Christopher D Morrison
email: morriscd@pbrc.edu

Leptin signaling in brain: A link between nutrition and cognition?

Keywords: hypothalamus, food intake, neuroprotection, hippocampus, cognitive function

Leptin is a protein hormone that acts within the hypothalamus to suppress food intake and decrease body adiposity, but it is increasingly clear that the hypothalamus is not the only site of leptin action, nor food intake the only biological effect of leptin. Instead, leptin is a pleiotropic hormone that impinges on many brain areas, and in doing so alters food intake, motivation, learning, memory, cognitive function, neuroprotection, reproduction, growth, metabolism, energy expenditure, and more. This diversity of function also means that a dysregulation of leptin secretion and signaling can have far reaching effects. To date research on leptin signaling has focused primarily on the hypothalamus, and the result is a relative lack of information regarding the impact of leptin signaling and leptin resistance in non-hypothalamic areas, despite a growing literature implicating leptin in the regulation of neuronal structure and function in the hippocampus, cortex and other brain areas associated with cognition.

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Introduction

The discovery of the protein hormone leptin ushered in an era in which a variety of neural circuits, both within and outside the hypothalamus, have been implicated in the regulation of food intake. Like many hormones, the initial description of leptin as a regulator of food intake and body adiposity focused the study of leptin biology, but it is now clear that leptin does not produce a singular effect (i.e, a reduction in food intake).

This review will highlight the pleiotropic effects of leptin on the brain, first reviewing the role of hypothalamic leptin signaling in the regulation of energy homeostasis, and then toward more recent data implicating leptin signaling in reward, motivation, learning and memory and neuroprotection. These latter effects appear to reflect leptin action in areas outside the hypothalamus, and indicate that leptin may have effects unrelated to its role as a signal of body adiposity.

Leptin as an adiposity signal that acts in the hypothalamus.

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In both humans and animals, physiological mechanisms monitor body adipose mass and react to changes in energy balance by altering ingestive behavior and energy expenditure to buffer against drastic changes in body adiposity and restore body weight and adiposity once the nutritional challenge dissipates [START_REF] Harris | Dynamics of recovery of body composition after overfeeding, food restriction or starvation of mature female rats[END_REF][START_REF] Sims | Endocrine and metabolic effects of experimental obesity in man[END_REF][START_REF] Sims | Experimental obesity in man[END_REF]. The protein hormone leptin represents an afferent signal within this homeostatic mechanism, with leptin being produced and secreted by adipocytes via mechanisms that are sensitive to both the chronic level of body adipose mass as well as current metabolic status [START_REF] Campfield | Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks[END_REF][START_REF] Considine | Serum immunoreactive-leptin concentrations in normal-weight and obese humans[END_REF][START_REF] Halaas | Weight-reducing effects of the plasma protein encoded by the obese gene[END_REF][START_REF] Ostlund | Relation between plasma leptin concentration and body fat, gender, diet, age, and metabolic covariates[END_REF]. Of the brain areas mediating leptin action, the hypothalamic arcuate nucleus (ARC) is most closely associated with leptin's effects on energy homeostasis. The ARC contains at least two populations of leptin-sensitive neurons. The first population contains the potent orexigenic peptides Neuropeptide Y (NPY) and Agouti-Related Protein (AgRP). Central injection of either NPY or AgRP leads to a profound increase in food intake, and acute ablation of the NPY/AgRP neurons in adult animals leads to significant hypophagia and weight loss [START_REF] Gropp | Agouti-related peptide-expressing neurons are mandatory for feeding[END_REF][START_REF] Luquet | NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates[END_REF]. Thus NPY/AgRP neurons represent important drivers of food intake, and leptin's action to suppress food intake is mediated, at least in part, by its ability to inhibit these neurons. Adjacent to NPY/AgRP neurons are a population of neurons containing proopiomelanocortin (POMC) and cocaine and amphetamine regulated transcript (CART). POMC is a large precursor protein which is processed into a variety of smaller products, notably alpha-melanocyte stimulating hormone (α-MSH). Central injection of α-MSH or its stable analog Melanotan II (MTII) produces a marked suppression of food intake, while genetic deletion of POMC results in an obese phenotype [START_REF] Krude | Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans[END_REF][START_REF] Yaswen | Obesity in the mouse model of proopiomelanocortin deficiency responds to peripheral melanocortin[END_REF]. Selective deletion of leptin receptors within POMC neurons also results in an obese phenotype [START_REF] Balthasar | Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis[END_REF], although the effect is much less robust than loss of POMC or deletion of leptin receptors in all neurons. Thus POMC neurons exert a tonic inhibitory influence on food intake, and leptin's action to suppress food intake is at least partly mediated by an inhibition of POMC neurons. A model has therefore emerged in which these two populations of neurons exert opposing effects on feeding behavior and energy metabolism, and simultaneously respond in an opposing fashion to leptin.

The mechanism by which leptin regulates these neurons involves changes in both gene expression and acute membrane activity [START_REF] Cowley | Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus[END_REF][START_REF] Schwartz | Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus[END_REF][START_REF] Spanswick | Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels[END_REF]. These observations suggest that leptin engages multiple signaling pathways to

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impact neuronal function, and recent data clearly supports this observation [START_REF] Munzberg | Molecular and anatomical determinants of central leptin resistance[END_REF]. The leptin receptor is a type 1 cytokine receptor traditionally proposed to signal through the Janus Kinase -Signal Transducer and Activator of transcription (Jak-Stat) pathway. Jak-Stat signaling is critical for leptin action on ARC neurons; leptin induces Stat3 activation in both POMC and NPY neurons [START_REF] Elias | Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area[END_REF][START_REF] Hakansson | Transcription factor STAT3 in leptin target neurons of the rat hypothalamus[END_REF][START_REF] Munzberg | Role of signal transducer and activator of transcription 3 in regulation of hypothalamic proopiomelanocortin gene expression by leptin[END_REF] and loss of leptin's ability to activate Stat3 leads to obesity [START_REF] Bates | STAT3 signalling is required for leptin regulation of energy balance but not reproduction[END_REF][START_REF] Xu | Inactivation of signal transducer and activator of transcription 3 in proopiomelanocortin (Pomc) neurons causes decreased pomc expression, mild obesity, and defects in compensatory refeeding[END_REF]. However, leptin also engages other intracellular pathways, including ERK, PI3K and cAMP/PDE3B [START_REF] Niswender | Intracellular signalling. Key enzyme in leptin-induced anorexia[END_REF][START_REF] Zhao | A phosphatidylinositol 3-kinase phosphodiesterase 3Bcyclic AMP pathway in hypothalamic action of leptin on feeding[END_REF]. Of these, PI3K signaling has received the most attention, as loss of PI3K signaling attenuates leptin-induced inhibition of food intake, regulation of neuropeptide expression, and stimulation of sympathetic nervous system activity [START_REF] Niswender | Intracellular signalling. Key enzyme in leptin-induced anorexia[END_REF][START_REF] Morrison | Leptin inhibits hypothalamic Npy and Agrp gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling[END_REF][START_REF] Rahmouni | Intracellular mechanisms involved in leptin regulation of sympathetic outflow[END_REF].

It should also be noted that these arcuate neurons respond to a variety of signals in addition to leptin.

Insulin also acts in the brain to suppress NPY neurons and stimulate POMC neurons [START_REF] Plum | Central insulin action in energy and glucose homeostasis[END_REF][START_REF] Woods | Insulin as an adiposity signal[END_REF]. Direct brain insulin injections suppress food intake, while selective deletion of brain insulin receptor leads to obesity [START_REF] Bruning | Role of brain insulin receptor in control of body weight and reproduction[END_REF][START_REF] Woods | Chronic intracerebroventricular infusion of insulin reduces food intake and body weight of baboons[END_REF]. Thus insulin and leptin have overlapping physiological effects, and this overlap is mimicked by a degree of overlap in the intracellular signaling pathways they regulate, although this regulation and interaction appears to be highly complex. In addition to leptin and insulin these neuronal populations also detect and respond to a variety of additional nutrient signals, including hormonal signals such at ghrelin and PYY [START_REF] Kohno | Ghrelin directly interacts with neuropeptide-Ycontaining neurons in the rat arcuate nucleus: Ca2+ signaling via protein kinase A and N-type channeldependent mechanisms and cross-talk with leptin and orexin[END_REF][START_REF] Riediger | Peptide YY directly inhibits ghrelin-activated neurons of the arcuate nucleus and reverses fasting-induced c-Fos expression[END_REF], as well as fuels such as glucose, fatty acids and amino acids [START_REF] Cota | Hypothalamic mTOR signaling regulates food intake[END_REF][START_REF] Levin | Glucosensing neurons do more than just sense glucose[END_REF][START_REF] Morrison | Amino acids inhibit Agrp gene expression via an mTORdependent mechanism[END_REF][START_REF] Obici | Central administration of oleic acid inhibits glucose production and food intake[END_REF][START_REF] Van Den Top | Integration of metabolic stimuli in the hypothalamic arcuate nucleus[END_REF].

While the arcuate nucleus has received the most attention as a site mediating leptin's regulation of food intake, leptin appears to also act in additional brain areas both within and outside the hypothalamus. Recent work indicates that leptin activates at least one population of neurons (SF-1 containing neurons) within the ventromedial nucleus (VMN), and that specific deletion of leptin receptor in SF-1 neurons increases food intake and body adiposity [START_REF] Dhillon | Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis[END_REF][START_REF] Bingham | Selective loss of leptin receptors in the ventromedial hypothalamic nucleus results in increased adiposity and a metabolic syndrome[END_REF] . These data are consistent with previous work demonstrating effects of exogenous leptin on VMN neurons and reductions in food intake in response to local VMH leptin injection [START_REF] Jacob | The effect of leptin is enhanced by microinjection into the ventromedial hypothalamus[END_REF][START_REF] Elmquist | Leptin activates neurons in ventrobasal hypothalamus and brainstem[END_REF], although the latter studies are complicated by the close anatomical proximity of the VMN to the ARC. There is also strong evidence that leptin suppresses food intake by acting directly in areas of the caudal brainstem, in particular a

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population of neurons within the nucleus of the solitary tract (NTS). The NTS is classically associated with satiety and meal termination, and local injections of leptin into the brainstem produce reductions in food intake that are similar in magnitude to injections into the forebrain [START_REF] Grill | Distributed neural control of energy balance: contributions from hindbrain and hypothalamus[END_REF][START_REF] Grill | Evidence that the caudal brainstem is a target for the inhibitory effect of leptin on food intake[END_REF].

