Silent information regulation 2 inhibition contributes to reduced α-synuclein toxicity. Pharmacological inhibition or siRNA-mediated inhibition of SIRT2 decreases the number of α-synuclein inclusions in a cellular model of PD (Outeiro et al., 2007). SIRT2 deacetylates α-synuclein, and knockdown of SIRT2 suppresses α-synuclein aggregation and toxicity in a mouse model of PD (de Oliveira et al., 2017). The acetylation of α-synuclein promotes the clearance of α-synuclein inclusions via autophagy and exerts neuroprotective effects in cultured neurons, whereas blocking the acetylation of α-synuclein causes the loss of nigral dopaminergic neurons. Other studies showed that deletion of SIRT2 reduces MPTP-induced neuronal damage in TH-positive cells through increased acetylation of FOXO3a and reduces expression of a proapoptotic factor, Bim, thus blocking the apoptotic pathway (Liu et al., 2012). These results suggest that SIRT2 inhibition may have beneficial effects for PD.

#### Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons in the brain and spinal cord. ALS is a uniformly fatal disorder and causes death about 2–5 years after onset, mostly because of respiratory paralysis. Mutations in various genes have been identified in ALS, including superoxide dismutase (SOD1) (Rosen et al., 1993) and TAR DNA-binding protein 43 (TDP43) (Arai et al., 2006; Neumann et al., 2006; Kabashi et al., 2008; Sreedharan et al., 2008; Van Deerlin et al., 2008). It has been reported that sirtuin expression is altered in both mouse models of as well as patients with ALS (Kim et al., 2007; Lee et al., 2012; Korner et al., 2013; Buck et al., 2017). Treating with resveratrol or lentivirus-mediated forced expression of SIRT1 protects against neurodegeneration in the SOD1 mutant mouse model of ALS (Kim et al., 2007; Han et al., 2012; Mancuso et al., 2014). SOD mutant mice that show consistent pan-neural expression of exogenous SIRT1 have extended lifespans compared to those without this expression (Watanabe et al., 2014).

SIRT3 has also received attention for its role in ALS. Expression of SIRT3 decreases in the spinal cord and brain stem during the progression of diseases in SOD1G93A mouse, a widely used mouse model of ALS (Buck et al., 2017). In contrast, the mitochondrial isoform of SIRT3 is increased in the muscle and spinal cords of SOD1G93A mice (Salvatori et al., 2017). SIRT3 expression attenuates mitochondrial fragmentation and cell death in neurons from SOD1G93A mice (Song et al., 2013). Although inhibition of SIRT2 is thought to have beneficial effects on neurodegenerative diseases such as PD, the deletion of SIRT2 did not appear to effect the disease course of SOD1G93A mice (Taes et al., 2013). Additional studies will be invaluable to unravel the role of other sirtuins in ALS.

#### SIRT1 Activator and Clinical Trials

Since accumulating studies have demonstrated the protective effects of SIRT1 against neurodegenerative diseases, potent SIRT1 activators are currently in clinical trials1 . Clinical trials that are both underway and have been completed have used various proprietary formulations of resveratrol (i.e., SRT501) to treat AD. In one study, treating AD subjects with resveratrol for 52 weeks stabilized the progressive decline in CSF Aβ40 and plasma Aβ40 compared to placebo (Turner et al., 2015). Resveratrol decreased CSF MMP9 and induced adaptive immunity (Moussa et al., 2017), suggesting that resveratrol is beneficial for AD subjects. In addition, there is currently a clinical trial testing resveratrol in HD subjects2 . However, since SIRT1 is expressed in various tissues, the risk of adverse effects could be considered. To ensure its safety, additional resveratrol and other SIRT1 activator clinical studies are warranted.

### CONCLUSION

Sirtuins mediate diverse functions in the CNS. The evidence of the beneficial effects of SIRT1 obtained from animal models and human studies imply that SIRT1 activation can be a potential therapeutic treatment for neurodegenerative diseases. Neuronal degeneration in various traumatic injury and neurological disorders, such as SCI, stroke, and AD, is often accompanied by inflammation. It has been reported that activation of SIRT1 also contributes to the suppression of inflammatory responses. Therefore, SIRT1 activation would be a unique strategy in that it is able to control both neurons and inflammatory cells.

1https://clinicaltrials.gov/ct2/results?cond=&term=resveratrol&cntry=&state= &city=&dist=

2https://clinicaltrials.gov/ct2/results?cond=Huntington+Disease&term= resveratrol&cntry=&state=&city=&dist=

However, there have also been reports in several animal models of neurodegenerative diseases that SIRT1 activation does not have neuroprotective effects. These conflicting results may be due to a number of factors, including the cell-type-specific alteration of SIRT1 expression following injury.

Understanding the more detailed molecular mechanisms of sirtuins in regulating neuroprotection and degeneration as well as the precise expression patterns of sirtuins following neuronal pathology will contribute to the development of novel anti-neurodegenerative therapeutics. For example, combination treatment with SIRT1 activators and NAD may provide a synergetic strategy that contributes to neuroprotection.

The observation that SIRT2 activity promotes neurodegeneration in a PD model suggests that sirtuins have family-dependent functions. Further studies linking neurodegenerative diseases and sirtuin members, especially

#### REFERENCES

SIRT4, 5, and 7, will be helpful to reveal the tissue or diseasespecificity of the role of sirtuins. Highly selective compounds targeting specific sirtuins may therefore serve as attractive candidates for a variety of neurological conditions.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### FUNDING

This work was supported by KAKENHI from the Japan Society for the Promotion of Science (JSPS) (17H05767) to YF.

mouse models. Front. Mol. Neurosci. 10:156. doi: 10.3389/fnmol.2017. 00156

by its nuclear translocation. Proc. Natl. Acad. Sci. U.S.A. 105, 15599–15604. doi: 10.1073/pnas.0800612105

amyotrophic lateral sclerosis via HSF1/HSP70i chaperone system. Mol. Brain 7:62. doi: 10.1186/s13041-014-0062-1

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Fujita and Yamashita. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Essential Role of SIRT1 in Hypothalamic-Pituitary Axis

#### Masaaki Yamamoto1 and Yutaka Takahashi 2 \*

1 Pituitary Center, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, United States, 2 Division of Diabetes and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan

The endocrine system plays an essential role in the physiological adaptation to malnutrition. The adaptive response of various hormones directs the energy utilization toward the survival functions and away from growth and reproduction. Particularly, the hypothalamic pituitary axis plays an integral and a central role in the regulation of endocrine organs. Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase that is activated in response to calorie restriction (CR). SIRT1 is involved in cellular processes via the deacetylation of histone as well as various transcription factors and signal transduction molecules and thereby modulates the endocrine/metabolic functions. There is much evidence to demonstrate clearly that SIRT1 in the hypothalamus, pituitary gland, and other target organs modifies the synthesis, secretion, and activities of hormones and in turn induces the adaptive responses. In this review, we discussed the role of SIRT1 in the hypothalamic pituitary axis and its pathophysiological significance.

