Current Drug Targets (v.12, #9)

New therapeutic strategies that focus on disease-relevant molecular events and interfere with specific signaling pathwayshave raised considerable expectations for the treatment of many human diseases. Forkhead box O (FOXO) proteins areemerging as transcriptional integrators of pathways that regulate a variety of cellular processes and have been considered aspotential therapeutic targets for a broad range of human health conditions, including obesity, diabetes, hypertriglyceridemia,aging, infertility, muscle atrophy, inflammation, immune diseases and cancer [1] (see reviews in this issue). The four membersof the mammalian Foxo family of proteins“Foxo1, Foxo3a, Foxo4 and Foxo6”belong to class O of the forkhead/wingedhelix transcription factors (Fox) and function as transcriptional regulators in the cell nucleus [2].Foxo proteins influence the transcription of an ever-growing list of target genes through direct binding to their consensusDNA sequence or via protein-protein interactions with other transcription factors and coactivators [3]. Foxo factors are ancient,evolutionarily conserved targets of insulin-like signaling and have evolved to respond to multiple intracellular and extracellularstimuli with engagement of adaptive gene expression programs. The activity of Foxo factors is regulated by a sophisticatedsignaling network that integrates metabolic, mitogenic and stress signals resulting in a specific pattern of posttranscriptionalmodifications. Phosphorylation, acetylation, methylation, glycosylation and ubiquitination modulate FoxO function and controlnuclearcytoplasmic shuttling, DNA binding and protein-protein interactions. Foxo proteins have been recently established asbona fide tumor suppressors and correspondingly the abrogation of Foxo function is a key feature of many tumor cells.Contrary to other tumor suppressors like p53 or PTEN whose functions are abrogated via genetic or epigenetic changes,inactivation of FOXOs occurs mostly due to posttranscriptional up-regulation of their inhibitory inputs. That offers a widerange of possibilities for restoring FOXO activity e.g. with small molecule inhibitors targeting up-regulated FOXO repressors[4-6]. However, as Foxo factors regulate a broad variety of cellular functions some of which are seemingly opposing such asapoptosis and resistance against oxidative stress, their therapeutic activation or inhibition may lead to undesirable clinicaloutcome. Therapeutic interference with FOXO functions might have both beneficial effects in one disease setting while havingdeleterious effects in another. A number of potential therapeutic limitations could arise particularly from chronic modulation ofFOXO function. Selective inhibition of FOXO1 activity in the liver might ameliorate metabolic abnormalities associated withobesity and diabetes, while at the same time promote hepatic fibrosis (see Kim et al., this issue [7]). Therapies aimed atreactivating FOXO activity could provide exciting opportunities for innovative treatments for cancer patients [8]. Accordingly,the cytostatic and cytotoxic effects of a diverse spectrum of anti-cancer drugs are mediated through the activation of FOXOfactors. Paradoxically, FOXO proteins also contribute to drug resistance by driving the expression of genes important for drugefflux as well as DNA repair and cell survival pathways in drug resistant cancers (see Wilson et al., this issue). In addition,while FOXO proteins exert many if not all the properties attributed to tumor suppressors they may promote the maintenance ofthe very few tumor-initiating cells that regenerate the disease [9, 10] (see Ghaffari et al., this issue).Based on our current knowledge on the regulation and functions of FOXO proteins the success of their exploitation in theclinic critically relies on the capability to engage specific FOXO-dependent transcriptional programs. The development oftherapeutic agents, drug combinations and treatment regimes that modulate specific subsets of FOXO target genes at the rightplace at the right time constitute a major challenge for basic and drug discovery research.In this special issue of CDT leading experts in the field discuss controversies and advances in our understanding of FOXObiology and its biomedical applications. Huarui Lu and Haojie Huang focus on the therapeutic potential of the FOXO familymember FOXO1 for the treatment of diseases such as cancer, diabetes and muscular atrophy. Although Foxo1 null mice exhibitembryonic lethal phenotypes with marked evidence of vascular defects precluding the analysis of the role of this isoform in adult tissues several genetically modified mouse models have been developed that shed light on the role of FOXO1 in humandiseases.Dae Hyun Kim, Ting Zhang, Steven Ringquist and H. Henry Dong discuss the hypothesis that selective inhibition ofFOXO1 in the liver would ameliorate hypertriglyceridemia, a common lipid disorder associated with obesity and type 2 diabetisand a hallmark of metabolic syndrome.Miranda S. C. Wilson, Jan J. Brosens, Helma D. C. Schwenen and Eric W.-F. Lam review evidence indicating that targetingthe FOXO-FOXM1 axis could be a viable strategy for treatment of cancer and for overcoming drug resistance.Saghi Ghaffari, Safak Yalcin, Maite Rielland and Xin Zhang describe the critical relationship between the FOXO drivendetoxification of reactive oxygen species (ROS) and the regulation of quiescence in stem cell populations including tumorinitiatingcells and explore the therapeutic implications.....