Lastly, it should it should also be recognized that the suppression of food intake is only one component of leptin's overall effect on energy homeostasis. In essence, the primary effect of brain leptin signaling could be viewed as a reduction in body adiposity, with the suppression of food intake (energy intake) being one component in an array of physiological changes that collectively lead to reduced body adiposity [START_REF] Harris | Leptin--much more than a satiety signal[END_REF][START_REF] Unger | The physiology of cellular liporegulation[END_REF]. One example is the acute effect of leptin on sympathetic outflow and energy expenditure. Relatively early on in the study of leptin it was recognized that the reduction in body weight induced by leptin involved more than changes in food intake [START_REF] Collins | Role of leptin in fat regulation[END_REF], with leptin-deficient animals exhibit increased energy efficiency such that excess adiposity occurs even when hyperphagia is prevented [START_REF] Coleman | Increased metabolic efficiency in obese mutant mice[END_REF]. These observations were extended when it was clearly described that leptin increases energy expenditure and fat oxidation [START_REF] Hwa | Leptin increases energy expenditure and selectively promotes fat metabolism in ob/ob mice[END_REF][START_REF] Wang | Metabolic responses to intracerebroventricular leptin and restricted feeding[END_REF][START_REF] Shimabukuro | Direct antidiabetic effect of leptin through triglyceride depletion of tissues[END_REF], at least in part via an activation of the sympathetic nervous system [START_REF] Dunbar | Intracerebroventricular leptin increases lumbar and renal sympathetic nerve activity and blood pressure in normal rats[END_REF][START_REF] Haynes | Receptor-mediated regional sympathetic nerve activation by leptin[END_REF], and a resultant activation of brown adipose tissue (BAT) [START_REF] Cusin | Chronic central leptin infusion enhances insulin-stimulated glucose metabolism and favors the expression of uncoupling proteins[END_REF][START_REF] Scarpace | Leptin induction of UCP1 gene expression is dependent on sympathetic innervation[END_REF][START_REF] Commins | Norepinephrine is required for leptin effects on gene expression in brown and white adipose tissue[END_REF], although leptin decreases body adiposity and increases energy expenditure even in the absence of BATderived UCP1 [START_REF] Ukropec | Leptin is required for uncoupling protein-1independent thermogenesis during cold stress[END_REF]. These observations are consistent with leptin regulating sympathetic outflow and metabolism in other tissues, particularly skeletal muscle and adipose tissue [START_REF] Shimabukuro | Direct antidiabetic effect of leptin through triglyceride depletion of tissues[END_REF][START_REF] Haynes | Receptor-mediated regional sympathetic nerve activation by leptin[END_REF][START_REF] Commins | Norepinephrine is required for leptin effects on gene expression in brown and white adipose tissue[END_REF][START_REF] Minokoshi | Leptin stimulates fattyacid oxidation by activating AMP-activated protein kinase[END_REF][START_REF] Rosenbaum | Low-dose leptin reverses skeletal muscle, autonomic, and neuroendocrine adaptations to maintenance of reduced weight[END_REF].

In addition to an activation of the sympathetic nervous system, leptin also regulates energy homeostasis is via effects on neuroendocrine hormone secretion [START_REF] Ahima | Leptin and the neuroendocrinology of fasting[END_REF], particularly the secretion of thyroid hormone, gonadotropins, growth hormone, and ACTH. Leptin acts locally in the hypothalamus to stimulate TRH neurons and TRH expression [59] [60, 61], indicating that leptin regulation metabolic rate includes a stimulatory effect on the thyroid axis [START_REF] Lechan | The TRH neuron: a hypothalamic integrator of energy metabolism[END_REF]. Although somewhat less direct, leptin also alters energy balance by regulating reproduction. Decreases in body adiposity and periods of negative energy balance are associated with impaired reproductive function in a variety of species [START_REF] Schneider | Energy balance and reproduction[END_REF], and this effect is principally mediated by an inhibition of hypothalamic gonadotropin releasing hormone (GnRH) secretion [START_REF] Bronson | Food-restricted, prepubertal, female rats: rapid recovery of luteinizing hormone pulsing with excess food, and full recovery of pubertal development with gonadotropin-releasing hormone[END_REF][START_REF] Foster | Effect of restricted nutrition on puberty in the lamb: patterns of tonic luteinizing hormone (LH) secretion and competency of the LH surge system[END_REF][START_REF] Mcshane | Effects of dietary energy on ovarian function, estrogen suppression of luteinizing hormone and follicle-stimulating hormone, and competency of the gonadotropin surge[END_REF]. By acting in the hypothalamus, leptin

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hastens the onset of puberty in mice [START_REF] Chehab | Early onset of reproductive function in normal female mice treated with leptin[END_REF][START_REF] Ahima | Leptin accelerates the onset of puberty in normal female mice[END_REF][START_REF] Yura | Accelerated puberty and late-onset hypothalamic hypogonadism in female transgenic skinny mice overexpressing leptin[END_REF] and improves reproductive function in settings of negative energy balance or leptin deficiency [START_REF] Barash | Leptin is a metabolic signal to the reproductive system[END_REF][START_REF] Gruaz | Chronic administration of leptin into the lateral ventricle induces sexual maturation in severely food-restricted female rats[END_REF][START_REF] Wade | Leptin facilitates and inhibits sexual behavior in female hamsters[END_REF]. Likewise, leptin acts directly within the brain to stimulate gonadotropin secretion [START_REF] Finn | The stimulatory effect of leptin on the neuroendocrine reproductive axis of the monkey[END_REF][START_REF] Henry | Intracerebroventricular infusion of leptin elevates the secretion of luteinising hormone without affecting food intake in long-term food-restricted sheep, but increases growth hormone irrespective of bodyweight[END_REF][START_REF] Nagatani | Evidence for GnRH regulation by leptin: leptin administration prevents reduced pulsatile LH secretion during fasting[END_REF], possibly via the inhibition of NPY neurons [START_REF] Barker-Gibb | The role of neuropeptide Y (NPY) in the control of LH secretion in the ewe with respect to season, NPY receptor subtype and the site of action in the hypothalamus[END_REF][START_REF] Mcdonald | Neuropeptide-Y suppresses pulsatile secretion of luteinizing hormone in ovariectomized rats: possible site of action[END_REF][START_REF] Morrison | Luteinizing hormone and growth hormone secretion in ewes infused intracerebroventricularly with neuropeptide Y[END_REF]. Leptin deficient humans exhibit impaired reproduction and a failure to attain puberty, and this deficit is fully reversed by leptin replacement [START_REF] Farooqi | Clinical and molecular genetic spectrum of congenital deficiency of the leptin receptor[END_REF].

When taken together, these data provide a model for the neural basis of leptin-dependent regulation of energy homeostasis. The traditional view is thus one in which leptin, acting as an adiposity signal, regulates a defined populations of neurons in the hypothalamus to alter food intake and other components of energy homeostasis. While an abundant literature supports the core of this model, it is also apparent that leptin has a more diverse effect on the brain, with implications not only on the regulation of food intake, but on brain areas and neural systems that might be independent of the regulation of energy homeostasis.

Leptin and food intake: Reward and motivation.

It is well recognized that nutritional status influences motivated behavior. For instance, food deprivation increases motivation to attain rewarding stimuli, including food, drugs of abuse, and electrical stimulation of specific brain areas [START_REF] Bell | Food-deprivation increases cocaine-induced conditioned place preference and locomotor activity in rats[END_REF][START_REF] Carr | Feeding, drug abuse, and the sensitization of reward by metabolic need[END_REF][START_REF] Figlewicz | Modulation of food reward by adiposity signals[END_REF]. These observations collectively demonstrate that negative energy balance enhances the desire for rewarding stimuli. Interestingly, leptin attenuates the effects of negative energy balance in these models [START_REF] Figlewicz | Leptin reverses sucrose-conditioned place preference in food-restricted rats[END_REF][START_REF] Fulton | Modulation of brain reward circuitry by leptin[END_REF][START_REF] Shalev | Leptin attenuates acute food deprivation-induced relapse to heroin seeking[END_REF], indicating that leptin does not simply act in the brain to reduce the consumption of food, but that it also acts more generally to reduce the motivation to acquire rewards. Although areas of the hypothalamus (i.e. lateral hypothalamus) are associated with these responses, areas outside the hypothalamus are also involved. The mesolimbic dopamine system is critical for the motivation to obtain pleasurable stimuli [START_REF] Berridge | Parsing reward[END_REF][START_REF] Berridge | Food reward: brain substrates of wanting and liking[END_REF]. Dopaminergic projections from the ventral tegmental area to the nucleus accumbens are particularly important, as manipulation of this dopamine system powerfully influences instrumental performance for and consumption of drugs or food [START_REF] Cannon | Reward without dopamine[END_REF][START_REF] Pecina | Hyperdopaminergic mutant mice have higher "wanting" but not "liking" for sweet rewards[END_REF][START_REF] Wyvell | Intra-accumbens amphetamine increases the conditioned incentive salience of sucrose reward: enhancement of reward "wanting" without enhanced "liking" or response reinforcement[END_REF]. Leptin directly impinges on this system, acting in the VTA to suppress

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food intake and modulate dopamine neurons [START_REF] Figlewicz | Expression of receptors for insulin and leptin in the ventral tegmental area/substantia nigra (VTA/SN) of the rat[END_REF][START_REF] Fulton | Leptin regulation of the mesoaccumbens dopamine pathway[END_REF][START_REF] Hommel | Leptin receptor signaling in midbrain dopamine neurons regulates feeding[END_REF], and also may indirectly influence this system via effects in the hypothalamus. Thus leptin-dependent suppression of food intake involves brain areas associated with the anticipation, memory or motivation, as supported by available data indicating that leptin alters behaviors related to food intake but independent of the actual consumption of food [START_REF] Figlewicz | Leptin reverses sucrose-conditioned place preference in food-restricted rats[END_REF][START_REF] Getchell | Leptin regulates olfactorymediated behavior in ob/ob mice[END_REF]. Lastly, brain imaging studies in leptin deficient humans have highlighted the profound effect that the absence of leptin has on the brain, with leptin deficient humans responding more strongly to food cues (i.e., images of foods) than normal individuals [START_REF] Farooqi | Leptin regulates striatal regions and human eating behavior[END_REF], and leptin treatment altering the neuronal response to food cues in a variety of brain areas [START_REF] Rosenbaum | Leptin reverses weight loss-induced changes in regional neural activity responses to visual food stimuli[END_REF][START_REF] Baicy | Leptin replacement alters brain response to food cues in genetically leptin-deficient adults[END_REF].

