Mitochondria Define Intestinal Stem Cell Differentiation Downstream of a FOXO/Notch Axis.

Differential WNT and Notch signaling regulates differentiation of Lgr5+ crypt-based columnar cells (CBCs) into intestinal cell lineages. Recently we showed that mitochondrial activity supports CBCs, while adjacent Paneth cells (PCs) show reduced mitochondrial activity. This implies that CBC differentiation into PCs involves a metabolic transition toward downregulation of mitochondrial dependency. Here we show that Forkhead box O (FoxO) transcription factors and Notch signaling interact in determining CBC fate. In agreement with the organoid data, Foxo1/3/4 deletion in mouse intestine induces secretory cell differentiation. Importantly, we show that FOXO and Notch signaling converge on regulation of mitochondrial fission, which in turn provokes stem cell differentiation into goblet cells and PCs. Finally, scRNA-seq-based reconstruction of CBC differentiation trajectories supports the role of FOXO, Notch, and mitochondria in secretory differentiation. Together, this points at a new signaling-metabolic axis in CBC differentiation and highlights the importance of mitochondria in determining stem cell fate.

Rapid and Selective Targeting of Heterogeneous Pancreatic Neuroendocrine Tumors.

Design of tissue-specific contrast agents to delineate tumors from background tissues is a major unmet clinical need for ultimate surgical interventions. Bioconjugation of fluorophore(s) to a ligand has been mainly used to target overexpressed receptors on tumors. However, the size of the final targeted ligand can be large, >20 kDa, and cannot readily cross the microvasculature to meet the specific tissue, resulting in low targetability with a high background. Here, we report a small and hydrophilic phenoxazine with high targetability and retention to pancreatic neuroendocrine tumor. This bioengineered fluorophore permits sensitive detection of ultrasmall (<0.5 mm) ectopic tumors within a few seconds after a single bolus injection, highlighting every tumor in the pancreas from the surrounding healthy tissues with reasonable half-life. The knowledge-based approach and validation used to develop structure-inherent tumor-targeted fluorophores have a tremendous potential to improve treatment outcome by providing definite tumor margins for image-guided surgery.

FoxO1 regulates leptin-induced mood behavior by targeting tyrosine hydroxylase.

PURPOSE: While leptin has been associated with various psycho-physiological functions, the molecular network in leptin-mediated mood regulation remains elusive. METHODS: Anxiolytic behaviors and tyrosine hydroxylase (TH) levels were examined after leptin administration. Functional roles of STAT3 and FoxO1 in regulation of TH expression were investigated using in vivo and in vitro systems. A series of animal behavioral tests using dopaminergic neuron-specific FoxO1 KO (FoxO1 KODAT) were performed and investigated the roles of FoxO1 in regulation of mood behaviors. RESULTS: Here, we show that administration of leptin induces anxiolytic-like phenotype through the activation of signal transducer and activator of transcription 3 (STAT3) and the inhibition of forkhead box protein O1 (FoxO1) in dopaminergic (DA) neurons of the midbrain. Specifically, STAT3 and FoxO1 directly bind to and exert opposing effects on tyrosine hydroxylase (TH) expression, where STAT3 acts as an enhancer and FoxO1 acts as a prominent repressor. Accordingly, suppression of the prominent suppressor FoxO1 by leptin strongly increased TH expression. Furthermore, our previous results showed that specific deletion of FoxO1 in DA neurons (FoxO1 KODAT) led to a profound elevation of TH activity and dopamine contents. Finally, FoxO1 KODAT mice exhibited enhanced leptin sensitivity as well as displayed reduced anxiety- and depression-like behaviors. CONCLUSIONS: This work establishes a novel molecular mechanism of mood behavior regulation by leptin and suggests FoxO1 suppression by leptin might be a key for leptin-induced behavioral manifestation in DA neurons.

FoxO1 in dopaminergic neurons regulates energy homeostasis and targets tyrosine hydroxylase.