#### Edited by:

Yang Yang, Northwest University, China

#### Reviewed by:

Akira Shimatsu, National Hospital Organization (NHO), Japan Jackson Cioni Bittencourt, Universidade de São Paulo, Brazil Wei-jiang Zhao, Shantou University Medical College, China

> \*Correspondence: Yutaka Takahashi<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology

Received: 27 June 2018 Accepted: 24 September 2018 Published: 23 October 2018

#### Citation:

Yamamoto M and Takahashi Y (2018) The Essential Role of SIRT1 in Hypothalamic-Pituitary Axis. Front. Endocrinol. 9:605. doi: 10.3389/fendo.2018.00605 Keywords: SIRT1, adaptation, calorie restriction, hypothalamus, pituitary

### INTRODUCTION

#### The Physiological Role of Sirtuin 1 (SIRT1)

SIR2 is the first sirtuin protein to be discovered in Saccharomyces cerevisae (yeast) (1). SIR2 orthologs are highly conserved throughout various species including mammals, plants, bacteria, worms, flies, and fishes. In mammals, the seven SIR2 orthologs are termed SIRT1-7 and are presumed to be ubiquitously expressed in all the tissues (2). Sirtuins were initially reported as protein deacetylases and ADP ribosyltransferases (3, 4). However, subsequent analyses revealed that sirtuins exhibit various enzymatic activities. For instance, SIRT5 exhibits lysine-desuccinylase and -demalonylase activities (5, 6). These enzymatic activities of sirtuins are generally activated in response to CR by the utilization of nicotine adenine dinucleotide (NAD+) as the co-substrate.

Regarding the intracellular localization of sirtuins, SIRT1, SIRT6, and SIRT7 are localized in the nucleus and mostly regulates protein function by the post-translational modification via histone deacetylation (7). Moreover, SIRT1 localizes in the cytosol and directly deacetylates various transcription factors and cofactors. SIRT2 is localized in the cytosol and nucleus and is involved in the metabolic process and cell cycle regulation (8–11). SIRT3, SIRT4, and SIRT5 are localized in the mitochondria and regulate various metabolic enzyme activities and mitochondrial oxidative stress (9). Therefore, in the organisms, sirtuins coordinate the cellular responses to adapt to CR in their corresponding cellular compartments in which they occur.

Among the seven sirtuins, SIRT1 is the most studied and wellcharacterized sirtuin with respect to its physiological functions. SIRT1 is involved in the production of various hormones and maintenance of homeostasis by regulating the function of histones and transcription factors, to adapt to malnutrition in the endocrine and metabolic systems. For instance, SIRT1 modulates forkhead box protein O1 (FOXO1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), signal transducer and activator of transcription (STAT3) (12–14).  Additionally, SIRT1 negatively regulates the mitochondrial uncoupling protein 2 (UCP2) expression and enhances the insulin secretion in response to glucose level elevation in the pancreas (16).

Recently, accumulating evidence suggests that SIRT1 plays important role in the homeostasis maintenance in the neuroendocrine system. Particularly, the hypothalamic pituitary axes play a central and an integral role in the neuroendocrine system.

### PHYSIOLOGY OF THE HYPOTHALAMUS AND PITUITARY GLAND

The neuroendocrine system, especially the hypothalamus and pituitary gland play an essential role in the homeostasis maintenance via orchestrating the endocrine system as a regulatory machinery. The hypothalamus–pituitary complex connects the nervous system with the endocrine system that regulates the systemic hormone secretion in a direct or indirect manner. The neural signaling of central nervous system modifies the hypothalamus–pituitary axis and results in the regulation of corresponding target hormonal secretion (17).

The hypothalamus is responsible for the homeostasis maintenance by regulating the body temperature, appetite, thirst, energy expenditure, behavior, and circadian rhythm of the organisms (18). Moreover, the hypothalamus synthesizes and secretes various hypothalamic hormones that in turn stimulate or inhibit the secretion of pituitary hormones (19). The medial preoptic nucleus regulates the release of gonadotropic hormones and thermogenesis (20). The putative anterior paraventricular (aPV), the paraventricular nucleus (PVN), and the supraoptic nucleus (SON) consist of parvicellular neurons that release corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), growth hormone releasing hormone (GHRH), and somatostatin (SST), and magnocellular neurons that produce vasopressin (AVP) and oxytocin (OXT) (21–24).

The pituitary gland consists of anterior posterior lobes and the anterior lobe is functionally subdivided into six cell types based on the production of a specific hormone by each cell type (25). The growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH) or follicular stimulating hormone (FSH) are secreted by the somatotroph, lactotroph, corticotroph, thyrotroph, and gonadotroph, respectively (26). The anterior pituitary hormones are mainly regulated by the release hormones or inhibitory hormones from the hypothalamus via the portal hypophyseal circulation into the anterior lobe and neural connection to the posterior lobe through the pituitary stalk (a physical and functional connection between the hypothalamus and pituitary gland) (27, 28). The hormones secreted by the pituitary gland circulate in the systemic blood flow and thereby stimulate the hormone secretion by each of their respective target organ. Generally, these pathways initiating from the hypothalamus to target organ via the pituitary gland are mainly classified into four axes depending on the target organ, namely, hypothalamus-pituitary-adrenal (HPA), hypothalamus-pituitary-thyroid (HPT), hypothalamuspituitary-gonadal (HPG), and somatotropic axes. In this review, we described the SIRT1 involvement in each of the aforementioned axes in the subsequent subsections.

#### SIRT1 in Hypothalamus

In the hypothalamus, SIRT1 is expressed in the SF1 neuron in the ventromedial hypothalamic nucleus (VMH) and in the POMC and AgRP neuron in the arcuate nucleus (ARH), respectively (29–31). POMC neuron specific SIRT1 knockout mice exhibited energy imbalance due to the altered sympathetic activity (30). Also, deletion of SIRT1 in SF1 neurons caused insulin resistance in skeletal muscle. On the contrary, SIRT1 overexpression in SF1 neurons prevented from diet-induced obesity and insulin resistance (29). Both targeted overexpression of SIRT1 in POMC or AgRP neurons prevented age-associated weight gain. However, SIRT1 overexpression in POMC neurons enhanced the energy expenditure mediated by increment of sympathetic activity in adipose tissue while SIRT1 overexpression in AgRP neurons suppressed food intake, indicating a presence of nuclear specific mechanisms (31). Also, food restriction increases SIRT1 protein levels in the dorsomedial (DMH) and lateral hypothalamic nuclei (LH) (32). Brain specific SIRT1 overexpressing mice exhibited extended life span mediated by enhanced neural activity in DMH and LH, through increased orexin type 2 receptor (Ox2r) expression (32). These data indicate that region specific expression of SIRT1 plays an important role in the regulation of appetite, energy expenditure, general metabolism, and life span.