FOXO1: A Potential Target for Human Diseases by Huarui Lu, Haojie Huang (1235-1244).
The forkhead box O (FoxO) transcription factors are known to be involved in many physiological andpathological processes including apoptosis, cell cycle arrest, stress resistance, glucose metabolism, cellular differentiationand development, and tumor suppression. The environmental cues, such as growth factors, nutrients, oxidative stress andirradiation, can either positively or negatively modulate FoxO proteins’ activities, thereby ensuring distinctivetranscription programs in the cell. The potent activities of FoxOs are tightly controlled by multiple mechanisms, whichinclude posttranslational modification such as phosphorylation, acetylation, methylation and ubiquitination, subcellularlocalization, and direct protein-protein interaction. Mounting evidence suggests that the human FOXO1 protein, afounding member of the FoxO family is likely involved in carcinogenesis, diabetes and other human diseases. Here wegive an overview of most recent findings regarding the regulation and function of FoxO1, its potential role in humandiseases and useful animal models for functional studies on FoxO1. Prospective ways in which the discoveries from thebasic research of FoxO1 can be utilized for drug targeting and development of novel therapeutics for human diseases arealso discussed.

Targeting FoxO1 for Hypertriglyceridemia by Dae Hyun Kim, Ting Zhang, Steven Ringquist, H. Henry Dong (1245-1255).
Hypertriglyceridemia is characterized by increased production and decreased clearance of triglyceride-richlipoproteins including very low-density lipoprotein (VLDL) and chylomicron. Due to its proatherogenic profile,hypertriglyceridemia contributes to the development of atherosclerosis and coronary artery disease. While thepathophysiology of hypertriglyceridemia remains poorly understood, its close association with obesity and type 2 diabetesimplicates insulin resistance in the pathogenesis of hypertriglyceridemia. However, the molecular basis linking insulinresistance to hypertriglyceridemia remains elusive. Preclinical studies show that FoxO1 plays a pivotal role in controllinginsulin-dependent regulation of microsomal triglyceride transfer protein (MTP) and apolipoprotein C-III (ApoC-III), twokey components that catalyze the rate-limiting steps in the production and clearance of triglyceride-rich lipoproteins.Under physiological conditions, FoxO1 activity is inhibited by insulin. In insulin resistant states, FoxO1 becomesderegulated, contributing to unbridled FoxO1 activity in the liver. This effect contributes to hepatic overproduction ofVLDL and impaired catabolism of triglyceride-rich particles, accounting for the pathogenesis of hypertriglyceridemia.These data spur the hypothesis that selective inhibition of FoxO1 activity in the liver would improve triglyceridemetabolism and ameliorate hypertriglyceridemia. In this article, we review the role of FoxO1 in insulin action and lipidmetabolism, and evaluate the therapeutic potential of targeting FoxO1 for treating hypertriglyceridemia in insulin resistantsubjects with obesity and type 2 diabetes.

FOXO and FOXM1 in Cancer: The FOXO-FOXM1 Axis Shapes the Outcome of Cancer Chemotherapy by Miranda S.C. Wilson, Jan J. Brosens, Helma D.C. Schwenen, Eric W.-F. Lam (1256-1266).
FOXO transcription factors, functioning downstream of the PI3K-PTEN-AKT (PKB) signalling cascade, areessential for cell proliferation, differentiation, DNA damage repair and apoptosis. Recent research indicates that therelated transcription factor FOXM1 is a direct target of repression by FOXO proteins. Inactivation of FOXO oroverexpression of FOXM1 is associated with tumorigenesis and cancer progression. In addition, the cytostatic andcytotoxic effects of a diverse spectrum of anti-cancer drugs, such as paclitaxel, doxorubicin, lapatinib, gefitinib, imatiniband cisplatin, are mediated through the activation of FOXO3a and/or the inhibition of its target FOXM1. Paradoxically,FOXO proteins also contribute to drug resistance by driving the expression of genes important for drug efflux as well asDNA repair and cell survival pathways in drug resistant cancers. Given its pivotal roles in drug sensitivity as well asresistance, targeting the FOXO-FOXM1 axis could be a viable strategy for treatment of cancer and for overcoming drugresistance. Studying the expression profiles of the components of the FOXO-FOXM1 axis and their cofactors in cancerpatients might also help to predict and monitor their clinical response to chemotherapy. A better understanding of themechanism by which FOXO and FOXM1 are regulated, as well as their roles in drug sensitivity and resistance, mayrender these proteins crucial prognostic markers and therapeutic targets for breast cancer and other malignancies.