Dopaminergic (DA) neurons are involved in the integration of neuronal and hormonal signals to regulate food consumption and energy balance. Forkhead transcriptional factor O1 (FoxO1) in the hypothalamus plays a crucial role in mediation of leptin and insulin function. However, the homoeostatic role of FoxO1 in DA system has not been investigated. Here we report that FoxO1 is highly expressed in DA neurons and mice lacking FoxO1 specifically in the DA neurons (FoxO1 KODAT) show markedly increased energy expenditure and interscapular brown adipose tissue (iBAT) thermogenesis accompanied by reduced fat mass and improved glucose/insulin homoeostasis. Moreover, FoxO1 KODAT mice exhibit an increased sucrose preference in concomitance with higher dopamine and norepinephrine levels. Finally, we found that FoxO1 directly targets and negatively regulates tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of the catecholamine synthesis, delineating a mechanism for the KO phenotypes. Collectively, these results suggest that FoxO1 in DA neurons is an important transcriptional factor that directs the coordinated control of energy balance, thermogenesis and glucose homoeostasis.

Regulation of autophagy and the ubiquitin-proteasome system by the FoxO transcriptional network during muscle atrophy.

Stresses like low nutrients, systemic inflammation, cancer or infections provoke a catabolic state characterized by enhanced muscle proteolysis and amino acid release to sustain liver gluconeogenesis and tissue protein synthesis. These conditions activate the family of Forkhead Box (Fox) O transcription factors. Here we report that muscle-specific deletion of FoxO members protects from muscle loss as a result of the role of FoxOs in the induction of autophagy-lysosome and ubiquitin-proteasome systems. Notably, in the setting of low nutrient signalling, we demonstrate that FoxOs are required for Akt activity but not for mTOR signalling. FoxOs control several stress-response pathways such as the unfolded protein response, ROS detoxification, DNA repair and translation. Finally, we identify FoxO-dependent ubiquitin ligases including MUSA1 and a previously uncharacterised ligase termed SMART (Specific of Muscle Atrophy and Regulated by Transcription). Our findings underscore the central function of FoxOs in coordinating a variety of stress-response genes during catabolic conditions.

Development of Resistance to EGFR-Targeted Therapy in Malignant Glioma Can Occur through EGFR-Dependent and -Independent Mechanisms.

Epidermal growth factor receptor (EGFR) is highly amplified, mutated, and overexpressed in human malignant gliomas. Despite its prevalence and growth-promoting functions, therapeutic strategies to inhibit EGFR kinase activity have not been translated into profound beneficial effects in glioma clinical trials. To determine the roles of oncogenic EGFR signaling in gliomagenesis and tumor maintenance, we generated a novel glioma mouse model driven by inducible expression of a mutant EGFR (EGFR*). Using combined genetic and pharmacologic interventions, we revealed that EGFR*-driven gliomas were insensitive to EGFR tyrosine kinase inhibitors, although they could efficiently inhibit EGFR* autophosphorylation in vitro and in vivo. This is in contrast with the genetic suppression of EGFR* induction that led to significant tumor regression and prolonged animal survival. However, despite their initial response to genetic EGFR* extinction, all tumors would relapse and propagate independent of EGFR*. We further showed that EGFR*-independent tumor cells existed prior to treatment and were responsible for relapse following genetic EGFR* suppression. And, the addition of a PI3K/mTOR inhibitor could significantly delay relapse and prolong animal survival. Our findings shed mechanistic insight into EGFR drug resistance in glioma and provide a platform to test therapies targeting aberrant EGFR signaling in this setting.

FoxO3 coordinates metabolic pathways to maintain redox balance in neural stem cells.

