Ana Ortega-Molina

Pten Positively Regulates Brown Adipose Function, Energy Expenditure, and Longevity

Aging in worms and flies is regulated by the PI3K/Akt/Foxo pathway. Here we extend this paradigm to mammals. Pten(tg) mice carrying additional genomic copies of Pten are protected from cancer and present a significant extension of life span that is independent of their lower cancer incidence. Interestingly, Pten(tg) mice have an increased energy expenditure and protection from metabolic pathologies. The brown adipose tissue (BAT) of Pten(tg) mice is hyperactive and presents high levels of the uncoupling protein Ucp1, which we show is a target of Foxo1. Importantly, a synthetic PI3K inhibitor also increases energy expenditure and hyperactivates the BAT in mice. These effects can be recapitulated in isolated brown adipocytes and, moreover, implants of Pten(tg) fibroblasts programmed with Prdm16 and Cebpβ form subcutaneous brown adipose pads more efficiently than wild-type fibroblasts. These observations uncover a role of Pten in promoting energy expenditure, thus decreasing nutrient storage and its associated damage.

Induction of p53-Dependent Senescence by the MDM2 Antagonist Nutlin-3a in Mouse Cells of Fibroblast Origin

Cellular senescence is emerging as an important in vivo anticancer response elicited by multiple stresses, including currently used chemotherapeutic drugs. Nutlin-3a is a recently discovered small-molecule antagonist of the p53-destabilizing protein murine double minute-2 (MDM2) that induces cell cycle arrest and apoptosis in cancer cells with functional p53. Here, we report that nutlin-3a induces cellular senescence in murine primary fibroblasts, oncogenically transformed fibroblasts, and fibrosarcoma cell lines. No evidence of drug-induced apoptosis was observed in any case. Nutlin-induced senescence was strictly dependent on the presence of functional p53 as revealed by the fact that cells lacking p53 were completely insensitive to the drug, whereas cells lacking the tumor suppressor alternative reading frame product of the CDKN2A locus underwent irreversible cell cycle arrest. Interestingly, irreversibility was achieved in neoplastic cells faster than in their corresponding parental primary cells, suggesting that nutlin-3a and oncogenic signaling cooperate in activating p53. Our current results suggest that senescence could be a major cellular outcome of cancer therapy by antagonists of the p53-MDM2 interaction, such as nutlin-3a.

The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development.

The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell–activating pathways.

PTEN in cancer, metabolism and aging

Recent reports on mice with systemic overexpression of the tumor-suppressor PTEN (phosphatase and tensin homolog) have expanded our understanding of its physiological functions. Pten transgenic mice present increased energy expenditure, decreased adiposity, improved insulin sensitivity upon high-fat feeding or with aging, and extended lifespan. This has led to new mechanistic insights about the role of PTEN in metabolism. Interestingly, PTEN promotes oxidative phosphorylation and decreases glycolysis, thus preventing the metabolic reprogramming characteristic of cancer cells, which might be relevant to PTEN-mediated cancer protection. PTEN also upregulates UCP1 expression in brown adipocytes, which enhances their nutrient burning capacity and decreases adiposity and associated pathologies. The newly discovered effects of PTEN on metabolism open new avenues for exploration relevant to cancer, obesity, diabetes, and aging.

Limited Role of Murine ATM in Oncogene-Induced Senescence and p53-Dependent Tumor Suppression

Recent studies in human fibroblasts have provided a new general paradigm of tumor suppression according to which oncogenic signaling produces DNA damage and this, in turn, results in ATM/p53-dependent cellular senescence. Here, we have tested this model in a variety of murine experimental systems. Overexpression of oncogenic Ras in murine fibroblasts efficiently induced senescence but this occurred in the absence of detectable DNA damage signaling, thus suggesting a fundamental difference between human and murine cells. Moreover, lung adenomas initiated by endogenous levels of oncogenic K-Ras presented abundant senescent cells, but undetectable DNA damage signaling. Accordingly, K-Ras-driven adenomas were also senescent in Atm-null mice, and the tumorigenic progression of these lesions was only modestly accelerated by Atm-deficiency. Finally, we have examined chemically-induced fibrosarcomas, which possess a persistently activated DNA damage response and are highly sensitive to the activity of p53. We found that the absence of Atm favored genomic instability in the resulting tumors, but did not affect the persistent DNA damage response and did not impair p53-dependent tumor suppression. All together, we conclude that oncogene-induced senescence in mice may occur in the absence of a detectable DNA damage response. Regarding murine Atm, our data suggest that it plays a minor role in oncogene-induced senescence or in p53-dependent tumor suppression, being its tumor suppressive activity probably limited to the maintenance of genomic stability.

PTEN recruitment controls synaptic and cognitive function in Alzheimer's models

Dyshomeostasis of amyloid-β peptide (Aβ) is responsible for synaptic malfunctions leading to cognitive deficits ranging from mild impairment to full-blown dementia in Alzheimers disease. Aβ appears to skew synaptic plasticity events toward depression. We found that inhibition of PTEN, a lipid phosphatase that is essential to long-term depression, rescued normal synaptic function and cognition in cellular and animal models of Alzheimers disease. Conversely, transgenic mice that overexpressed PTEN displayed synaptic depression that mimicked and occluded Aβ-induced depression. Mechanistically, Aβ triggers a PDZ-dependent recruitment of PTEN into the postsynaptic compartment. Using a PTEN knock-in mouse lacking the PDZ motif, and a cell-permeable interfering peptide, we found that this mechanism is crucial for Aβ-induced synaptic toxicity and cognitive dysfunction. Our results provide fundamental information on the molecular mechanisms of Aβ-induced synaptic malfunction and may offer new mechanism-based therapeutic targets to counteract downstream Aβ signaling.