Collectively, these data provide a unique view of the expanding role for leptin, and demonstrate that leptin exerts significant effects on food intake via actions via mechanisms that occur prior to and separate from the actual consumption of food, and that these effects are mediated by brain areas that are distinct from the hypothalamus.

Leptin as a signal promoting neuronal survival and cognition

Upon its discovery, leptin was heralded as a hormone acting in the brain as a signal of body adipose mass.

Yet in addition to regulating food intake and body weight homeostasis, leptin also acts within brain areas that are unrelated (at least directly) to an effect on energy homeostasis. Within these brain areas leptin alters neuronal/synaptic function and structure, and influences neuronal survival and proliferation. The result of these effects appears to be an improvement in learning, memory and other forms of cognition and a resistance to insults which impair cognitive performance.

Perhaps the first evidence for a role for leptin in cognition is the recognition that leptin receptors are expressed widely throughout the brain. Although leptin receptors, particularly the signaling form of the receptor (Long form -LepRb) are densely expressed within medial areas of the hypothalamus, leptin receptors are also expressed in many brain areas, including regions associated with learning and memory, such as various cortical regions and the hippocampus [START_REF] Couce | Localization of leptin receptor in the human brain[END_REF][START_REF] Burguera | The long form of the leptin receptor (OB-Rb) is widely expressed in the human brain[END_REF][START_REF] Huang | Localization of leptin receptor mRNA expression in mouse brain[END_REF][START_REF] Shioda | Immunohistochemical localization of leptin receptor in the rat brain[END_REF][START_REF] Ur | Leptin immunoreactivity is localized to neurons in rat brain[END_REF]. Another clue to the role of leptin in the regulation of these

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brains areas stems from the clear effects that both diet and obesity have on cognitive function. Variations in caloric intake or diet composition can produce changes in gene expression within areas of the hippocampus and cortex [START_REF] Martin | Conserved and differential effects of dietary energy intake on the hippocampal transcriptomes of females and males[END_REF], indicating that these brain areas are not insulated from variations in nutritional or metabolic status. Similarly, it is increasingly apparent that alterations in nutritional status can alter cognitive function, and in particular that settings of obesity are associated with decreases in cognitive function [START_REF] Farr | Obesity and hypertriglyceridemia produce cognitive impairment[END_REF]. Based on these and other observations, the key question therefore is whether variations in leptin signaling contribute to changes to metabolic or nutritionally induced alterations in neuronal or cognitive function within areas outside the hypothalamus.

Effects on neuronal/synaptic function

Within these non-hypothalamic areas, leptin acts in part by directly regulating neural function, including local effects at the synapse [START_REF] Harvey | Leptin regulation of neuronal excitability and cognitive function[END_REF]. In rodent models of genetic leptin or leptin receptor deficiency, hippocampal neurons (CA1) exhibit impaired development of long term potentiation (LTP) and depression (LTD), and an associated reduction in CaMK II activity [START_REF] Li | Impairment of long-term potentiation and spatial memory in leptin receptor-deficient rodents[END_REF]. Consistent with this observation, leptin treatment of hippocampal neurons stimulates CaMK II phosphorylation and facilitates the development of LTP [START_REF] Oomura | Leptin facilitates learning and memory performance and enhances hippocampal CA1 long-term potentiation and CaMK II phosphorylation in rats[END_REF]. These changes in LTP and LTD are influenced by leptin dependent regulation of NMDA receptor function [START_REF] Shanley | Leptin enhances NMDA receptor function and modulates hippocampal synaptic plasticity[END_REF][START_REF] Durakoglugil | Leptin induces a novel form of NMDA receptor-dependent longterm depression[END_REF], as leptin enhances NMDA-induced increases in intracellular calcium levels and facilitates NMDA receptor-mediated synaptic transmission [START_REF] Shanley | Leptin enhances NMDA receptor function and modulates hippocampal synaptic plasticity[END_REF]. Leptin treatment in neonatal animals altered the expression of NR1, NR2B, synapsin 2A and synaptophysin in the hippocampus [START_REF] Walker | Long-lasting effects of elevated neonatal leptin on rat hippocampal function, synaptic proteins and NMDA receptor subunits[END_REF]. In addition to altering NMDA receptors, there is also evidence that leptin regulates the function of large-conductance, calcium activated K+ channels (BK channels) [START_REF] Shanley | Leptin inhibits rat hippocampal neurons via activation of large conductance calcium-activated K+ channels[END_REF][START_REF] O'malley | Leptin-induced dynamic alterations in the actin cytoskeleton mediate the activation and synaptic clustering of BK channels[END_REF]. Lastly, these effects of leptin are not limited to hippocampal neurons, as leptin receptors are expressed in cerebellar neurons at the both the somatic plasma membrane and the synapse, and treatment of these neurons with leptin facilitated NR2B NMDA receptor mediated calcium influx [START_REF] Irving | Leptin enhances NR2B-mediated N-methyl-D-aspartate responses via a mitogen-activated protein kinase-dependent process in cerebellar granule cells[END_REF].

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Just as leptin regulates hypothalamic neurons via a variety of signaling pathways, evidence suggests that leptin uses multiple signaling pathways to regulate non-hypothalamic neurons. For leptin dependent effects on synaptic function and activity, a large abundance of data implicates leptin-dependent activation of phosphotidylinositol 3-kinase (PI3K) signaling. PI3K is implicated in leptin-dependent regulation of LTP and LTD, NMDA receptors, and actin cytoskeleton dependent clustering of BK channels [START_REF] Shanley | Leptin enhances NMDA receptor function and modulates hippocampal synaptic plasticity[END_REF][START_REF] Durakoglugil | Leptin induces a novel form of NMDA receptor-dependent longterm depression[END_REF][START_REF] Shanley | Leptin inhibits rat hippocampal neurons via activation of large conductance calcium-activated K+ channels[END_REF][START_REF] O'malley | Leptin-induced dynamic alterations in the actin cytoskeleton mediate the activation and synaptic clustering of BK channels[END_REF]]. Yet other signaling systems are also implicated, including leptin activation of MAPK and Src signaling [START_REF] Shanley | Leptin enhances NMDA receptor function and modulates hippocampal synaptic plasticity[END_REF][START_REF] Irving | Leptin enhances NR2B-mediated N-methyl-D-aspartate responses via a mitogen-activated protein kinase-dependent process in cerebellar granule cells[END_REF]. Taken together, the above data indicates that leptin directly regulates the signaling and synaptic activity of neurons within the hippocampus, cortex and cerebellum. It is thus highly likely that these signaling effects contribute to the cognitive and behavioral effects of leptin that will be discussed below.

Effects on neuronal structure and plasticity

In addition to direct effects on neuronal/synaptic function, leptin also appears to influence neurons by altering their structure and plasticity. Initial evidence for this effect stemmed from the observation that rodent models of leptin deficiency exhibit smaller brains, that these alterations in brain development are evident in utero, and that leptin replacement in these models serves to increase brain size, protein and DNA [START_REF] Ahima | Regulation of neuronal and glial proteins by leptin: implications for brain development[END_REF][START_REF] Steppan | A role for leptin in brain development[END_REF][START_REF] Udagawa | The role of leptin in the development of the cerebral cortex in mouse embryos[END_REF][START_REF] Udagawa | Leptin deficiency causes pycnotic change in fetal cingulate cortical cells[END_REF].

More recent studies provide evidence for neuroproliferative effects within the fetal cortex, and indicate that leptin is associated with a maintenance and differentiation of neural stem cells, glial-restricted progenitor cells and/or neuronal lineage cells [START_REF] Udagawa | Roles of leptin in prenatal and perinatal brain development[END_REF]. Interestingly, similar observations have been made in humans, where fasting plasma leptin levels in normal humans are correlated with grey matter volume in several brain areas [START_REF] Pannacciulli | Relationships between plasma leptin concentrations and human brain structure: a voxel-based morphometric study[END_REF] , and leptin replacement in leptin deficient humans leads to gross changes in brain structure, particularly an increase in grey matter volume in discrete areas [START_REF] Matochik | Effect of leptin replacement on brain structure in genetically leptin-deficient adults[END_REF]. These very provocative observations thus provide strong evidence that leptin, and particularly the absence of leptin, has fundamental effects on brain structure.

In addition to these general effects of leptin, there have been more specific observations of leptin dependent changes in neuronal structure in a variety of brain areas, including the hypothalamus. Leptin

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deficiency appears to reduce the density of axonal projections from leptin sensitive neurons, and this structural defect can be restored by leptin treatment during a critical period of neonatal life [START_REF] Bouret | Trophic action of leptin on hypothalamic neurons that regulate feeding[END_REF]. Consistent with this observation, leptin acts on arcuate nucleus explants to promote neurite outgrowth [START_REF] Bouret | Trophic action of leptin on hypothalamic neurons that regulate feeding[END_REF]. Similar observations have also been made within areas of the hippocampus, cortex and cerebellum. Leptin was shown to act in cerebellar neurons to promote neurite outgrowth and increase the complexity of the neurite arbor [START_REF] Oldreive | Neurotrophic effects of leptin on cerebellar Purkinje but not granule neurons in vitro[END_REF], in hippocampal cells to enhance the motility and density of dendritic filopodia [START_REF] O'malley | Leptin promotes rapid dynamic changes in hippocampal dendritic morphology[END_REF], and in cortical neurons to stimulate growth cone morphogenesis [START_REF] Valerio | Leptin increases axonal growth cone size in developing mouse cortical neurons by convergent signals inactivating glycogen synthase kinase-3beta[END_REF]. Lastly, there is also evidence in the hypothalamus that leptin influences the number of both inhibitory and stimulatory synaptic inputs onto neurons within the arcuate nucleus. Mice that are deficient for leptin exhibit a skewed distribution of synaptic input onto feeding related neurons, and leptin treatment induces a relatively rapid reorganization of the synaptic input to these ARC neurons [START_REF] Pinto | Rapid rewiring of arcuate nucleus feeding circuits by leptin[END_REF]. In summary, these data collectively support a model in which leptin acts to influence neuronal structure and plasticity. As such, these changes provide an additional means by which leptin might regulate neuronal function.