Forkhead Box O (FoxO) transcription factors act in adult stem cells to preserve their regenerative potential. Previously, we reported that FoxO maintains the long-term proliferative capacity of neural stem/progenitor cells (NPCs), and that this occurs, in part, through the maintenance of redox homeostasis. Herein, we demonstrate that among the FoxO3-regulated genes in NPCs are a host of enzymes in central carbon metabolism that act to combat reactive oxygen species (ROS) by directing the flow of glucose and glutamine carbon into defined metabolic pathways. Characterization of the metabolic circuit observed upon loss of FoxO3 revealed a drop in glutaminolysis and filling of the tricarboxylic acid (TCA) cycle. Additionally, we found that glucose uptake, glucose metabolism and oxidative pentose phosphate pathway activity were similarly repressed in the absence of FoxO3. Finally, we demonstrate that impaired glucose and glutamine metabolism compromises the proliferative potential of NPCs and that this is exacerbated following FoxO3 loss. Collectively, our findings show that a FoxO3-dependent metabolic programme supports redox balance and the neurogenic potential of NPCs.

ZNF365 promotes stalled replication forks recovery to maintain genome stability.

The ZNF365 locus is associated with breast cancer risk in carriers of mutated BRCA1 and BRCA2, which are important molecules required for DNA damage response. Previously, we demonstrated that ZNF365 is necessary for timely resolution of replication intermediates of genomic fragile sites and, thus, for suppression of genomic instability; however, the mechanism underlying the function of ZNF365 on damaged DNA and stalled replication forks remains unknown. Here, we demonstrate that ZNF365 is induced by DNA double-strand break (DSB) signals, is involved in the homologous recombination (HR) repair pathway, and maintains genome integrity during DNA replication. On the mechanistic level, ZNF365 interacts with poly(ADP-ribose) polymerase (PARP) 1 to tether MRE11 to the DNA end resection site. Loss of ZNF365 results in delayed mitotic progression and exit due to increased replication stress, ultimately leading to cytokinesis failure, re-duplication of centrosomes, and increased aneuploidy. Collectively, these results suggest an HR repair-dependent function of ZNF365 in preventing genomic instability.

ZNF365 promotes stability of fragile sites and telomeres.

Critically short telomeres activate cellular senescence or apoptosis, as mediated by the tumor suppressor p53, but in the absence of this checkpoint response, telomere dysfunction engenders chromosomal aberrations and cancer. Here, analysis of p53-regulated genes activated in the setting of telomere dysfunction identified Zfp365 (ZNF365 in humans) as a direct p53 target that promotes genome stability. Germline polymorphisms in the ZNF365 locus are associated with increased cancer risk, including those associated with telomere dysfunction. On the mechanistic level, ZNF365 suppresses expression of a subset of common fragile sites, including telomeres. In the absence of ZNF365, defective telomeres engage in aberrant recombination of telomere ends, leading to increased telomere sister chromatid exchange and formation of anaphase DNA bridges, including ultra-fine DNA bridges, and ultimately increased cytokinesis failure and aneuploidy. Thus, the p53-ZNF365 axis contributes to genomic stability in the setting of telomere dysfunction.

Endothelial FoxO1 is an intrinsic regulator of thrombospondin 1 expression that restrains angiogenesis in ischemic muscle.

Peripheral artery disease (PAD) is characterized by chronic muscle ischemia. Compensatory angiogenesis is minimal within ischemic muscle despite an increase in angiogenic factors. This may occur due to the prevalence of angiostatic factors. Regulatory mechanisms that could evoke an angiostatic environment during ischemia are largely unknown. Forkhead box O (FoxO) transcription factors, known to repress endothelial cell proliferation in vitro, are potential candidates. Our goal was to determine whether FoxO proteins promote an angiostatic phenotype within ischemic muscle. FoxO1 and the angiostatic matrix protein thrombospondin 1 (THBS1) were elevated in ischemic muscle from PAD patients, or from mice post-femoral artery ligation. Mice with conditional endothelial cell-directed deletion of FoxO proteins (Mx1Cre (+), FoxO1,3,4 (L/L) , referred to as FoxOΔ) were used to assess the role of endothelial FoxO proteins within ischemic tissue. FoxO deletion abrogated the elevation of FoxO1 and THBS1 proteins, enhanced hindlimb blood flow recovery and improved neovascularization in murine ischemic muscle. Endothelial cell outgrowth from 3D explant cultures was more robust in muscles derived from FoxOΔ mice. FoxO1 overexpression induced THBS1 production, and a direct interaction of endogenous FoxO1 with the THBS1 promoter was detectable in primary endothelial cells. We provide evidence that FoxO1 directly regulates THBS1 within ischemic muscle. Altogether, these findings bring novel insight into the regulatory mechanisms underlying the repression of angiogenesis within peripheral ischemic tissues.