Regulation of the tumor suppressor PTEN by SUMO

The crucial function of the PTEN tumor suppressor in multiple cellular processes suggests that its activity must be tightly controlled. Both, membrane association and a variety of post-translational modifications, such as acetylation, phosphorylation, and mono- and polyubiquitination, have been reported to regulate PTEN activity. Here, we demonstrated that PTEN is also post-translationally modified by the small ubiquitin-like proteins, small ubiquitin-related modifier 1 (SUMO1) and SUMO2. We identified lysine residue 266 and the major monoubiquitination site 289, both located within the C2 domain required for PTEN membrane association, as SUMO acceptors in PTEN. We demonstrated the existence of a crosstalk between PTEN SUMOylation and ubiquitination, with PTEN-SUMO1 showing a reduced capacity to form covalent interactions with monoubiquitin and accumulation of PTEN-SUMO2 conjugates after inhibition of the proteasome. Moreover, we found that virus infection induces PTEN SUMOylation and favors PTEN localization at the cell membrane. Finally, we demonstrated that SUMOylation contributes to the control of virus infection by PTEN.

The PTEN/NRF2 axis promotes human carcinogenesis.

AIMS A recent study conducted in mice reported that liver-specific knockout of tumor suppressor Pten augments nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcriptional activity. Here, we further investigated how phosphatase and tensin homolog deleted on chromosome 10 (PTEN) controls NRF2 and the relevance of this pathway in human carcin ogenesis. RESULTS Drug and genetic targeting to PTEN and phosphoproteomics approaches indicated that PTEN leads to glycogen synthase kinase-3 (GSK-3)-mediated phosphorylation of NRF2 at residues Ser(335) and Ser(338) and subsequent beta-transducin repeat containing protein (β-TrCP)-dependent but Kelch-like ECH-associated protein 1 (KEAP1)-independent degradation. Rescue experiments in PTEN-deficient cells and xerographs in athymic mice indicated that loss of PTEN leads to increased NRF2 signature which provides a proliferating and tumorigenic advantage. Tissue microarrays from endometrioid carcinomas showed that 80% of PTEN-negative tumors expressed high levels of NRF2 or its target heme oxygenase-1 (HO-1). INNOVATION These results uncover a new mechanism of oncogenic activation of NRF2 by loss of its negative regulation by PTEN/GSK-3/β-TrCP that may be relevant to a large number of tumors, including endometrioid carcinomas. CONCLUSION Increased activity of NRF2 due to loss of PTEN is instrumental in human carcinogenesis and represents a novel therapeutic target.

CREBBP Inactivation Promotes the Development of HDAC3-Dependent Lymphomas

Somatic mutations in CREBBP occur frequently in B-cell lymphoma. Here, we show that loss of CREBBP facilitates the development of germinal center (GC)-derived lymphomas in mice. In both human and murine lymphomas, CREBBP loss-of-function resulted in focal depletion of enhancer H3K27 acetylation and aberrant transcriptional silencing of genes that regulate B-cell signaling and immune responses, including class II MHC. Mechanistically, CREBBP-regulated enhancers are counter-regulated by the BCL6 transcriptional repressor in a complex with SMRT and HDAC3, which we found to bind extensively to MHC class II loci. HDAC3 loss-of-function rescued repression of these enhancers and corresponding genes, including MHC class II, and more profoundly suppressed CREBBP-mutant lymphomas in vitro and in vivo Hence, CREBBP loss-of-function contributes to lymphomagenesis by enabling unopposed suppression of enhancers by BCL6/SMRT/HDAC3 complexes, suggesting HDAC3-targeted therapy as a precision approach for CREBBP-mutant lymphomas. SIGNIFICANCE Our findings establish the tumor suppressor function of CREBBP in GC lymphomas in which CREBBP mutations disable acetylation and result in unopposed deacetylation by BCL6/SMRT/HDAC3 complexes at enhancers of B-cell signaling and immune response genes. Hence, inhibition of HDAC3 can restore the enhancer histone acetylation and may serve as a targeted therapy for CREBBP-mutant lymphomas. Cancer Discov; 7(1); 38-53. ©2016 AACR.See related commentary by Höpken, p. 14This article is highlighted in the In This Issue feature, p. 1.

Pharmacological Inhibition of PI3K Reduces Adiposity and Metabolic Syndrome in Obese Mice and Rhesus Monkeys

Genetic inhibition of PI3K signaling increases energy expenditure, protects from obesity and metabolic syndrome, and extends longevity. Here, we show that two pharmacological inhibitors of PI3K, CNIO-PI3Ki and GDC-0941, decrease the adiposity of obese mice without affecting their lean mass. Long-term treatment of obese mice with low doses of CNIO-PI3Ki reduces body weight until reaching a balance that is stable for months as long as the treatment continues. CNIO-PI3Ki treatment also ameliorates liver steatosis and decreases glucose serum levels. The above observations have been recapitulated in independent laboratories and using different oral formulations of CNIO-PI3Ki. Finally, daily oral treatment of obese rhesus monkeys for 3 months with low doses of CNIO-PI3Ki decreased their adiposity and lowered their serum glucose levels, in the absence of detectable toxicities. Therefore, pharmacological inhibition of PI3K is an effective and safe anti-obesity intervention that could reverse the negative effects of metabolic syndrome in humans.