Effects on neuronal survival and proliferation.

It has long been known that various growth factors act on neurons to protect against neurodegeneration and cell death [START_REF] Signore | Leptin neuroprotection in the CNS: mechanisms and therapeutic potentials[END_REF], and a rather large literature has accumulated specifically implicating leptin as a neuroprotective signal [START_REF] Tang | Leptin as a neuroprotective agent[END_REF]. Within a variety of non-neural cells, including cancer cells, leptin has been show to inhibit apoptotic cell death [START_REF] Fujita | Leptin inhibits stress-induced apoptosis of T lymphocytes[END_REF][START_REF] Hoda | Leptin acts as a mitogenic and antiapoptotic factor for colonic cancer cells[END_REF][START_REF] Howard | Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice[END_REF][START_REF] Shimabukuro | Protection against lipoapoptosis of beta cells through leptin-dependent maintenance of Bcl-2 expression[END_REF][START_REF] Somasundar | Differential effects of leptin on cancer in vitro[END_REF]. In neurons in vitro, leptin appears to attenuate cell death induced by the removal of serum or neurotrophines [START_REF] Guo | Leptin-mediated cell survival signaling in hippocampal neurons mediated by JAK STAT3 and mitochondrial stabilization[END_REF][START_REF] Russo | Antiapoptotic effects of leptin in human neuroblastoma cells[END_REF], to improve cell survival in models of ischemic stroke [START_REF] Zhang | Neuroprotective effects of leptin against ischemic injury induced by oxygen-glucose deprivation and transient cerebral ischemia[END_REF][START_REF] Zhang | Leptin protects hippocampal CA1 neurons against ischemic injury[END_REF],

to protect against glutamatergic excitotoxicity [START_REF] Guo | Leptin-mediated cell survival signaling in hippocampal neurons mediated by JAK STAT3 and mitochondrial stabilization[END_REF][START_REF] Dicou | Neuroprotective effects of leptin in vivo and in vitro[END_REF], to protect against oxidative stress [START_REF] Guo | Leptin-mediated cell survival signaling in hippocampal neurons mediated by JAK STAT3 and mitochondrial stabilization[END_REF], and to promote the proliferation of hippocampal progenitor cells [START_REF] Garza | Leptin increases adult hippocampal neurogenesis in vivo and in vitro[END_REF]. These in vitro effects of leptin are replicated by in vivo experiments demonstrating that leptin attenuates the loss of dopamine neurons in a chemically induced model of Parkinson's disease [START_REF] Weng | Leptin protects against 6hydroxydopamine-induced dopaminergic cell death via mitogen-activated protein kinase signaling[END_REF], that leptin deficient mice are more sensitive to middle cerebral artery

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occlusion (MCAO) but that leptin treatment decreases infarct volume and animal recovery following MCAO [START_REF] Zhang | Neuroprotective effects of leptin against ischemic injury induced by oxygen-glucose deprivation and transient cerebral ischemia[END_REF][START_REF] Valerio | Leptin Is Induced in the Ischemic Cerebral Cortex and Exerts Neuroprotection Through NF-{kappa}B/c-Rel-Dependent Transcription[END_REF], and that leptin reduces symptoms of chemically induced epileptic seizures [START_REF] Xu | Leptin inhibits 4-aminopyridine-and pentylenetetrazole-induced seizures and AMPARmediated synaptic transmission in rodents[END_REF]. These effects of leptin appear to stem from leptin's activation of intracellular signaling pathways associated with growth factor signaling, including the activation of Stat3, PI3K/Akt and ERK/MAPK [133-136, 138, 139, 141]. Interestingly some evidence also implicates an effect of leptin on the NF-KappaB/c-Rel signaling pathway, as the ability of leptin to improve function following MCAO was attenuated in c-Rel deficient mice, and leptin stimulated the antiapoptotic Bcl-xl in cortical neurons via a c-Rel dependent mechanism [START_REF] Valerio | Leptin Is Induced in the Ischemic Cerebral Cortex and Exerts Neuroprotection Through NF-{kappa}B/c-Rel-Dependent Transcription[END_REF]. Taken together, these data provide compelling support for leptin as a neuroprotective signal, although the physiological implications of this relationship are not fully clear. Are physiological increases (or decreases) in leptin relevant to neuronal health? Are these effects in any way related to leptin's role as a nutritional signal? Additional work is clearly required to test whether leptin's neuroprotective actions have any role in neurodegenerative processes, particularly those related to nutrition and metabolism.

Effects on cognition and behavior

The above observations provide strong support for a role for leptin in a variety of brain areas and signaling systems that appear, at least on the surface, to be unrelated to its role in energy homeostasis. In addition, these molecular and cellular examples for leptin-dependent effects on synaptic function, neuronal structure, and neuroprotection also provide a mechanistic basis for the remarkable observation that leptin acts within the brain to support and promote cognitive function. The relationship between leptin and cognitive function exists on multiple levels. First, correlational evidence provides a link between altered, particularly reduced, circulating leptin levels and impaired cognition in humans [START_REF] Gunstad | Serum leptin levels are associated with cognitive function in older adults[END_REF][START_REF] Holden | Serum leptin level and cognition in the elderly: Findings from the Health ABC Study[END_REF][START_REF] Huang | Low CSF leptin levels are associated with worse learning and memory performance in HIV-infected men[END_REF], with examples including a correlation between high leptin and improved cognition in the elderly and an association between low leptin levels and impaired learning and memory in HIV infected men. Secondly, genetic models of leptin suggest that leptin deficiency impairs cognitive performance [START_REF] Li | Impairment of long-term potentiation and spatial memory in leptin receptor-deficient rodents[END_REF][START_REF] Paz-Filho | Leptin replacement improves cognitive development[END_REF][START_REF] Ohta | Conditioned taste aversion learning in leptin-receptor-deficient db/db mice[END_REF][START_REF] Winocur | Memory impairment in obese Zucker rats: an investigation of cognitive function in an animal model of insulin resistance and obesity[END_REF]. The most provocative of these is the human example, where a leptin

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deficient individual with reduced cognitive performance at baseline exhibited a marked improvement in neurocognitive tests following leptin treatment [START_REF] Paz-Filho | Leptin replacement improves cognitive development[END_REF]. Lastly, there is also direct evidence for an effect of leptin treatment to improve cognitive performance in rodents [START_REF] Oomura | Leptin facilitates learning and memory performance and enhances hippocampal CA1 long-term potentiation and CaMK II phosphorylation in rats[END_REF]. These experiments particularly implicate the hippocampus, as local injections of leptin into the hippocampus improved memory retention [START_REF] Farr | Effects of leptin on memory processing[END_REF], although at least one experiment has failed to detect an effect of hippocampal leptin on spatial memory [START_REF] Paulus | Central nervous effects of leptin and insulin on hippocampal leptin and insulin receptor expression following a learning task in Wistar rats[END_REF].

While these observations provide a direct link between leptin and cognition, it is currently unclear whether these behavioral effects of leptin are mediated by discrete brain regions (hippocampus vs cortex), or whether these cognitive changes are mediated by any or all of the diverse effects of leptin on synaptic function, neuronal structure and plasticity, and neuroprotection. Nevertheless, these observations provide an exciting new direction for leptin research, and clearly indicate that leptin is much more than an adiposity signal that acts within the hypothalamus to regulate food intake.

Implications for leptin resistance

Shortly after the discovery of leptin, it was recognized that leptin deficiency was not a common cause of obesity. Instead, most obese individuals exhibit high circulating leptin levels coincident with ongoing hyperphagia [START_REF] Considine | Serum immunoreactive-leptin concentrations in normal-weight and obese humans[END_REF]. Obese humans and animals are also relatively unresponsive to exogenously administered leptin, and this syndrome of reduced sensitivity to endogenous and exogenous leptin has been termed leptin resistance [START_REF] El-Haschimi | Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity[END_REF]. Leptin resistance appears to occur on at least two fronts, with the first being a reduced relative transport of circulating leptin across the blood brain barrier [START_REF] Banks | Impaired transport of leptin across the blood-brain barrier in obesity is acquired and reversible[END_REF], and the second a reduction in intracellular signaling downstream of the leptin receptor. The existence of leptin resistance, at least within the hypothalamus, is thus well accepted, and considerable research effort continues to focus on hypothalamic resistance, both to define the cellular mechanisms underlying leptin resistance and to determine the role of hypothalamic leptin resistance in the development of obesity [START_REF] Myers | Mechanisms of leptin action and leptin resistance[END_REF][START_REF] Ladyman | Leptin resistance during pregnancy in the rat[END_REF][START_REF] Morrison | Leptin resistance and the response to positive energy balance[END_REF][START_REF] Scarpace | Elevated leptin: consequence or cause of obesity?[END_REF]. Yet absent within this discussion is the resistance may vary based on biological endpoint. For instance, there is evidence that leptin-dependent effects on sympathetic outflow may persist despite an attenuation of leptin's suppression of food intake [START_REF] Rahmouni | Role of selective leptin resistance in diet-induced obesity hypertension[END_REF][START_REF] Prpic | Differential mechanisms and development of leptin resistance in A/J versus C57BL/6J mice during diet-induced obesity[END_REF].

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These data therefore provide support for the hypothesis that leptin signaling and leptin resistance vary across brain areas.

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Although virtually no work has specifically tested whether leptin resistance develops within brain areas associated with learning, memory or other aspects of cognitive function, a collective consideration of the work reviewed above provides a foundation for this hypothesis. First, increasing age, obesity and metabolic dysfunction each negatively impact these brain areas and lead to impaired cognitive function. Second, leptin signaling, at least within the hypothalamus, is impaired coincident with age, obesity and metabolic dysfunction.