#### The HPA Axis

The HPA axis is an important neuroendocrine system that plays an essential role in the survival, stress response, metabolism, appetite, immunoreaction, mood, and behavior of the organisms. Corticotrophin releasing hormone (CRH) secreted by the PVN in the hypothalamus initiates stress response in the HPA axis via the production of proopiomelanocortin (POMC) by the pituitary corticotroph. POMC is a precursor polypeptide of ACTH and alpha-melanocyte stimulating hormone (αMSH) that are cleaved by prohormone convertases 1 and 2 (PC1 and PC2), respectively. ACTH induced by corticotroph stimulates the glucocorticoid production by the adrenal cortex.

The SIRT1 expression in the hypothalamic POMC neuron is enhanced by fasting and is associated with the reduced αMSH levels, although the significance to the HPA axis is unknown (33). On the other hand, another group reported that POMC neuron specific ablation of SIRT1 did not alter POMC, ACTH, and αMSH levels (30). The precursor pro-CRH is posttranslationally processed by the PC1 and PC2. SIRT1 increases the PC2 levels in the PVN that in turn increases the active-CRH production and results in the HPA axis activation (34). In mouse corticotroph cell line (AtT20 cells), treatment with resveratrol (a SIRT1 activating compound) increased the PC1 and PC2 levels. In accordance with this, Ex-527 (a SIRT1 specific inhibitor) treatment decreased the PC1 and PC2 levels in AtT20 cells (33). Although there have been no reports demonstrating the expression of SIRT1 in the corticotrophs, these data suggest that SIRT1 may indirectly modulates the HPA axis by regulating the PC1 and PC2 levels. Additionally, resveratrol increased the expression and prolonged the half-life of P450 side chain cleavage enzyme (P450scc) in the adrenal gland that results in an increase in glucocorticoid secretion by the adrenal cortex (35). Moreover, the hypothalamic SIRT1 plays an important role in the energy homeostasis maintenance associated with the HPA axis. These data clearly indicate that SIRT1 in the hypothalamus, pituitary, and adrenal gland regulates the HPA axis activation that acts as an adaptive response to starvation (**Table 1**A).

## The HPT Axis

The HPT axis is activated by the thyrotropin-releasing hormone (TRH) secretion by the hypothalamus when the hypothalamus senses low levels of circulating thyroid hormone. The TRH induces thyroid-stimulating hormone (TSH) secretion by thyrotroph cells in the pituitary that in turn stimulates the thyroid hormone (TH) secretion by the thyroid to maintain normal TH levels (36). SIRT1 is expressed in thyrotroph cells and enhances TSH endocytosis via the deacetylation of phosphatidylinositol-4-phosphate 5-kinase type 1γ (PIP5K1γ), which is a main enzyme that synthesizes phosphatidylinositol 4,5-bisphosphate in thyrotroph cells (37). Tyrotroph-specific SIRT1 knockout mice showed decreased TSH secretion resulting in decreased metabolic rate (37). Additionally, SIRT1 knockout in whole body exhibited hypermetabolism caused by an increased oxygen consumption in the hepatic mitochondria leading to the decreased body weight while the mice manifested hyperphagia, reduced serum thyroxin level, and decreased nocturnal physical activity (38). Interestingly, thyroxin treatment suppressed the fasting-induced SIRT1 expression as thyroxin negatively regulates the SIRT1 level and activity in the liver via TH receptor-β (39). These data indicate that the HPT axis and SIRT1 interact with each other and adaptively regulate metabolism (**Table 1**B).

## The HPG Axis

The HPG axis is responsible for the reproduction, life cycle, and sexual dimorphism in the organisms. Gonadotropin-releasing hormone (GnRH) is secreted by GnRH neurons that diffusely localize and form a network named pulse-generator in the hypothalamus. The pulsatile secretion of GnRH stimulates the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion by pituitary gonadotroph cells and subsequently the gonads produce estrogen or testosterone (36, 40).

Although secondary effect of the general condition including body weight loss cannot be ruled out, SIRT1 knockout mice exhibited a diminished hypothalamic GnRH expression and in turn reduced serum LH and FSH levels and spermatogenesis arrest, suggesting an important role of SIRT1 in the HPG axis (41). Additionally, GnRH treatment decreases SIRT1 level via the miR-132/212 induction in the pituitary. This results in the downregulation of SIRT1-dependent FOXO1 deacetylation and a decrease in the FOXO1-mediated inhibition of Fshβ transcription that ultimately increases the Fshβ expression in rat primary pituitary cells and LβT2 cell line (42) (**Table 1**C).

### The Somatotropic Axis

In the somatotropic axis, GH releasing hormone (GHRH) that is secreted by the hypothalamus induces the GH secretion by pituitary somatotroph cells. Circulating GH binds to GH receptor on hepatocytes and results in the increased serum insulin like growth factor-I (IGF-I) level (36). Moreover, GH induces local IGF-I production in various tissues including bone, muscle, and fat tissue. In various species, numerous evidences demonstrated that the reduced function of somatotropic axis extends lifespan in

(A) HPA axis, (B) HPT axis, (C) HPG axis, (D) somatotrophic axis.

SIRT1 , Overexpression/activation of SIRT1. SIRT1 , Knockout/inhibition of SIRT1. GC, Glucocorticoid, T4, Thyroxine.

animal models (43–48). Regarding the underlying mechanisms, among the species which evolved from C. elegans to mouse, an evolutionarily conserved interplay between SIR2/SIRT1 and the somatotropic axis was reported in which SIR2/SIRT1 modulates the signaling molecules of somatotropic pathway (49–54).

which inhibits activation of STAT5. Excerpt from, Yamamoto et al. (60).

SIRT1 brain-specific knockout (BSKO) mice exhibited dwarfism with small pituitary and reduced GH-IGF-I levels while the other pituitary hormonal levels were unaltered (55). Interestingly, SIRT6 BSKO manifested similar phenotype as that of SIRT1 BSKO. Despite the significant reduction in the number of somatotroph cells and GH content in the pituitary gland of SIRT6 BSKO, the hypothalamic GHRH and somatotropin release–inhibiting factor (SRIF) levels remained unaltered (56). Although the precise mechanism remains unclear in these models, it is speculated that aberrations during the pituitary somatotroph development and GH synthesis were presumably caused by the hypothalamic dysfunction or feedback dysregulation between the hypothalamus and pituitary gland.