Regulation and Function of FoxO Transcription Factors in Normal and Cancer Stem Cells: What Have We Learned? by Xin Zhang, Maite Rielland, Safak Yalcin, Saghi Ghaffari (1267-1283).
Forkhead FoxO transcription factors exert critical biological functions in response to genotoxic stress. Inmammals four FoxOs proteins are known. FoxOs induce cell cycle arrest, repair damaged DNA, or initiate apoptosis bymodulating genes that control these processes. In particular, FoxO proteins are critical regulators of oxidative stress bymodulating the expression of several anti-oxidant enzyme genes. This function of FoxO is essential for the regulation ofstem and progenitor cell pool in the hematopoietic system and possibly in cellular systems. Overall functions of FoxOs areconsistent with their role as tumor suppressors as has been shown in animal models. As such, FoxOs are suppressed invarious cancer cells. However, recent reports strongly suggest that FoxOs are critical for the maintenance of leukemicstem cells. The diverse functions of FoxOs are orchestrated by tight regulations of expression and activity of its familymembers. Here we discuss the recent progress in understanding the function of FoxOs specifically in normal and cancerstem cells and what is known about the regulation of these proteins in various cell types and tissues including in thephysiological setting of primary cells in vivo. These studies underscore the importance of regulation of FoxO proteins andwhether these factors play distinct or redundant functions. Understanding how FoxOs are modulated is critical fordevising novel therapies based on targeted restoration/or inhibition of FoxO function in cancer and in other diseased cellsin which FoxOs have a key function.

Forkhead O transcription factors (FOXO) are critical for the regulation of cell cycle arrest, cell death, and DNAdamage repair. Inactivation of FOXO proteins may be associated with tumorigenesis, including breast cancer, prostatecancer, glioblastoma, rhabdomyosarcoma, and leukemia. Accumulated evidence shows that activation of oncogenicpathways such as phosphoinositide-3-kinase/AKT/IKK or RAS/mitogen-activated protein kinase suppresses FOXOtranscriptional activity through the phosphorylation of FOXOs at different sites that ultimately leads to nuclear exclusionand degradation of FOXOs. In addition, posttranslational modifications of FOXOs such as acetylation, methylation andubiquitination also contribute to modulating FOXO3a functions. Several anti-cancer drugs like paclitaxel, imatinib, anddoxorubicin activate FOXO3a by counteracting those oncogenic pathways which restrain FOXOs functions. In thisreview, we will illustrate the regulation of FOXOs and reveal potential therapeutics that target FOXOs for cancertreatment.

FOXO Transcription Factors and their Role in Disorders of the Female Reproductive Tract by Mark Christian, Eric W.-F. Lam, Miranda S.C. Wilson, Jan J. Brosens (1291-1302).
All fundamental reproductive events in the human ovary and uterus, including ovulation, implantation andmenstruation, are dependent upon profound tissue remodelling, characterized by cyclical waves of cell proliferation,differentiation, recruitment of inflammatory cells, apoptosis, tissue breakdown and regeneration. Although the rise andfall in ovarian hormones, estradiol and progesterone, orchestrate these reproductive events, FOXO transcription factors,an evolutionary conserved subfamily of forkhead transcription factors, have emerged major downstream effectormolecules, capable of integrating hormonal cues with a variety of stress, growth factor and cytokine signal transductionpathways. The ability of FOXOs to regulate seemingly opposing cellular responses, ranging from cell cycle arrest andoxidative stress responses to differentiation and apoptosis, renders these transcription factors indispensable for cyclictissue remodelling in the reproductive tract. Aberrant expression or perturbed activity of FOXO transcription factors areincreasingly linked to prevalent reproductive disorders, such as endometriosis, endometrial cancer, primary ovarianinsufficiency and pregnancy failure, which in turn highlights their potential as therapeutic targets.

The Forkhead Box O (Foxo) proteins represent an evolutionarily conserved family of transcription factors thatplay an important role in regulating processes including metabolism, longevity, and cell death/survival. How is it that asingle transcription factor can initiate such divergent cellular responses? We will review the evidence that specific patternsof post-translational modifications play a key role in directing Foxo into various transcriptional readouts. This regulationappears to take on a two tiered regulatory model; with a group of well defined post-translational modifications regulatingnuclear localization and transcriptional activity while a second set of modifications regulate the transcriptional specificityof Foxo.