FoxOs in neural stem cell fate decision.

Neural stem cells (NSCs) persist over the lifespan of mammals to give rise to committed progenitors and their differentiated cells in order to maintain the brain homeostasis. To this end, NSCs must be able to self-renew and otherwise maintain their quiescence. Suppression of aberrant proliferation or undesired differentiation is crucial to preclude either malignant growth or precocious depletion of NSCs. The PI3K-Akt-FoxO signaling pathway plays a central role in the regulation of multiple stem cells including one in the mammalian brain. In particular, members of FoxO family transcription factors are highly expressed in these stem cells. As an important downstream effector of growth, differentiation, and stress stimuli, mammalian FoxO transcription factor family controls cellular proliferation, oxidative stress response, homeostasis, and eventual maintenance of long-term repopulating potential. The review will focus on the current understanding of FoxO function in NSCs as well as discuss their biological activities that contribute to determining neural stem cell fate.

STAR RNA-binding protein Quaking suppresses cancer via stabilization of specific miRNA.

Multidimensional cancer genome analysis and validation has defined Quaking (QKI), a member of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, as a novel glioblastoma multiforme (GBM) tumor suppressor. Here, we establish that p53 directly regulates QKI gene expression, and QKI protein associates with and leads to the stabilization of miR-20a; miR-20a, in turn, regulates TGFßR2 and the TGFß signaling network. This pathway circuitry is substantiated by in silico epistasis analysis of its components in the human GBM TCGA (The Cancer Genome Atlas Project) collection and by their gain- and loss-of-function interactions in in vitro and in vivo complementation studies. This p53-QKI-miR-20a-TGFß pathway expands our understanding of the p53 tumor suppression network in cancer and reveals a novel tumor suppression mechanism involving regulation of specific cancer-relevant microRNAs.

Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism.

Tumor maintenance relies on continued activity of driver oncogenes, although their rate-limiting role is highly context dependent. Oncogenic Kras mutation is the signature event in pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible Kras(G12D)-driven PDAC mouse model establishes that advanced PDAC remains strictly dependent on Kras(G12D) expression. Transcriptome and metabolomic analyses indicate that Kras(G12D) serves a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis and pentose phosphate pathways (PPP). These studies also reveal that oncogenic Kras promotes ribose biogenesis. Unlike canonical models, we demonstrate that Kras(G12D) drives glycolysis intermediates into the nonoxidative PPP, thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in PDAC.

Transcription factor FoxO1 is essential for enamel biomineralization.

The Transforming growth factor ß (Tgf-ß) pathway, by signaling via the activation of Smad transcription factors, induces the expression of many diverse downstream target genes thereby regulating a vast array of cellular events essential for proper development and homeostasis. In order for a specific cell type to properly interpret the Tgf-ß signal and elicit a specific cellular response, cell-specific transcriptional co-factors often cooperate with the Smads to activate a discrete set of genes in the appropriate temporal and spatial manner. Here, via a conditional knockout approach, we show that mice mutant for Forkhead Box O transcription factor FoxO1 exhibit an enamel hypomaturation defect which phenocopies that of the Smad3 mutant mice. Furthermore, we determined that both the FoxO1 and Smad3 mutant teeth exhibit changes in the expression of similar cohort of genes encoding enamel matrix proteins required for proper enamel development. These data raise the possibility that FoxO1 and Smad3 act in concert to regulate a common repertoire of genes necessary for complete enamel maturation. This study is the first to define an essential role for the FoxO family of transcription factors in tooth development and provides a new molecular entry point which will allow researchers to delineate novel genetic pathways regulating the process of biomineralization which may also have significance for studies of human tooth diseases such as amelogenesis imperfecta.