Moreover, the direct role of SIRT1 in pituitary somatotroph cells was reported (57). The SIRT1 activation in somatotroph cells suppressed the GHRH-induced GH secretion in in vivo and in vitro studies. SIRT1 deacetylates glycogen synthase kinase 3 beta (GSK3β) and cAMP response element-binding protein (CREB). Deacetylated-GSK3β gets activated and inactivates CREB via protein phosphatase-1. Deacetylated-CREB exhibits a decrease in its activity and is unable to activate the transcription of POU domain, class 1, transcription factor 1 that results in the GH

#### REFERENCES

synthesis impairment (57). These data demonstrate that SIRT1 regulates the somatotropic axis in the hypothalamus and pituitary gland.

During starvation condition, it is well-known that the reduced serum IGF-I level is observed despite the elevated GH level. Moreover, it is reported that exogenous GH treatment did not increase the serum IGF-I level in starving individuals (58). This is considered as GH resistance status in the liver (59). In the organisms, it is considered that GH resistance is an adaptive response to survive during starvation and malnutrition conditions. These responses include the decreased IGF-I level inhibits growth and the elevated GH level causes insulin resistance and free fatty acid mobilization to avoid hypoglycemia. Therefore, we hypothesized that SIRT1 might negatively regulate the GH-dependent IGF-I production during starved condition in the liver. We demonstrated that hepatic SIRT1 directly deacetylates STAT5 and suppresses the tyrosine phosphorylation of STAT5 via GH that results in the impairment of GH signaling during fasting condition (60) (**Figure 1**).

Considering that the somatotropic axis utilizes energy to promote body growth, SIRT1 signaling basically utilizes energy to improve the survival of organisms during CR. Conclusively, SIRT1 plays an important role in switching from growth to survival mode in order to adapt to malnutrition by modulating the somatotropic axis at various steps (**Table 1**D).

### CONCLUSION

These studies clearly demonstrated that SIRT1 is involved in the regulatory mechanism of hypothalamus-pituitary axis with respect to the homeostasis maintenance. The determination of a crosstalk between the neuroendocrine system and SIRT1 function is crucial as both of them play an important role in the homeostasis maintenance as well as the regulation of lifespan.

### AUTHOR CONTRIBUTIONS

MY drafted the manuscript and MY and YT were responsible for the conception and design of this review.

### FUNDING

This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (23591354, 23659477, 26670459 [YT]), and, a grant from the Japan Agency for Medical Research and Development (AMED) (17bm0804012h0001), and by the Uehara Memorial Foundation and the Naito Foundation [YT].

3. Imai S, Armstrong CM, Kaeberlein M, Guarente L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature (2000) 403:795–800. doi: 10.1038/350 01622

4. Faraone-Mennella MR. A new facet of ADP-ribosylation reactions: SIRTs and PARPs interplay. Front Biosci. (2015) 20:458–73. doi: 10.274 1/4319

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Yamamoto and Takahashi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# SIRT1 Is a Potential Drug Target for Treatment of Diabetic Kidney Disease

Yifei Zhong1 \*, Kyung Lee2 and John Cijiang He2,3 \*

*1 Division of Nephrology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China, 2 Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York City, NY, United States, 3 Renal Section, James J. Peters VA Medical Center, Bronx, NY, United States*

Multiple studies have demonstrated a critical role of Sirtuin-1 (SIRT1) deacetylase in protecting kidney cells from cellular stresses. A protective role of SIRT1 has been reported in both podocytes and renal tubular cells in multiple kidney disease settings, including diabetic kidney disease (DKD). We and others have shown that SIRT1 exerts renoprotective effects in DKD in part through the deacetylation of transcription factors involved in the disease pathogenesis, such as p53, FOXO, RelA/p65NF-κB, STAT3, and PGC1α/PPARγ. Recently we showed that the podocyte-specific overexpression of SIRT1 attenuated proteinuria and kidney injury in an experimental model of DKD, further confirming SIRT1 as a potential target to treat kidney disease. Known agonists of SIRT1 such as resveratrol diminished diabetic kidney injury in several animal models. Similarly, we also showed that puerarin, a Chinese herbal medicine compound, activates SIRT1 to provide renoprotection in mouse models of DKD. However, as these are non-specific SIRT1 agonists, we recently developed a more specific and potent SIRT1 agonist (BF175) that significantly attenuated diabetic kidney injury in type 1 diabetic OVE26 mice. We also previously reported that MS417, a bromodomain inhibitor that disrupts the interaction between the acetyl-residues of NF-κB and bromodomain-containing protein 4 (BRD4) also attenuates DKD. These results suggest that SIRT1 agonists and bromodomain inhibitors could be potential new therapuetic treatments against DKD progression.

Keywords: SIRT1, acetylation, diabetic kidney disease, bromodomain inhibitor, podocytes

#### INTRODUCTION

Sirtuin family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, a homolog of yeast Sir2 (silent mating type information regulation 2), has been shown to play an important role in a variety of cellular functions. The mammalian Sir2 ortholog, Sirtuin-1 (SIRT1) is upregulated by caloric restriction and mediates the longevity effect of calorie restriction by regulation of glucose and lipid metabolism (1, 2). At the cellular level, SIRT1 regulates variety of processes including autophagy (3), energetic homeostasis (2), mitochondrial biogenesis (4), and apoptosis (5). A large body of evidence suggests that SIRT1 plays a major role in various kidney diseases by providing protection against cellular stresses associated with kidney injury (6–8). Here, we provide an overview of the role of SIRT1 in kidney cells in the context of diabetic kidney disease (DKD), with a focus on its role on the regulation of transcription factor activation. The review also discusses the potential new therapies by targeting SIRT1 pathway for DKD.

#### Edited by:

*Yang Yang, Northwest University, China*

#### Reviewed by:

*Fan Peng, Fourth Military Medical University, China Agnieszka Swiatecka-Urban, University of Pittsburgh, United States Belinda Jim, Jacobi Medical Center, United States*

#### \*Correspondence:

*Yifei Zhong<EMAIL_ADDRESS>John Cijiang He<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology*

Received: *03 July 2018* Accepted: *01 October 2018* Published: *17 October 2018*

#### Citation:

*Zhong Y, Lee K and He JC (2018) SIRT1 Is a Potential Drug Target for Treatment of Diabetic Kidney Disease. Front. Endocrinol. 9:624. doi: 10.3389/fendo.2018.00624*