The promotion of cellular survival, dedifferentiation, and uncontrolled proliferation via the suppression ofapoptotic effectors is a fundamental characteristic of tumor cells. As substrates that are negatively regulated by oncogenicsignaling cascades driven by AKT, SGK (serum- and glucocorticoid-inducible kinase), IkB kinase (IKK), ERK, andcyclin-dependent kinases (CDK), forkhead box-class O (FOXO) transcription factors have emerged as bona fide tumorsuppressors. These transcription factors indeed regulate a variety of cellular responses and themselves are regulated byreversible phosphorylation, acetylation, ubiquitination and miRNAs. This review will discuss our current understanding ofmechanisms for FOXO regulation and the potential implications for therapeutically restoring FOXO transcriptionalactivity.

The Complex Biology of FOXO by Maria Monsalve, Yolanda Olmos (1322-1350).
FOXO transcription factors control proliferation, apoptosis, differentiation and metabolic processes. Loss ofFOXO function has been identified in several human cancers, and results in increased cellular survival and apredisposition to neoplasia, especially in epithelial cancer. FOXO factors are therefore bona fide tumor suppressors, andtheir potential use as therapeutic targets in cancer has been a matter of debate. Importantly, FOXO factors can alsopositively regulate cell survival through the activation of several detoxification genes, complicating its putativetherapeutic potential. Targeting of FOXO factors has also been proposed for the treatment of metabolic dysfunctions suchas diabetes mellitus, immunological disorders and neurodegeneration, as well as for the prevention of aging bymaintaining the hematopoyetic stem cells niche. But again, data has accumulated that cautions against the potential use ofthe FOXO activators in these settings. Therefore, greater understanding of the regulation of FOXO target specificity isstill needed to boost its use as a therapeutic target.The four members of the FOXO family (FOXO1, FOXO3A, FOXO4 and FOXO6) have distinct but overlapping cellularfunctions, although they seem to bind a common set of DNA sites. This fact together with the observation that FOXOs areonly partially dependent on their DNA binding activity to regulate their target genes highlights the fact that the interactionof the FOXOs with other transcription factors is crucial for the FOXO-mediated transcriptional programs.In this review, we provide an overview of recent progress in the understanding of the modulation of FOXO activity andtarget specificity by transcription factors and coactivators.

Dietary Nitrite in Nitric Oxide Biology: A Redox Interplay with Implications for Pathophysiology and Therapeutics by Barbara S. Rocha, Bruno Gago, Cassilda Pereira, Rui M. Barbosa, Silvina Bartesaghi, Jon O. Lundberg, Rafael Radi, Joao Laranjinha (1351-1363).
Until recently, nitrite has been considered a stable and inert metabolite of nitric oxide (•NO) metabolism. Thisview is now changing as it has been shown that nitrite can be reduced back to •NO and thus one may consider a reversibleinteraction regarding •NO:nitrite couple. Not only physiological regulatory actions have been assigned to nitrite but alsomay represent, in addition to nitrate, the largest •NO reservoir in the body. This notion has obvious importance whenconsidering that •NO is a ubiquitous regulator of cell functions, ranging from neuromodulation to the regulation ofvascular tone. Particularly in the stomach, following ingestion of nitrate and food or beverages-containing polyphenols, arich chemistry occurs in which •NO, •NO-derived species and nitroso or nitrated derivatives may be formed. Most of thesemolecules may play an important role in vivo. For instance, it has been shown that polyphenol-catalyzed nitrite reductionto •NO may induce local vasodilation and that ethanol (from wine) reacts with •NO-derived species yielding nitrosoderivatives endowed with •NO-donating properties. Thus, this review reveals new pathways for the biological effects ofdietary nitrite encompassing its interaction with dietary components (polyphenols, red wine, lipids), yielding productswith impact on human physiology and pathology, namely cardiovascular, urinary and gastrointestinal systems. Noveltherapeutic strategies are therefore expected to follow the elucidation of the mechanisms of nitrite biology.

The advent of the biological era has seen many improvements in the management of inflammatory boweldisease (IBD). These agents, however, are not a ubiquitous panacea as they are neither universally available nor are theyuniversally efficacious in the short or long-term. There is, therefore, still a need for other therapies and it is important toremember about the medications that have been effective in the past. The use of azathioprine and 6-mercoptopurine hasbeen the mainstay of long-term therapy for many IBD patients for many years. Their role as steroid sparing agents and inthe maintenance of remission is well recognized, and with the advent of metabolite testing their use has been refined.Methotrexate is a second line immunomodulator with less impressive data but still with observed benefits in Crohn’sdisease (CD) and two newer immunosuppressive agents, mycophenylate mofetil and tacrolimus have sparked someinterest as they appear to be efficacious in some patients.As IBD is a chronic incurable condition that primarily presents in young patients, the treating clinician’s goal is to induceand maintain long-term remission. So when one agent is ineffective, or unavailable, other agents need to be considered.This review aims to provide clinicians with practical and up to date knowledge about the use of the immunomodulators inthe management of IBD, which is vital in order to offer the best management for their patients.