Third, animals with genetically impaired leptin signaling show defects in cognitive function. Fourth, exogenous leptin administration improves cognitive function and protects neurons against damage and degeneration, including an effect of leptin when directly administered into the hippocampus. Taken together, these observations indicate that leptin signaling (or the loss thereof) in the cortex or hippocampus is well positioned to contribute to age and/or diet-associated declines in cognitive function. However, future studies must be designed to clearly test whether and when leptin resistance develops in these non-hypothalamic areas, and then to determine if maintenance of leptin signaling ameliorates the effects of diet or age.

Conclusions

Leptin has received considerable attention as a nutritional signal acting in the hypothalamus to suppress food intake. Yet leptin is a pleiotropic hormone, impinging on a variety of brain areas to influence satiety, motivation, learning, memory, cognitive function, reproduction, growth, metabolism, and additional effects not discussed here [START_REF] Harle | Leptin is a link between adipose tissue and inflammation[END_REF][START_REF] Matarese | Leptin in immunology[END_REF][START_REF] Karsenty | Convergence between bone and energy homeostases: leptin regulation of bone mass[END_REF]. This diversity of function indicates that the dysregulation of leptin secretion and signaling that occurs in settings of both undernutrition and obesity can have far reaching effects. While to date research on leptin resistance has focused primarily on the hypothalamus and emphasized obesity and diabetes, it is increasingly apparent that leptin also acts within areas such as the cortex and hippocampus to influence neuronal function and promote cognition. As such, alterations in leptin signaling within these non-hypothalamic brain areas may provide a unique mechanism linking obesity and diabetes to impaired cognitive function.

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. Leptin sensitive brain areas in the regulation of energy homeostasis and cognition. Leptin-dependent regulation of energy homeostasis is mediated by a complex interaction of brain areas which contribute to leptin's suppression of feeding behavior and stimulation of energy expenditure. Contrastingly, relatively little is known about leptin's potential role in the regulation of cognition, but there is clear evidence for a direct effect of leptin within areas of the cortex and hippocampus. It remains unclear whether an interaction exists between those areas influencing energy homeostasis and those influencing cognition, but the existence of well established neuroanatomical connections indicate that such interactions are likely.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Mesolimbic Brainstem

Energy Homeostasis Cognition

Leptin

Hypothalamus

Cortex, Hippocampus

Mesolimbic Dopamine System

Brainstem

  R I P T consideration that leptin resistance within non-hypothalamic areas might also occur, and that this resistance, by altering neuronal function within brain areas such as the cortex and hippocampus, might also alter behavior and cognition.The study of leptin resistance has focused primarily on the failure of exogenous leptin reduce food intake and activate key signaling molecules (Stat3) within neurons of the hypothalamic arcuate nucleus. This ARCspecific leptin resistance is well described, and occurs in response to high fat diets, age, pregnancy and lactation, seasonal variations, and even chronic leptin exposure[START_REF] Ladyman | Leptin resistance during pregnancy in the rat[END_REF][START_REF] Scarpace | Hypothalamic leptin resistance is associated with impaired leptin signal transduction in aged obese rats[END_REF][START_REF] Pal | Leptin signaling in the hypothalamus during chronic central leptin infusion[END_REF][START_REF] Krol | Photoperiod regulates leptin sensitivity in field voles, Microtus agrestis[END_REF][START_REF] Krol | Altered expression of SOCS3 in the hypothalamic arcuate nucleus during seasonal body mass changes in the field vole, Microtus agrestis[END_REF][START_REF] White | HF diets increase hypothalamic PTP1B and induce leptin resistance through both leptin-dependent and independent mechanisms[END_REF][161]. But does leptin resistance occur in brain areas besides the arcuate nucleus? In addressing the question of region-specific leptin resistance, Munzberg et al.,[START_REF] Munzberg | Region-specific leptin resistance within the hypothalamus of dietinduced obese mice[END_REF] compared leptin-induced phosphorylation of Stat3 in a variety of brain areas in mice fed a high diet, while Ladyman et al.,[START_REF] Ladyman | Region-specific reduction in leptin-induced phosphorylation of signal transducer and activator of transcription-3 (STAT3) in the rat hypothalamus is associated with leptin resistance during pregnancy[END_REF] conducted a similar study in pregnant, leptin resistant rats. These experiments detected a marked reduction of leptin-dependent Stat3 phosphorylation in the ARC in leptin resistant animals, but persistent leptin signaling within other brain areas both within and outside the hypothalamus. The conclusion was therefore that leptin resistance occurs specifically in the hypothalamic arcuate nucleus, and to date few if any studies have followed up on these observations by focusing on leptin resistance in nonhypothalamic areas. However, it should be noted that these anatomical studies were based solely on leptin activation of Stat3, and did not account for changes in other signaling pathways. Yet the majority of studies indicate that leptin-dependent changes in neuroprotection, proliferation, synaptic function and structural plasticity are mediated by pathways such as the PI3K/Akt pathway and ERK/MAPK signaling[108, 109, 111-113, 123, 133-136, 138, 139, 141]. Thus, it remains possible that leptin signaling is altered within these brain areas, despite a normal or persistent regulation of Stat3. Available evidence already exists to suggest that leptin

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M.D./ Z Gunnur Dikmen

Taner Ozgurtas

Sergei M Gryaznov

Ph.D Brittney-Shea Herbert
email: brherber@iupui.edu

Targeting critical steps of cancer metastasis and recurrence using telomerase template antagonists

Keywords: telomerase, metastasis, cancer stem cells, angiogenesis, thio-phosphoramidate oligonucleotides, targeted cancer therapy

Metastasis, tumor relapse, and drug resistance remain major obstacles in the treatment of cancer. Therefore, more research on the mechanisms of these processes in disease is warranted for improved treatment options. Recent evidence suggests that the capability to sustain tumor growth and metastasis resides in a subpopulation of cells, termed cancer stem cells or tumorinitiating cells. Continuous proliferation and self-renewal are characteristics of stem/progenitor cells. Telomerase and the maintenance of telomeres are key players in the ability of stem and cancer cells to bypass senescence and be immortal. Therefore, telomerase inhibitors have the therapeutic potential for reducing tumor relapse by targeting cancer stem cells and other processes involved in metastasis. Herein we review the role of telomerase in the immortal phenotype of cancer and cancer stem cells, targeting telomerase in cancer, and discuss other opportunities for telomerase inhibitors to target critical steps in cancer metastasis and recurrence.

invasion and metastasis [START_REF] Hanahan | The hallmarks of cancer[END_REF]. Advances in diagnostics and treatment have significantly reduced mortality from primary cancers by successfully hitting some of the cancer hallmarks.

Unfortunately, most deaths from cancer are due to metastases and tumor relapse in which the tumors display resistance to standard therapies [START_REF] Fidler | The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited[END_REF]. Within the last few decades, an expansion of research on cancer metastasis, angiogenesis, and replicative potential has led to increased understanding of these processes and new developments in therapeutic targeting and potential biomarkers.

Metastasis is the ability of the cancer to spread from its origin to distant locations within the body and continue to grow [reviewed in 2]. Metastasis depends on the cancer cells acquiring increased motility and invasiveness. More specifically, the main steps in metastasis include: proliferation and angiogenesis; detachment and local invasion into the stroma; detachment and circulation of tumor cells; adherence of tumor cells at secondary sites (on capillary endothelial cells or a basement membrane); extravasation; and, proliferation/survival and angiogenesis at distant sites. As described in more detail later in this review, angiogenesis is an early step in the formation of a metastasis in which extensive vascularization must occur to allow tumors to feed and grow [START_REF] Fidler | The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited[END_REF]. Several growth factors as well as the endothelial cell population play key roles in sustaining angiogenesis and therefore can be subjected to targeting by therapeutics.

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The hallmark of limitless replicative potential in cells is driven by the maintenance of telomeres. Telomeres are hexameric DNA sequences (TTAGGG) found at the ends of chromosomes to prevent end to end fusions and help to maintain genomic stability [START_REF] Blackburn | Telomeres and telomerase: the path from maize, Tetrahymena and yeast to human cancer and aging[END_REF]. The telomere is also a complex structure consisting of a 3′ G-rich overhang, which wraps around to form a protective telomere loop (t-loop), and its associated capping proteins, collectively termed the telosome or shelterin [START_REF] Blackburn | Telomeres and telomerase: the path from maize, Tetrahymena and yeast to human cancer and aging[END_REF][START_REF] Kim | Telomeres, aging and cancer: In search of a happy ending[END_REF][START_REF] Liu | Telosome, a mammalian telomere-associated complex formed by multiple telomeric proteins[END_REF][START_REF] De Lange | Shelterin: the protein complex that shapes and safeguards human telomeres[END_REF]. Telomeres in the majority of normal human cells shorten with each cellular division due to the "end-replication problem." Since DNA polymerase can only initiate synthesis in the 5'→3' direction with a preformed primer, it cannot fully synthesize the 3' terminal end of the lagging strand of linear DNA (which is based on a series of Okazaki fragments). The result is an end-replication problem in which terminal DNA is lost with each round of division. Once telomeres have shortened to a certain length, cells with intact cellular checkpoint controls (e.g., p53/Rb) undergo replicative senescence [reviewed in [START_REF] Blackburn | Telomeres and telomerase: the path from maize, Tetrahymena and yeast to human cancer and aging[END_REF][START_REF] Kim | Telomeres, aging and cancer: In search of a happy ending[END_REF][START_REF] Shay | Hallmarks of telomeres in ageing research[END_REF]. Cells that have lost critical checkpoint controls can bypass senescence and their telomeres continue to shorten. During this crisis period of cell proliferation and death due to short telomeres, cell death usually prevails which can limit cellular lifespan. In rare instances, the presence of critically short or dysfunctional telomeres can result in end fusions and genomic instability, which can lead to a cell acquiring mutations and the ability to progress towards cancer.

Cellular senescence is the ultimate and irreversible loss of replicative capacity occurring in primary somatic cell culture. The shortening of telomeres is thought to function as a replicometer that counts the finite number of cell divisions and triggers replicative senescence in normal diploid cells [START_REF] Passos | DNA damage in telomeres and mitochondria during cellular senescence: is there a connection?[END_REF]. In the majority of human malignancies, maintenance of telomeres is achieved by reactivation of telomerase, an enzyme that counteracts telomere shortening, overcomes senescence and leads to cell immortalization [START_REF] Hiyama | Telomerase detection in the diagnosis and prognosis of cancer[END_REF]. A smaller fraction of tumors (10-

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15%) use an alternative recombination-based mechanism termed ALT (alternative telomere lengthening) pathway [START_REF] Bryan | Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines[END_REF]. Although the mechanisms involved in the decision of a cell to activate either telomerase or ALT are unknown at present, the ALT pathway is more commonly activated in tumors of mesenchymal origin and the presence of the ALT phenotype is associated with poor prognosis [START_REF] Cairney | High level of telomerase RNA gene expression is associated with chromatin modification, the ALT phenotype and poor prognosis in liposarcoma[END_REF].