### ROLE OF SIRT1 IN REGULATION OF TRANSCRIPTION FACTOR ACETYLATION

Recent evidence suggests that transcription factor activation is regulated not only by protein phosphorylation, but also by acetylation. SIRT1 exerts biological effects not only through deacetylation of histones, but also deacetylation of various transcription factors that include p53, FOXO, RelA/p65, STAT3, PGC1α, and PPAR-γ (9), thereby leading to transcription repression. SIRT1 regulates p53 activity through deacetylation (10–13) and promotes cell survival through suppression of p53 dependent apoptosis in response to DNA damage and oxidative stress (5). SIRT1 was also shown to regulate the activities of FOXO family of transcription factors through deacetylation (14). Deacetylation of FOXO3 by SIRT1 enhances its ability to induce cell cycle arrest and resistance to oxidative stress, while inhibiting its ability to induce cell death (14, 15). We also showed that SIRT1 inhibits podocyte apoptosis through deacetylation of FOXO4 (16, 17). Several studies suggest that the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is also negatively regulated by SIRT1 (18–20). SIRT1 was found to cause the deacetylation and inactivation of STAT3 during caloric restriction (21). SIRT1 also exerts anti-inflammatory effects through the inhibition of NF-κB pathway. It was shown that the duration of nuclear NF-κB action is highly regulated by reversible acetylation (22, 23), and that SIRT1 inhibits the NF-κB signaling pathway through deacetylation of p65 (24). In addition, SIRT1 modulates cellular response to hypoxia via deacetylation of hypoxia-induced factor 1α (HIF-1α) (25–27). All these highlight an important transcription modulatory function by SIRT1 activity.

### SIRT1 PROVIDES RENOPROTECTION AGAINST DKD

Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease and end-stage renal failure in the US (28). Even with optimal therapy, the incidence of DKD remains high, as none of the currently available therapy can reverse or completely forestall the progression of DKD. Therefore, development of more effective treatment for DKD is urgently required. The important role of SIRT1 in DKD has been demonstrated by number of studies (**Table 1**). We have previous shown that SIRT1 expression is significantly reduced in human kidney with DKD and that its reduction is more pronounced in the glomerular compartment than in the tubular compartment (17). An association between single nucleotide polymorphisms within SIRT1 gene and DKD was observed in Japanese subjects with type 2 diabetes (40). However, the exact mechanism of regulation of SIRT1 expression in DKD remains unclear. On the cellular level, SIRT1 has been shown to regulate autophagy (41, 42) and oxidative stress response in the diabetic kidneys (35). Resveratrol wasshown to attenuate DKD through activation of AMPK/SIRT1 pathway (29, 31) and by modulating angiogenesis (43). Studies have demonstrated a clear role of SIRT1 in renal tubular cells in the setting of acute kidney injury (6, 44). In diabetic kidneys, it was shown that reduced proximal renal tubular SIRT1 expression contributes to albuminuria by upregulation of the tight junction protein Claudin-1 in podocytes (32). Interestingly, reduction in Sirt1 expression in tubular cells induced hypomethylation of the claudin-1 gene in podocytes to promote its expression, while overexpression of Sirt1 in tubular cells induced hypermethylation of claudin-1 and downregulated expression in podocytes, indicating an important cross-talk between the two cell types and epigenetic regulation of Claudin-1 expression by SIRT1. Work from our laboratory also demonstrated a critical role of SIRT1 in podocyte injury in DKD. We showed that either knockdown or knockout of Sirt1 specifically in podocytes aggravated DKD injury in type 2 diabetic db/db mice (33) and in STZ-induced diabetic mice (34). Importantly, our recent study demonstrated that the podocyte-specific overexpression of SIRT1 was sufficient to significantly attenuate podocyte injury and to impede DKD progression in type1 diabetic OVE26 mice. Together, these studies clearly demonstrate a protective role of SIRT1 against DKD in experimental models of both type 1 and type 2 diabetes.

## RENOPROTECTIVE MECHANISMS OF SIRT1 IN DKD

As the cellular and molecular mechanisms of SIRT1 has been recently reviewed (42, 45, 46), as well as the role and mechanism of other sirtuins in kidney disease (47), this review is focused primarily on its modulation of transcription factor through deacetylation in the setting of DKD.

#### Effects of SIRT1 in Inflammation in Diabetic Kidneys Through NF-κB and STAT3 Deacetylation

Many studies suggest that SIRT1 regulates activity of several transcription factors that regulate kidney cell homeostasis and are involved in pathogenesis of DKD through deacetylation. Systems biology analysis of microarray data suggests that JAK-STAT and NF-κB are key inflammatory pathways activated in diabetic kidneys (48, 49). Recently, we showed that the acetylation of STAT3 and RelA/p65 is increased in kidneys from diabetic patients and mouse models (33). More importantly, we demonstrated that the podocyte-specific knockout of Sirt1 in db/db mice led to higher levels of p65 and STAT3 acetylation and resulted in greater degree of proteinuria and kidney injury than in control db/db mice, implicating SIRT1 as a key inhibitor of the NF-κB- and STAT3-induced inflammatory responses in DKD (33). In addition, we found that expression of the key proinflammatory factors mediated by NF-kB and Stat3 were also increased in the kidney of Sirt1 knockout db/db mice, further confirming a key role of Sirt1 in regulation of inflammation in the diabetic kidney.

### Effects of SIRT1 in Cell Death in Diabetic Kidneys Through p53 and FOXO4 Deacetylation

Several lines of evidence indicate that p53 mediates apoptosis of both podocytes and tubular epithelial cells in DKD (50–52). SIRT1 has been shown to promote cell survival by suppressing p53-dependent apoptosis in response to DNA damage and

TABLE 1 | Summary of the *in vivo* studies of SIRT1 in DKD.

oxidative stress (5). The interplay of SIRT1-p53 pathway also controls cellular senescence (53–55). We reported previously that advanced glycation endproducts (AGEs) induce podocyte apoptosis through FOXO4-mediated Bim expression and that acetylation of FOXO4 is critical for mediating this effect (17). Overexpression of SIRT1 inhibited AGE-induced FOXO4 acetylation and podocyte apoptosis.

### Effects of SIRT1 in Mitochondrial Dysfunction and Fibrosis in Diabetic Kidneys Through of PGC-1α and Smad3 Deacetylation

SIRT1 has also been shown to regulate PGC-1α activity and to play an important role for maintenance of mitochondrial function in podocytes (56). The PGC-1α in regulation of mitochondrial function has been well described for neurodegenerative disorders (57). Both mitochondrial injury and cellular senescence are key pathological processes mediating kidney injury (58–60). Consistent with this, we have shown recently SIRT1 deficiency in podocytes aggravates agingrelated kidney disease through enhanced cells senescence and mitochondrial dysfunction (61). Although the effects of SIRT1 on Smad3 acetylation remain to be determined, resveratrol was shown to affect acetylation but not phosphorylation of Smad3 to inhibit TGF-β1-induced up-regulation of collagen IV and fibronectin mRNA levels in vitro and renal fibrosis in the model of unilateral ureteral obstruction (UUO) in vivo (62). Therefore, it is plausible that increased SIRT1 activity may also attenuate renal fibrosis in DKD. Taken together, these studies suggest that SIRT1, as a negative regulator of inflammation, cellular senescence and mitochondrial dysfunction, is a key repressor of DKD pathogenesis.

