positively and negatively regulate metabolism. Genomic organization allows a plethora of different strategies to regulate transcription. Importantly, large evidence suggests that the quality of diet and the caloric regimen can influence the epigenetic state of our genome and that certain metabolic pathways are also epigenetically controlled reveling a tight crosstalk between metabolism and epigenomes. Here we focus our attention on methylation-based epigenetic reactions, on how different metabolic pathways control these activities, and how these can influence metabolism. Altogether, the recent discoveries linking these apparent distant areas reveal that an exciting field of research is emerging.

PRC2 preserves intestinal progenitors and restricts secretory lineage commitment

Chromatin modifications shape cell heterogeneity by activating and repressing defined sets of genes involved in cell proliferation, differentiation and development. Polycomb‐repressive complexes (PRCs) act synergistically during development and differentiation by maintaining transcriptional repression of common genes. PRC2 exerts this activity by catalysing H3K27 trimethylation. Here, we show that in the intestinal epithelium PRC2 is required to sustain progenitor cell proliferation and the correct balance between secretory and absorptive lineage differentiation programs. Using genetic models, we show that PRC2 activity is largely dispensable for intestinal stem cell maintenance but is strictly required for radiation‐induced regeneration by preventing Cdkn2a transcription. Combining these models with genomewide molecular analysis, we further demonstrate that preferential accumulation of secretory cells does not result from impaired proliferation of progenitor cells induced by Cdkn2a activation but rather from direct regulation of transcription factors responsible for secretory lineage commitment. Overall, our data uncover a dual role of PRC2 in intestinal homeostasis highlighting the importance of this repressive layer in controlling cell plasticity and lineage choices in adult tissues.