The activity of telomerase to synthesize and maintain telomeres is reliant on the expression of both the RNA component (hTR) and the catalytic subunit (hTERT), the regulation of which is tightly coordinated on multiple levels by transcriptional, post-transcriptional, translational, and epigenetic mechanisms [START_REF] Cong | Human telomerase and its regulation[END_REF][START_REF] Forsyth | Telomerase and differentiation in multicellular organisms: turn it off, turn it on, and turn it off again[END_REF]. During early embryonic development, hTERT is transcriptionally repressed and subsequently most human somatic cells have undetectable telomerase activity. However, germline cells, stem cells, and progenitor cells exhibit telomerase activity [START_REF] Cong | Actions of human telomerase beyond telomeres[END_REF]. Ectopic expression of hTERT in normal human cells leads to extension of life-span or immortalization of many cell types [START_REF] Bodnar | Extension of life-span by introduction of telomerase into normal human cells[END_REF]. Additionally, constitutive expression of TERT in cancer-resistant mice, engineered by enhanced expression of the tumor suppressors p53, p16, and p19ARF, produces a systemic delay in aging accompanied by extension of the median life span [START_REF] Loba | Telomerase Reverse Transcriptase Delays Aging in Cancer-Resistant Mice[END_REF]. In summary, telomerase plays a critical role in the immortal phenotype of cells that have indefinite proliferative potential, such as stem/progenitor cells (including the putative cancer stem cells) and cancer cells.

This review focuses on the targeting of telomerase and the immortal phenotype in cancer using telomerase template antagonists. One such antagonist, GRN163L, has been shown to act as a potent telomerase inhibitor with additional properties that may affect cancer cell adhesion, new vessel formation, metastasis, and drug resistance. Furthermore, telomerase has been implicated to possess extracurricular activities in replicative aging and cell survival beyond

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telomere maintenance [START_REF] Cong | Actions of human telomerase beyond telomeres[END_REF]. Telomerase may be directly or indirectly involved in apoptosis (via mitochondria homeostasis or membrane potential), DNA damage response, stem cell and organism fitness, and regulation of chromatin architecture or gene expression (including genes involved in cell signaling, cell cycle, metabolism, cell death/differentiation, and growth factors) [START_REF] Cong | Actions of human telomerase beyond telomeres[END_REF]. Therefore, while one should caution the use of telomerase as a universal and safe target for cancer therapy if normal cells may be affected, there may be increased therapeutic potential for telomerase antagonists in the fight against cancer metastasis and recurrence. Based on emerging data, we discuss the potential of telomerase antagonists for targeting cancer stem cells, hypothesized to play a major role in tumor recurrence and metastasis, as well as targeting angiogenesis, a critical step in metastasis.

Targeting telomerase and the immortal phenotype in cancer using telomerase template antagonists

Telomerase and the maintenance of telomeres are key players in the ability of cells to bypass senescence and be immortal. . This process can be disrupted when the extended telomere is dissociated from telomerase [START_REF] Harley | Telomerase and cancer therapeutics[END_REF]. The hTR is not translated into peptides; therefore, oligonucleotides complementary to the hTR template region block the active site of hTERT reverse transcriptase and act as competitive enzyme inhibitors (or template antagonists). Furthermore, since hTERT may have other functions in cell survival independent of its activity [START_REF] Cong | Actions of human telomerase beyond telomeres[END_REF], targeting hTR and designing direct enzymatic inhibitors will maintain hTERT expression in the cell. Since 1995, many laboratories have shown, through extensive research, the proof of principle that targeting hTR results in the inhibition of telomerase activity, progressive telomere shortening, and subsequent death of immortalized or cancer cells in vitro [reviewed in [START_REF] Harley | Telomerase and cancer therapeutics[END_REF][START_REF] Shay | Telomerase therapeutics for cancer: challenges and new directions[END_REF][START_REF] Kelland | Targeting the limitless replicative potential of cancer: the telomerase/telomere pathway[END_REF]. Preclinical animal studies have also demonstrated that telomerase template antagonists are effective in reducing the primary growth in a variety of cancers (lung, prostate, breast, ovarian, glioblastoma, leukemia, lymphoma, myeloma, for example). Furthermore, several studies suggest that telomerase template antagonists can reduce the metastasis index in animal models [START_REF] Dikmen | In vivo inhibition of lung cancer by GRN163L: a novel human telomerase inhibitor[END_REF][START_REF] Hochreiter | Telomerase template antagonist GRN163L disrupts telomere maintenance, tumor growth, and metastasis of breast cancer[END_REF][START_REF] Jackson | Antiadhesive effects of GRN163L--an oligonucleotide N3'-P5' thiophosphoramidate targeting telomerase[END_REF]. However, the mechanisms for the reduction in metastases are not completely understood. The direct telomereshortening effects of GRN163L on the tumor cells themselves might be sufficient to explain these effects on metastasis as massive numbers of cell divisions are required for metastases to form from single invading cells. On the other hand, reduction in cell growth due to targeting the shortest telomere in a cell may result in a bystander effect on the population, or potential telomere-independent effects by the telomerase template antagonists..

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Several methods for targeting the hTR to inhibit telomerase activity have been tested [START_REF] Shay | Telomerase therapeutics for cancer: challenges and new directions[END_REF].

GRN163L: a potent telomerase template antagonist with additional properties?

The telomerase inhibitor GRN163L is a 13-mer oligonucleotide N3′→P5′-thiophosphoramidate lipid conjugate and represents the latest generation of modified oligonucleotides targeting hTR [START_REF] Herbert | Lipid modification of GRN163, an N3'-P5' thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition[END_REF]. The molecule contains two functionally important domains, which are an oligonucleotide thio-phosphoramidate and a lipid group. These moieties are connected via a short amino glycerol linker (Figure 1). The oligonucleotide portion of the conjugate has the nucleoside sequence 5′ -TAGGGTTAGACAA-3′, which is complementary to the template region of hTR and designated as GRN163. The 3′-terminal adenosine contains a 3′amino group (estimated pKa value of ~ 9), which should be mainly protonated under physiological conditions at pH 7.4. All the composing nucleosides contain 3′-amino groups and the non-bridging oxygen atom of the internucleoside phosphoramidate monoester groups uniformly substituted with a sulfur atom (see chemical structure in Figure 2B). Hence, all the internucleoside linkages of this molecule are N3′→P5′-thio-phosphoramidates. This is one of the key distinguishing features of GRN163L compared to other oligonucleotides, containing either natural phosphodiester, or phosphorothioate, or N3′→P5′ phosphoramidate internucleoside groups -see Figures 2C, 2D and 2A respectively, and the text below. GRN163L also contains

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one phosphorothioate diester group tethering the lipid amino glycerol and the oligonucleotide domains (see chemical structure in Figure 1). All the internucleoside thio-phosphoramidate as well as the linking phosphorothioate groups are ionized and negatively charged under physiological conditions. GRN163L is currently synthesized and used as all-sodium salt with molecular weight of 4895 g/mole, and 4609 g/mole for the all-acid form. The compound is also well-soluble either in water or in saline solution, making this compound translatable to the clinic.

The presence of thio-phosphoramidate internucleoside linkages in GRN163L provides for high thermodynamic stability of its complex with the targeted telomerase hTR [START_REF] Pongracz | Oligonucleotide N3′→P5′ thiophosphoramidates: synthesis and properties[END_REF][START_REF] Gryaznov | Oligonucleotide N3'-->P5' phosphoramidates as antisense agents[END_REF][START_REF] Gryaznov | Telomerase inhibitors--oligonucleotide phosphoramidates as potential therapeutic agents[END_REF]. Thus, the melting temperature (T m ) of a duplex formed by GRN163L and a complementary RNA strand is 69.5 °C under close to physiological conditions, in PBS. For comparison, T m of the duplexes formed by either iso-sequential phosphodiester DNA (Figure 2C) or phosphorothioate (Figure 2D) 13-mer oligonucleotides is 45.2 °C or 40.5 °C, correspondingly. Moreover, thiophosphoramidate linkages are very resistant to cellular nuclease hydrolysis, providing for a long half-life of the intact GRN163L in vitro and in vivo. At the same time, in cell-free assays, an N3′→P5′ phosphoramidate oligonucleotide is approximately 5-10 times less potent of a telomerase inhibitor than its iso-sequential thio-phosphoramidate counterpart, GRN163 (compare and2B). This difference in activity was attributed to possible secondary (to Watson-Crick type hydrogen bonding) interactions between sulfur atoms of the sugar-phosphate backbone and the hTERT subunit of telomerase [START_REF] Pongracz | Oligonucleotide N3′→P5′ thiophosphoramidates: synthesis and properties[END_REF][START_REF] Gryaznov | Oligonucleotide N3'-->P5' phosphoramidates as antisense agents[END_REF][START_REF] Gryaznov | Telomerase inhibitors--oligonucleotide phosphoramidates as potential therapeutic agents[END_REF]. Importantly, the presence of the covalently conjugated 5′-palmitic lipid group provides for better cellular up-take and higher bioavailability of GRN163L vs. GRN163 molecules. In the majority of cancer cell lined tested, IC 50 values for telomerase inhibition were at least 5-10 times lower for GRN163L than that for forms an all-strands parallel G-quadruplex structure, which efficiently binds to the HIV envelope glycoprotein gp120, and thus inhibits both cell-to-cell and virus-to-cell infections [START_REF] Ecker | Novel guanosine quartet structure binds to the HIV envelope and inhibits envelope mediated cell fusion[END_REF]. Our preliminary physico-chemical data indicate that, in addition to the formation of thermodynamically stable and sequence-specific duplexes with hTR, GRN163L is capable of forming rather stable G-quadruplex complexes under close to physiological conditions. This was determined using thermal dissociation experiments, where the change in the UV-absorption spectra was monitored at a 295 nm wavelength, which is characteristic for dissociationassociation transitions of guanine quartets (Figure 3). Moreover, the thermal dissociation experiments were conducted in practically iso-molar sodium (NaCl in PBS) and potassium (0.15M KCl) containing buffer solutions, where strong G-quadruplex stabilizing effects of potassium ions were observed. The melting temperatures of the G-quadruplexes formed by GRN163L are 63.9 °C or 89.7 °C in the presence of sodium or potassium ions, respectively (Figure 3). The parental iso-sequential oligonucleotide, GRN163, which lacks the 5′-lipid group, forms noticeably less stable G-quadruplexes, with T m of 49.7 °C and 74.5 °C, respectively, under similar experimental conditions. GRN163L does not form stable G-quadruplex complexes in low

Figures 2A
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ionic strength solutions, i.e., in 10 mM sodium phosphate buffer, pH 7.4, or in de-ionized water (Figure 3).