### SIRT1 IS A POTENTIAL DRUG TARGET FOR TREATMENT OF DKD

Given that SIRT1 is a key mediator in thwarting the progression of DKD and other kidney diseases, development of therapeutic

FIGURE 1 | Role of SIRT1 in DKD pathogenesis. This schema summarizes how Sirt1 mediates podocyte injury in DKD. The data suggest that Sirt1 expression is reduced in the diabetic glomeruli including podocytes. Reduced SIRT1 expression leads to increased acetylation and activation of transcription factors, such as NF-κB, STAT3, p53, and PGC1α, leading to exacerbated inflammation, senescence/apoptosis, and mitochondrial dysfunction of kidney cells such as podocytes (shown in blue arrows). All these processes interact each other and contribute to the progression of DKD (shown in red arrows). Therefore, SIRT1 agonists or inhibition of transcription factor acetylation through use of bromodomain inhibitors will reverse these diseased processes and could be developed to treat DKD. ( ): stimulation; ( ): inhibition; ( ): interaction.

strategies to specifically restore SIRT1 activity is warranted. In support of this, we recently demonstrated that increased SIRT1 expression in podocytes attenuated albuminuria and glomerular injury in OVE26 diabetic mice (39). As SIRT1 expression is reduced in diseased kidneys, identifying the molecular basis of its suppression in diabetic kidneys and to interfere in this process may be an avenue of therapeutic approach. We previously showed that increased advanced glycation endproducts (AGEs) in in the diabetic milieu contribute to reduced SIRT1 expression in podocytes (17). Inhibition of AGE formation by pyridoxamine in vivo restored SIRT1 expression the glomeruli of db/db mice and attenuated podocyte injury and progression of DKD (17). Similar observations of SIRT1 reduction by AGEs were made in mesangial cells in vitro (63).

Another approach would be to stimulate SIRT1 activity through SIRT1 agonists. Resveratrol is a well-known SIRT1 agonist that has shown to improve DKD in several animal models (30, 31). However, recent reports indicate that resveratrol may not be a SIRT1-specific (64), nor are other purported SIRT1 agonists, such as SRT1720, SRT2183, and SRT1460 (65). We also showed that puerarin, an extract from a Chinese herbal medicine, attenuates diabetic kidney injury through activation of SIRT1 and suppression of NOX4 expression in podocytes in experimental diabetic mouse model (62). Other herbal medicines or compounds have also been reported to improve DKD through activation of SIRT1 (36, 38). Metformin is reported to improve podocyte function by activating SIRT1 (66). However, SIRT1 independent effects on podocytes or in DKD by non-specific SIRT1 agonists or metformin cannot be ruled out. Recently, we developed a new potent and selective SIRT1 agonist, BF175 (39). In cultured podocytes BF175 increased SIRT1-mediated activation of PGC1-α and protected against high glucosemediated mitochondrial injury. In vivo, administration of BF175 for 6 weeks in type 1 diabetic OVE26 mice resulted in a marked reduction in albuminuria and in glomerular injury in a manner similar to podocyte-specific SIRT1 overexpression. BT175 treatment also attenuated diabetes-induced podocyte loss and reduced oxidative stress in glomeruli of OVE26 mice. Therefore, BT175 and its analogs could be developed as novel therapeutic strategy to treat DKD. However, these approaches of targeting SIRT1 are not without limitations. As discussed above, the specificity of the SIRT1 agonists remains a concern. Given the heterogeneity of SIRT1 function, the ever expanding list of its substrates, and the different effects of deacetylation on its target protein functions, it's possible that the beneficial effects of SIRT1 are mixed with potentially pernicious side effects.

As SIRT1 exerts its renoprotective effects through deacetylation of key transcription factors (TFs) involved in DKD, another therapeutic approach may be to directly regulate the transcription factor acetylation through bromodomain inhibitors (BrDi). Acetylated lysines of the key TFs involved in DKD pathogenesis such as p65 NF-κB interact with proteins containing bromodomains (67), and BrDi could suppress their acetylation in a more specific manner. For instance, NF-κB transcriptional activity is dependent upon its acetylation at lysine 310 (Lys310), and Lys310-acetylated p65 NF-κB recruits the BET protein BRD4 in complex with positive transcription elongation factor b (p-TEFb) and RNA polymerase II that together form a productive transcriptional machinery complex (68). We reported that a BET-specific BrDi MS417 suppresses TNF-αinduced acetylation of p65 NF-κB and the expression of NF-κB target genes in kidney cells in vitro and attenuates proteinuria and glomerulosclerosis in a mouse model of HIV-associated nephropathy in vivo. MS417 also inhibited AGE-induced acetylation of p65 NF-κB in podocytes in vitro and mitigated proteinuria in diabetic db/db mice. Therefore, MS417 or other BrDi might be another class of potential drug candidates to treat DKD patients.

In summary, SIRT1 has significant renoprotective effects against podocyte injury in DKD (**Figure 1**), and SIRT1 agonists and bromodomain inhibitors are promising candidates as therapeutic approach in treatment of DKD patients.

### Clinical Perspectives

Since a large amount of evidence suggest that Sirt1 is a key molecule involving in the pathogenesis of DKD and the expression of Sirt1 is suppressed in human diabetic kidney, enhancing the SIRT1-induced transcription factor deacetylation via SIRT1 agonists or bromodomain inhibitors may serve as potential therapies for human DKD.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### ACKNOWLEDGMENTS

YZ is supported by National Natural Science Foundation of China (2015-81573768), Shanghai Shuguang Scholar (2016- 6SG37), Shanghai Three Years' Pilot Project (ZY3-CCCX-2-1002, ZY3-CCCX-3-2001), and Award from Chen Yiping's National Research Studio for Famous Chinese Medicine Practitioners; KL is supported by NIH 1R01DK117913; and JH is supported by NIH 1R01DK078897, NIH 1R01DK088541.

### REFERENCES

programs in human diabetic nephropathy. Diabetes (2006) 55:2993–3003. doi: 10.2337/db06-0477

aged mice following transient ischemic injury. PLoS ONE (2013) 8:e70464. doi: 10.1371/journal.pone.0070464

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Zhong, Lee and He. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# High Levels of SIRT1 Expression as a Protective Mechanism Against Disease-Related Conditions

Birsen Elibol 1 and Ulkan Kilic2 \*

*1 Department of Medical Biology, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey, 2 Department of Medical Biology, Faculty of Medicine, University of Health Sciences, Istanbul, Turkey*

SIRT1 protein, a member of Silent Information Regulator 2 (Sir2) protein family, have gained considerable attention as epigenetic regulators for a great area in the human physiology. Changes in sirtuin expression are critical in several diseases, including metabolic syndrome, cardiovascular diseases, cancer and neurodegeneration. Here, we provide an overview of the association of the increasing level of SIRT1 protein for regulating some disease related conditions such as obesity, cardiovascular diseases and neurodegeneration. This review also provides a detailed molecular understanding of the interaction of the some basic molecules with increasing SIRT1 levels rather than reduction of the SIRT1 expression. In this context, the current approaches to enhancing the expression of SIRT1 points the importance of epigenetics in several age-related diseases to provide a healthy aging by developing novel therapies which can prevent or damp the progression of some diseases.