PTEN is a major tumor suppressor in pancreatic ductal adenocarcinoma and regulates an NF-κB-cytokine network.

Initiation of pancreatic ductal adenocarcinoma (PDAC) is driven by oncogenic KRAS mutation, and disease progression is associated with frequent loss of tumor suppressors. In this study, human PDAC genome analyses revealed frequent deletion of the PTEN gene as well as loss of expression in primary tumor specimens. A potential role for PTEN as a haploinsufficient tumor suppressor is further supported by mouse genetic studies. The mouse PDAC driven by oncogenic Kras mutation and Pten deficiency also sustains spontaneous extinction of Ink4a expression and shows prometastatic capacity. Unbiased transcriptomic analyses established that combined oncogenic Kras and Pten loss promotes marked NF-κB activation and its cytokine network, with accompanying robust stromal activation and immune cell infiltration with known tumor-promoting properties. Thus, PTEN/phosphoinositide 3-kinase (PI3K) pathway alteration is a common event in PDAC development and functions in part to strongly activate the NF-κB network, which may serve to shape the PDAC tumor microenvironment.

FoxO family members in cancer.

The PI3K-Akt-FoxO signaling pathway plays a central role in diverse physiological processes including cellular energy storage, growth, and survival, among others. As an important effector of this pathway, FoxO is involved in versatile activities that protect organisms from stress and aging. Recent studies on mammalian FoxO have established a direct role for this transcription factor family in cellular proliferation, oxidative stress response, and tumorigenesis. The review will focus on the recent developments pertaining to the function of FoxO as well as discuss the various contexts in which FoxO exerts distinct biological activity such as drug resistance and autophagy in cancer pathogenesis and therapy.

IL-7 engages multiple mechanisms to overcome chronic viral infection and limit organ pathology.

Understanding the factors that impede immune responses to persistent viruses is essential in designing therapies for HIV infection. Mice infected with LCMV clone-13 have persistent high-level viremia and a dysfunctional immune response. Interleukin-7, a cytokine that is critical for immune development and homeostasis, was used here to promote immunity toward clone-13, enabling elucidation of the inhibitory pathways underlying impaired antiviral immune response. Mechanistically, IL-7 downregulated a critical repressor of cytokine signaling, Socs3, resulting in amplified cytokine production, increased T cell effector function and numbers, and viral clearance. IL-7 enhanced thymic output to expand the naive T cell pool, including T cells that were not LCMV specific. Additionally, IL-7 promoted production of cytoprotective IL-22 that abrogated liver pathology. The IL-7-mediated effects were dependent on endogenous IL-6. These attributes of IL-7 have profound implications for its use as a therapeutic in the treatment of chronic viral diseases.

FoxO transcription factors promote cardiomyocyte survival upon induction of oxidative stress.