It is estimated that the special arrangement of the G-quadruplex formed by GRN163L is structurally similar to that formed by ISIS5320, with all four strands being aligned in parallel orientation. This arrangement, we believe, is the most thermodynamically stable oligonucleotide strand orientation, due to potential lipid-lipid stabilizing hydrophobic interactions. High resolution NMR or X-ray structural data is currently not yet available, in part due to the difficulties with crystallization of GRN163L. Formation of this G-quadruplex is driven by the presence of the GGG-motif close to the 5'-terminus in the molecule. The 5′-C 16 palmate lipid groups further stabilizes this four-stranded complex via hydrophobic interactions. A similar type of DNA duplex and triplex stabilizing effects via lipophylic interactions was previously reported [START_REF] Gryaznov | Modulation of oligonucleotide duplex and triplex stability via hydrophobic interactions[END_REF]. The formed duplex with hTR is apparently more thermodynamically stable than the Gquadruplex structure of GRN163L alone.

Additionally, there is an apparent correlation between G-quadruplex forming ability of oligonucleotide phosphoramidates and their effects on cellular morphology and cell adhesion properties. GRN163L has been shown to be capable of inducing a rounding-effect on cellular morphology and prevent cell attachment to a substrate for various cell lines in a telomerase activity-independent manner [START_REF] Jackson | Antiadhesive effects of GRN163L--an oligonucleotide N3'-P5' thiophosphoramidate targeting telomerase[END_REF][START_REF] Dikmen | Telomerase targeted oligonucleotide thio-phosphoramidates in T24-luc bladder cancer cells[END_REF]. However, its mismatch control compound (5'-Palm-TAGGTGTAAGCAA, mismatch sequences in italics), having the same nucleoside composition, but lacking the GGG-motif (and therefore unable to form stable G-quadruplex, with T m of 29.2 °C in PBS), does not affect the cell adhesion and morphology properties under similar experimental conditions. GRN163L was observed to not have an effect on cell growth,

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morphology, or adhesion in normal fibroblasts, nonmalignant cell lines, or normal tissues in animal models [17 and relevant references therein, [START_REF] Jackson | Antiadhesive effects of GRN163L--an oligonucleotide N3'-P5' thiophosphoramidate targeting telomerase[END_REF][START_REF] Dikmen | Telomerase targeted oligonucleotide thio-phosphoramidates in T24-luc bladder cancer cells[END_REF].

Status of telomerase template antagonists in clinical trials

GRN163L is the most advanced direct enzymatic telomerase inhibitor that has reached cancer therapeutic clinical trials [START_REF] Harley | Telomerase and cancer therapeutics[END_REF]. As described earlier, the chemistry of GRN163L allows its potential to be transitioned to the clinic. GRN163L exhibits a higher melting temperature, formation of stable duplexes with single-stranded RNA, resistance to degradation and drug efflux mechanisms (drug resistance), and higher affinity and specificity for its target. GRN163L entered clinical trials for cancer patients in 2005 and currently there are six trials for multiple tumor types (chronic lymphocytic leukemia, multiple myeloma, lung, breast, and advanced solid tumors). Preliminary toxicity findings for the phase I trial of patients with advanced solid tumors in escalating dose cohorts (0.4 to 4.8 mg/kg per week) were common low-grade adverse effects [START_REF] Molckovsky | First-in-class, first-in-human phase I results of targeted agents: Highlights of the 2008 American Society of Clinical Oncology meeting[END_REF]. These effects include fatigue, anemia, gastrointestinal side effects, and increased partial thromboplastin time. Dose-limiting toxicities include thrombocytopenia (4.8 mg/kg) and one death due to unknown causes (at 3.2 mg/kg) [START_REF] Molckovsky | First-in-class, first-in-human phase I results of targeted agents: Highlights of the 2008 American Society of Clinical Oncology meeting[END_REF]. Correlative studies include investigating the number of circulating tumor cells, which may constitute the "seeds" for metastasis, after treatment with GRN163L. Recently, GRN163L entered a Phase I/II clinical trial for patients with locally recurrent or metastatic breast cancer (Trial ID: NCT00732056, www.clinicaltrials.gov). This trial is in combination with paclitaxel, a mitotic inhibitor, and bevacizumab, an anti-angiogenic agent and monoclonal antibody against vascular endothelial growth factor (VEGF). Important to note is that these early stage trials do not address combination effects of GRN163L with other forms of therapy; therefore, the future design of

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combination trials is eagerly anticipated. The use of anti-telomerase agents in clinical trials is still in its earliest stages for complete analyses of outcomes, efficacy, tolerance, and toxicity on normal tissues [START_REF] Molckovsky | First-in-class, first-in-human phase I results of targeted agents: Highlights of the 2008 American Society of Clinical Oncology meeting[END_REF].

New therapeutic potential for telomerase antagonists: targeting critical steps in cancer metastasis and recurrence

Targeting telomerase in cancer stem cells: a potential target for tumor recurrence and metastasis

Recent evidence has suggested that the capability to sustain tumor growth and metastasis may reside in a small number of cells, termed cancer stem cells or tumor-initiating cells [START_REF] Marx | Mutant stem cells may seed cancer[END_REF][START_REF] Pardal | Applying the principles of stem-cell biology to cancer[END_REF][START_REF] Reya | Stem cells, cancer, and cancer stem cells[END_REF].

The cancer stem cell population is proposed to be highly tumorigenic, to contain self-renewal properties, to differentiate to give rise to a diverse progeny and a heterogeneous tumor, to possess efficient efflux mechanisms for drug resistance, and to undergo continuous proliferation.

Cancer stem cells have been recently identified in several cancers based on the expression of stem cell-like cell surface markers as has been historically utilized for hematological cell/cancer cell types [START_REF] Al-Hajj | Prospective identification of tumorigenic breast cancer cells[END_REF][START_REF] Locke | Retention of intrinsic stem cell hierarchies in carcinoma-derived cell lines[END_REF][START_REF] Ponti | Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties[END_REF]. Continuous proliferation and self-renewal upon mitogen stimulation, in addition to telomerase activity, are also characteristics of normal stem/progenitor cells. As a putative stem/progenitor cell population with similar characteristics for indefinite replicative potential, cancer stem cells should be subjected to similar telomere maintenance and telomerase regulation as other stem/progenitor cells. Interestingly, while it has been accepted that stem/progenitor cells express telomerase activity, stem/progenitor cells actually exhibit gradual telomere shortening during their life-span in culture. So while the expression of telomerase in these cells may not be sufficient to prevent telomere shortening, the expression of low levels of

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ACCEPTED MANUSCRIPT 14 telomerase activity in these cells may permit their continuous cell turnover and overall survival or function [START_REF] Cong | Actions of human telomerase beyond telomeres[END_REF]. On the other hand, recent evidence has shown that the adult stem cell compartment is enriched with the longest telomeres of a tissue [START_REF] Flores | The longest telomeres: a general signature of adult stem cell compartments[END_REF]. Consistent with in vitro data, the telomeres were observed to shorten during age in the normal adult stem cell compartment and this coincides with a decrease in stem cell function during aging. Recent findings with breast cancer cells suggest that isolated side populations of CD44 + /CD24 -/low cells, which show similar characteristics and cell surface markers of stem/progenitor cells, also exhibit telomerase activity and short telomere lengths similar to the parental cancer cell line [START_REF] Ponti | Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties[END_REF]. These results suggest that the stem cell-like tumor initiating cells may be prime targets for telomerase inhibitor treatment as part of the therapeutic regimen for cancer.

Li et al. have shown that the intrinsic resistance to chemotherapy in breast cancer patients, which can result in tumor relapse, resides in a small subpopulation of cancer stem cells [START_REF] Li | Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy[END_REF].

Since a characteristic of stem cells is efflux (such as in the efflux of the Hoechst dye) and drug resistance [START_REF] Li | Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy[END_REF][START_REF] Hirschmann-Jax | A distinct "side population" of cells with high drug efflux capacity in human tumor cells[END_REF], it is imperative to develop novel therapy that can effectively enter these cancer stem cells and reduce tumor re-growth. Therefore, it will be important to determine whether oligonucleotide telomerase template antagonists, such as GRN163L, can be effectively taken up by the putative cancer stem cells. Emerging evidence suggests that GRN163L can target drug-resistant cancer cells and restore sensitivity to therapy that may be due to both telomeredependent and independent mechanisms [START_REF] Hochreiter | Telomerase template antagonist GRN163L disrupts telomere maintenance, tumor growth, and metastasis of breast cancer[END_REF][START_REF] Goldblatt | Lipidconjugated telomerase template antagonists sensitize resistant HER2-positive breast cancer cells to trastuzumab[END_REF]. Goldblatt et al. have shown that GRN163L can effectively inhibit telomerase in trastuzumab-resistant breast cancer cells, reduce cell growth in vitro, and restore sensitivity to these breast cancer cells to trastuzumab [START_REF] Goldblatt | Lipidconjugated telomerase template antagonists sensitize resistant HER2-positive breast cancer cells to trastuzumab[END_REF]. Therefore, telomerase inhibitors have the potential to target telomerase, telomere maintenance, drug resistance, and the replicative potential of cancer stem cells for effective cancer therapy.