Edited by:

*Yang Yang, Northwest University, China*

#### Reviewed by:

*Suowen Xu, University of Rochester, United States Bin Geng, Fu Wai Hospital, China Boon-Seng Wong, Singapore Institute of Technology, Singapore*

> \*Correspondence: *Ulkan Kilic<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology*

Received: *31 July 2018* Accepted: *27 September 2018* Published: *15 October 2018*

#### Citation:

*Elibol B and Kilic U (2018) High Levels of SIRT1 Expression as a Protective Mechanism Against Disease-Related Conditions. Front. Endocrinol. 9:614. doi: 10.3389/fendo.2018.00614* Keywords: SIRT1 expression, oxidative stress, metabolic diseases, cardiovascular diseases, neurodegenerative diseases

#### INTRODUCTION

Sirtuin 1 (SIRT1) which is encoded by the SIRT1 gene is the most conserved mammalian nicotinamide adenine dinucleotide (NAD+) dependent histone deacetylase (1). Besides its role being a target for histone and non-histone proteins, SIRT1 functions as a transcription factor for many different physiological processes (2). According to the previous experiments which were performed using yeast, worms and flies as model organisms, sirtuins were accepted as evolutionarily conserved epigenetic mediators of longevity (3–5). In addition to the key role on extending life by regulating the response to some conditions such as fasting, caloric restriction and exercise, SIRT1 regulates many endocrine functions, protects organism from oxidative stress-related cellular events, promotes DNA stability, and decreases various age-related disorders, such as neurodegenerative disease, metabolic abnormalities, and cancer (6–9).

SIRT1 protein is expressed in most of the body parts including brain, heart, kidney, liver, pancreas, spleen, skeletal muscle, endothelial tissue and white adipose tissue. By expression and activation of SIRT1, modulation of its downstream pathways occurs by targeting several cellular proteins, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), peroxisome proliferators-activated receptor-gamma (PPAR-γ) and its coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), protein tyrosine phosphatase (PTP), forkhead transcriptional factors (the FoxO subgroup), adenosine monophosphate activated protein kinase (AMPK), CRE-binding protein regulated transcription coactivator 2 (CRTC2),

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endothelial nitric oxide synthase (eNOS), p53, myogenic differentiation (MyoD), liver X receptor (LXR), and transcription factor E2F1 (10, 11). Through its deacetylation activity, SIRT1 modulates functions of these critical molecules and shows its critical and multifaceted roles in cellular physiology (**Figure 1**).

Alterations of the level of SIRT1 expression were determined in several diseases including metabolic diseases, neurodegenerative diseases, cancer and aging. Whereas an increase in the expression of the SIRT1 protein was observed in cancer (12, 13), reductions in the SIRT1 level was more common in other diseases such as Alzheimer's Diseases (AD), Parkinson Disease (PD), obesity, diabetes, and cardiovascular diseases (14–18). Recent developments elucidated the relation between downregulation of SIRT1 levels and disease progression as an increase in the oxidative stress and inflammation (16, 17). For example, due to a significant decrease in SIRT1 levels which correlated with an increase in the oxidative stress parameters, accumulation of Tau proteins in AD, enhancement of acetylated p53 expression levels in coronary artery disease and increase in the fatty acid oxidation in obesity were observed in the patients (15, 17, 19, 20).

Previous studies showed that SIRT1 overexpression significantly increased cell viability, decreased cell apoptosis and reduced the release of pro-inflammatory cytokines (21–24). In addition, the regulation of metabolism and longevity by SIRT1 occurs through controlling the maturation of hypothalamic peptide hormones (25, 26). Specificity for SIRT1 increases in the relevant metabolic pathways in the hypothalamic circuitries which is also associated with altered downstream factors of SIRT1 such as FoxO transcription factors (27, 28). In the light of this information, we reviewed recent findings related to the association of the increasing level of SIRT1 protein rather than reduction of the SIRT1 expression and regulation of some disease related conditions such as obesity, cardiovascular diseases and neurodegeneration. The overarching aim of this paper is to provide a basis for hypothesizing that the level of SIRT1 are mechanistically increased to overcome the dysfunction of SIRT1 activity in the diseased conditions.

#### SIRT1 AND METABOLIC DISEASES

SIRT1 protein protects the functions of adipose tissue and liver in several aspects (29, 30) such as glucose homeostasis and fat metabolism against severe obesity (31, 32). It is also involved in energy balance and stress. Insulin sensitivity is increased in the pancreatic beta cells which have insulin resistance due to overexpression of SIRT1 (30, 33). The activity of PPARγ which have a role in the storage of glucose and fatty acid in adipose tissue is repressed by SIRT1 (34). During short term fasting, the CRTC2 is also depressed by SIRT1 and thus gluconeogenesis is declined in the liver tissue. During long term fasting, SIRT1 expression deacetylates and activates the PGC-1α to decrease adiposity and lipogenesis and to increase fatty acid oxidation (35– 37). In addition, SIRT1 deacetlylates sterol regulatory element binding protein (SREBP), farnesoid X receptor (FXR), as well as liver X receptor (LXR) to increase bile acid production and to reverse cholesterol transport (30, 38). Thus, SIRT1 can be called as a "Master Metabolic Regulator" (30). Indeed, the dysregulation of energy sensing may cause inflammation and insulin resistance. Because of prevention of pro-inflammatory responses, SIRT1 behaves as a positive regulator of insulin in the adipose tissue (39). In one of the recent study, after feeding with high dietary fructose, the liver of rats were investigated in response to SIRT1 expression as a main energy sensing protein (40). However, they demonstrated a significant increase in the SIRT1 expression in the fructose-induced inflammation suggesting compensatory rise in the level of SIRT1 to decline the inflammation-related metabolic reactions (40). In addition, overexpression of SIRT1 in obesity which was formed by high-fat diet protects lipidinduced inflammation and hepatic steatosis while providing better glucose tolerance (41). These favorable effects of SIRT1 may be related with the activation of the antioxidant enzymes and stimulation of PGC1α to decrease the level of pro-inflammatory cytokines (41).