Transcriptional regulatory mechanisms of cardiac oxidative stress resistance are not well defined. FoxO transcription factors are critical mediators of oxidative stress resistance in multiple cell types, but cardioprotective functions have not been reported previously. FoxO function in oxidative stress resistance was investigated in cultured cardiomyocytes and in mice with cardiomyocyte-specific combined deficiency of FoxO1 and FoxO3 subjected to myocardial infarction (MI) or acute ischemia/reperfusion (I/R) injury. Induction of oxidative stress in cardiomyocytes promotes FoxO1 and FoxO3 nuclear localization and target gene activation. Infection of cardiomyocytes with a dominant-negative FoxO1(Δ256) adenovirus results in a significant increase in reactive oxygen species and cell death, whereas increased FoxO1 or FoxO3 expression reduces reactive oxygen species and cell death. Mice generated with combined conditional deletion of FoxO1 and FoxO3 specifically in cardiomyocytes were subjected to I/R or MI. Loss of FoxO1 and FoxO3 in cardiomyocytes results in a significant increase in infarct area with decreased expression of the antiapoptotic molecules, PTEN-induced kinase1 (PINK1) and CBP/P300-interacting transactivator (CITED2). Expressions of the antioxidants catalase and manganese superoxide dismutase-2 (SOD2) and the autophagy-related proteins LC3II and Gabarapl1 also are decreased following I/R compared with controls. Mice with cardiomyocyte-specific FoxO deficiency subjected to MI have reduced cardiac function, increased scar formation, induction of stress-responsive signaling, and increased apoptotic cell death relative to controls. These data support a critical role for FoxOs in promoting cardiomyocyte survival during conditions of oxidative stress through induction of antioxidants and cell survival pathways.

Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice.

An ageing world population has fuelled interest in regenerative remedies that may stem declining organ function and maintain fitness. Unanswered is whether elimination of intrinsic instigators driving age-associated degeneration can reverse, as opposed to simply arrest, various afflictions of the aged. Such instigators include progressively damaged genomes. Telomerase-deficient mice have served as a model system to study the adverse cellular and organismal consequences of wide-spread endogenous DNA damage signalling activation in vivo. Telomere loss and uncapping provokes progressive tissue atrophy, stem cell depletion, organ system failure and impaired tissue injury responses. Here, we sought to determine whether entrenched multi-system degeneration in adult mice with severe telomere dysfunction can be halted or possibly reversed by reactivation of endogenous telomerase activity. To this end, we engineered a knock-in allele encoding a 4-hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-oestrogen receptor (TERT-ER) under transcriptional control of the endogenous TERT promoter. Homozygous TERT-ER mice have short dysfunctional telomeres and sustain increased DNA damage signalling and classical degenerative phenotypes upon successive generational matings and advancing age. Telomerase reactivation in such late generation TERT-ER mice extends telomeres, reduces DNA damage signalling and associated cellular checkpoint responses, allows resumption of proliferation in quiescent cultures, and eliminates degenerative phenotypes across multiple organs including testes, spleens and intestines. Notably, somatic telomerase reactivation reversed neurodegeneration with restoration of proliferating Sox2(+) neural progenitors, Dcx(+) newborn neurons, and Olig2(+) oligodendrocyte populations. Consistent with the integral role of subventricular zone neural progenitors in generation and maintenance of olfactory bulb interneurons, this wave of telomerase-dependent neurogenesis resulted in alleviation of hyposmia and recovery of innate olfactory avoidance responses. Accumulating evidence implicating telomere damage as a driver of age-associated organ decline and disease risk and the marked reversal of systemic degenerative phenotypes in adult mice observed here support the development of regenerative strategies designed to restore telomere integrity.

Biosynthesis of plant-specific flavones and flavonols in Streptomyces venezuelae.

Recently, recombinant Streptomyces venezuelae has been established as a heterologous host for microbial production of flavanones and stilbenes, a class of plant-specific polyketides. In the present work, we expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides including flavones and flavonols. A plasmid with the synthetic codon-optimized flavone synthase I gene from Petroselium crispum was introduced to S. venezuelae DHS2001 bearing a deletion of the native pikromycin polyketide synthase gene, and the resulting strain generated flavones from exogenously fed flavanones. In addition, a recombinant S. venezuelae mutant expressing a codon-optimized flavanone 3beta-hydroxylase gene from Citrus siensis and a flavonol synthase gene from Citrus unshius also successfully produced flavonols.