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Potential for telomerase antagonists to target angiogenesis

Angiogenesis is required for invasive tumor growth and metastasis and constitutes an important point in the control of cancer progression. During tumor progression and metastasis, environmental and genetic changes induce an "angiogenic switch" through upregulation of angiogenic factors or downregulation of angiogenesis inhibitors. Frequently, overexpression of angiogenic factors, such as vascular endothelial growth factor (VEGF), allows tumors to make the angiogenic switch. Anti-angiogenic therapy targets proliferating endothelial cells instead of cancer cells and may reverse this "angiogenic switch" preventing growth of tumor vasculature [START_REF] Folkman | Role of angiogenesis in tumor growth and metastasis[END_REF][START_REF] Pang | Clinical implications of angiogenesis in cancer[END_REF]. The correlation between increased angiogenesis and poor outcome has been established in many types of tumors such as breast cancer, renal cancer, prostate cancer, colon cancer and melanoma [START_REF] Pang | Clinical implications of angiogenesis in cancer[END_REF][START_REF] Gasparini | Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool[END_REF].

Telomerase, which is highly expressed in advanced stages of most cancers, may also contribute to tumor progression through still undefined cell growth-promoting functions [START_REF] Cong | Actions of human telomerase beyond telomeres[END_REF].

Vascular endothelial cells were observed to express hTERT mRNA by in situ hybridization on human astrocytic tumor sections [START_REF] Pallini | Evidence for telomerase involvement in the angiogenesis of astrocytic tumors: expression of human telomerase reverse transcriptase messenger RNA by vascular endothelial cells[END_REF]. Additionally, a significant correlation was observed between the level of hTERT mRNA expression and the proliferation rate of the endothelial cells within the tumor vasculature as well as the histological grade of the tumor. Since hTERT expression by vascular endothelial cells is absent in nonneoplastic granulation tissues and in normal brain, it is possible that diffusible angiogenic factor(s) released by the tumor might act on the endothelial cells [START_REF] Pallini | Evidence for telomerase involvement in the angiogenesis of astrocytic tumors: expression of human telomerase reverse transcriptase messenger RNA by vascular endothelial cells[END_REF][START_REF] Falchetti | Glioblastoma induces vascular endothelial cells to express telomerase in vitro[END_REF]. When telomerase activity was inhibited in glioblastoma cell lines via retroviral transduction of an small-interfering RNA (siRNA) targeting hTERT mRNA,

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decreased cell proliferation, increased apoptosis, and reduced angiogenesis was observed in the hTERT-siRNA expressing xenograft tumors compared with controls [START_REF] Pallini | Telomerase inhibition by stable RNA interference impairs tumor growth and angiogenesis in glioblastoma xenografts[END_REF]. Furthermore, telomerase expression has been shown to correlate with VEGF expression in breast carcinomas [START_REF] Kirkpatrick | The mRNA expression of hTERT in human breast carcinomas correlates with VEGF expression[END_REF]. The increase in hTERT, hTR and VEGF expression during malignancy may also be attributed to hypoxia stimulation of transcription [START_REF] Anderson | Hypoxic regulation of telomerase gene expression by transcriptional and post-transcriptional mechanisms[END_REF]. Zaccagnini et al. showed that telomerase activation is essential for the VEGF dependent vascularization of ischemic tissue [START_REF] Zaccagnini | Telomerase mediates vascular endothelial growth factor dependent responsiveness in a rat model of hind limb ischemia[END_REF].

Adenovirus-mediated transfer of the wild-type hTERT gene in ischemic rats induced development of new capillaries whereas delivery of a dominant negative mutant of TERT failed to promote capillarogenesis. Additionally, the inhibition of telomerase by 3'-azido-3'deoxythymidine (AZT) in VEGF-treated endothelial cells strongly reduced capillary formation and failed to prevent apoptosis [START_REF] Zaccagnini | Telomerase mediates vascular endothelial growth factor dependent responsiveness in a rat model of hind limb ischemia[END_REF]. Similarly, when Falchetti et al. inhibited telomerase activity of human endothelial cells, either by RNA interference (RNAi) or by a dominant negative allele of hTERT, tubule formation was abolished and survival of the endothelial cells in tumor xenografts were significantly reduced [START_REF] Falchetti | Inhibition of telomerase in the endothelial cells disrupts tumor angiogenesis in glioblastoma xenografts[END_REF].

All these findings suggest a contribution of telomerase activity to angiogenesis. The expression of telomerase by tumor vascular cells and endothelial progenitor cell proliferation appears to be an early event during the tumor progression. In this view, anti-telomerase therapy would be effective against new vessel formation by decreasing proliferation of the endothelial progenitor cells in addition to inhibiting neoplastic cell proliferation. It has been demonstrated

that telomerase-deficient (Terc -/-) mice with critically short telomeres show a striking impairment in angiogenesis. Therefore, cancer therapies based on eliciting telomere shortening (i.e., by telomerase inhibition) would contribute to tumor size reduction not only by a cytolytic effect but also by slowing or preventing angiogenesis [START_REF] Franco | Decreased B16F10 Melanoma Growth and Impaired Vascularization in Telomerase deficient Mice with Critically Short Telomeres[END_REF]. Alternatively, the emerging evidence of GRN163L's additional properties as an anti-cancer agent, in addition to its effects as an antitelomerase agent, may also play a role in this context. In support of this concept, the chorioallantoic membrane (CAM) assay can be used to determine the in vivo effects of GRN163L on blood vessel growth. The CAM assay is well established and widely used as an in vivo model to examine angiogenesis and anti-angiogenesis by topical administration of new study agents [START_REF] Ribatti | Effect of exogenous heparin on the vasculogenesis of the chorioallantoic membrane[END_REF]. It is an alternative approach to small animal models and provides a natural environment of growing blood vessels [START_REF] Ribatti | Chorioallantoic membrane capillary bed: A useful target for studying angiogenesis and anti-angiogenesis in vivo[END_REF][START_REF] Storgard | Angiogenesis assays in the chick CAM[END_REF]. Preliminary findings of the CAM assay data suggest that GRN163L at 5-10 mg/kg doses inhibits in vivo angiogenesis without a cytotoxic effect. As shown in Figure 4, physiological angiogenesis was observed in the form of some allantoic vessels in the mismatch control oligonucleotide-treated eggs, whereas a significant reduction in CAM area was appreciable macroscopically in the GRN163L treatment group after 48 hr incubation. Although 5-10 mg/kg GRN163L was well tolerated in these eggs, higher doses (20mg/kg) caused toxicity in 40% of the eggs. Considering these data and its role as a potent telomerase inhibitor, GRN163L may have therapeutic potential as an anti-angiogenic agent in the treatment of cancer; however, further animal studies are needed to show that GRN163L administration is safe and effective.

Conclusions and therapeutic perspectives

Although the main role of telomerase in aging and cancer has been well documented, accumulating evidence suggests that telomerase has important roles in angiogenesis, metastasis and cancer stem cells in addition to its classical function in telomere length maintenance.

Continued research will identify the novel biochemical properties of telomerase which will

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provide additional insights in aging and cancer. GRN163L, as a potential G-quadruplex forming oligonucleotide, may have additional anti-cancer effects beyond its function as a telomerase inhibitor (i.e., anti-metastatic). assay. The anti-angiogenic effect of GRN163L on vascular development was evaluated using the CAM assay [START_REF] Ribatti | Effect of exogenous heparin on the vasculogenesis of the chorioallantoic membrane[END_REF]. For the assay, Atak-S fertilized chicken eggs were obtained from Tavukculuk Institution (Ankara, Turkey) and incubated at 37 o C and 85-90 % relative humidity throughout the experiment. On day six, the eggs were divided in 4 groups, each group consisting of six eggs, and a window was opened in each egg to allow subsequent access to the CAM.

GRN163L and Mismatch control oligonucleotide solutions were prepared in PBS at 3 different concentrations (5 mg/kg, 10 mg/kg, and 20 mg/kg). GRN163L solution (100 μl) was placed on the surface of each CAM (dotted circle, time 0 hr), whereas control CAMs received only mismatch solution, and then all eggs were sealed with transparent tape. Following 48 hrs, CAMs were screened and photographed with a digital camera (Konica Minolta, Tokyo, Japan). The dotted circle at 48 hrs indicates the placement of the solution at time 0 hr.

A

  C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T Introduction In 2000, Hanahan and Weinberg listed "hallmarks" of cancer that must be considered in order to understand the underlying determinants of carcinogenesis: a) self-sufficiency in growth signals; b) insensitivity to growth-inhibitory (anti-growth) signals; c) evasion of programmed cell death (apoptosis); d) limitless replicative potential; e) sustained angiogenesis; and f) tissue

  These include antisense oligonucleotides, peptide nucleic acids (PNAs) and chemically modified PNAs, 2'-O-methyl RNA (2'-O-MeRNA) and 2'-O-methoxyethyl RNA (MOE RNA), phosphorothioate (PS), N3'-P5' phosphoramidate (NP), and N3'-P5' thio-phosphoramidate (NPS) DNA. Further development of NPS oligonucleotides has led to the first telomerase template antagonist, GRN163L, to reach clinical trials.

Figure 5 Figure legends Figure 1 .

 5legends1 Figure legends

Figure 2 .

 2 Figure 2. General chemical structure of internucleoside linkages for representative synthetic oligonucleotides evaluated as telomerase substrates and inhibitors. A)

Figure 3 .

 3 Figure 3. Thermal dissociation and association curves for G-quadruplex formed by GRN163L. Thermal dissociation experiments were conducted using a Cary 1E spectrophotometer. Change in UV absorbance was recorded at 295 nm. A temperature gradient

Figure 4 .

 4 Figure 4. The oligonucleotide telomerase template antagonist GRN163L has potential to reduce vascular development as demonstrated by the chorioallantoic membrane (CAM)

Figure 5 . 5 Outcome: reduce tumor relapse and metastases

 55 Figure 5. Strategies for targeting metastasis and the immortal phenotype of cancer/cancer stem cells using telomerase template antagonists.

Acknowledgements

We thank Dr. Brenda Grimes and the anonymous reviewers for their critical review and suggestions for the manuscript. BH acknowledges research support from the Mary Kay Ash Charitable Foundation, Indiana University Simon Cancer Center, and the Indiana Genomics Initiative (INGEN). INGEN of Indiana University is supported in part by the Lilly Endowment Inc. ZGD acknowledges research support from Hacettepe University Research Foundation and TUBİTAK (The Scientific and Technological Research Council of Turkey).

Herbert receives research support from the Geron Corporation.