In some neurodegenerative diseases, a number of neuropeptide systems in the hypothalamus are affected from activity of SIRT1 which indicate an impact on metabolism. For example, SIRT1 upregulates the level of orexin receptor specifically in the lateral hypothalamic area and the ventromedial nucleus of the hypothalamus, whereas the expression of orexin and melanin-concentrating hormone is reduced in the hypothalamus due to inhibition of the active state of orexin neurons (25, 42, 43). In addition, SIRT1 also regulates the expression of BDNF in the brain. It was found that increased SIRT1 level diminished BDNF signaling which resulted in severe hyperphagia and obesity both in humans and animals (44, 45). These results showed that compensatory increase in the SIRT1 level to cope with the disease outcomes such as oxidative stress brings some additional metabolic dysfunctions in the body due to altered peptides in the endocrine system.

In addition to the obesity, SIRT1 has a role in the hepatic energy metabolism by modulating it nutritionally and hormonally. This modulation is mostly occurred through the deacetylation of metabolic regulators (46). Previous studies also showed that obese patients with non-alcoholic fatty-liver disease (NAFLD), which is the most common liver disease caused by elevated hepatic lipids, inflammation and oxidative stress, had high plasma levels of SIRT1 producing a potential against the physiological mechanisms related to NAFLD (47). In this type of disease, the action mechanism of SIRT1 acted through the modulation of PPARα activity and fatty acid oxidation (48).

On the other hand, it was found that increase in the SIRT1 activity upregulates genes-related metabolic functions, promotes insulin sensitivity and reduces inflammatory gene expressions in the adipose tissue of diet-induced obese animals (49). In addition, we found a polymorphism in the promoter region of SIRT1 gene in obese children drawing attention to the association between altered SIRT1 activity and the risk of obesity (50). Previous reports also showed the protective role of SIRT1 on the development of osteoarthritis by upregulation of cartilage extracellular matrix genes and downregulation of matrix-degrading enzymes (51, 52). In addition, increase in the SIRT1 activity had a protective effect against osteoarthritis in

animal models (53, 54). Therefore, the investigators suggested that the increase both in the activity and expression of SIRT1 might be a protective strategy for progression of osteoarthritis via the modulation of the NF-κB pathway (55).

### SIRT1 AND AGE-RELATED NEURODEGENERATIVE DISEASES

The relationships between SIRT1 and age were investigated in the previous studies related to interaction of lifespan elongation and calorie restriction which is thought as an enhancer for SIRT1 activity (56, 57). Most of these studies showed that calorie intake restriction innervates the extension of life by the inducement of defense of cells against to free radicals and toxins for attenuation of apoptosis or amelioration of cell repair which are desired factors in aging (58–60). That means, altered SIRT1 expression and activity is thought to be a potent way to keep the cells and organs properly functioning for longer times. The positive correlation between age and SIRT1 expression and/or activity may be a compensation against unexpected situations such as oxidative stress (61, 62). For example, in one of our previous study, it was noted higher level of SIRT1 protein in older people compared with the SIRT1 level in the younger people (62). It was thought that increased protein level of SIRT1 in older people may be a compensatory mechanism due to accumulation of oxidative stress-related products and elimination of antioxidant enzyme level in elderly (62). However, increase in the expression does not mean increase in the activity of protein. An oxidative stressdependent decrease in the SIRT1 activity was noted in aged animals that had high levels of SIRT1 protein (63, 64). The decline in the activity of SIRT1 may not be related directly with SIRT1 protein but also its downstream or upstream molecules such as a decline in NAD+ levels with aging (65).

One of the main risk factor for several neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) is age. The common underlying mechanisms of neurodegeneration are increase in the neuroinflammation, mitochondrial damages and oxidative stress (66, 67). In literature, it was shown that sirtuins' hyperactivity could reduce these negative outcomes both in vivo and in vitro due to its neuroprotective role (68–71). In the AD pathology, SIRT1 deacetylates substrates in favor of the non-amyloidogenic pathway or acts directly on the Aβ and Tau proteins (72). Molecular studies showed that SIRT1 activation prevents the accumulation of Aβ plaques and tau pathology through the NF-κB signaling pathway by upregulation of the ADAM10 gene, induction of the Notch pathway, and inhibition of the mTOR pathway (20, 73, 74). As shown in previous studies, SIRT1 epigenetically reprograms inflammation taking about AD formation at the earlier stages by altering transcription factors (24, 75, 76). In addition, it was observed that brains of AD patients have consistently reduced NAD+ levels and SIRT1 transcription and/or protein levels involved in chronic inflammation that can also be altered by increased levels of the activated proinflammatory transcription factor NF-κB (77–79). In one of our studies, we found a significant increase in the SIRT1 level of dementia patients (80). Furthermore, in the patients with Huntington's disease (HD), Baldo and his colleagues found higher expression of SIRT1 protein level in the most affected brain regions, especially hypothalamic regions important for metabolic regulation, compared to brain regions which were less affected from the mutant huntingtin protein (28). In PD, SIRT1 inhibits α-synuclein aggregation by deacetylating proteins such as heat shock proteins and PGC-1α and, therefore, it protects dopaminergic neurons against cell death which occur due to the formation of insoluble fibrils called Lewy bodies (81, 82). In the in vitro PD model, it was observed that an overexpression of SIRT1 due to application of toxin (rotenone or MPTP) which causes neurodegeneration was rescued cells from oxidative stress (16, 83). The neuroprotection against to PD occurred by the mechanism of decreasing in the expression of NF-κB and cleaved PARP-1. In a postmortem study, the levels of SIRT1 showed a slight increase in the dementia patients with Lewy bodies (16, 83). However, as seen in the AD, the activity of the SIRT1 protein also decreased in the PD patients producing neurodegeneration in correlation with possible higher oxidative stress, synaptic and cell loss, and neuroinflammation (16).

We thought that the high levels of SIRT1 protein might have a role in alleviating the oxidative stress that is significantly increased in neurodegeneration because an induction of the SIRT1 expression occurs when the organism encounters a biological stress such as aging or an age-related disorder due to the role function of SIRT1 as an important stress sensor molecule.

Also, in literature, it was stated that high levels of SIRT1 may increase the expression of genes related to neuronal protection (84–86). On the other hand, SIRT1 behaves as a double edged sword in response to inflammation which is a cause of neurodegeneration. That means, low levels of SIRT1 cause early acute inflammation-related damages to tissues by increasing NF-kB, and high levels of SIRT1 during late inflammation cause immunosuppression and increased the rate of death (87). In a previous study, investigators observed that increase in the expression level of SIRT1 cannot protect the brain from neurodegeneration without increasing the activity level of SIRT1. For example, Ciriello and his colleagues observed a significant decrease in the level of phosphorylated SIRT1, the active form of SIRT1, in the patients with multiple sclerosis (88). Interestingly, a significant negative correlation between phosphorylated and non-phosphorylated forms of SIRT1 was observed explaining both the SIRT1 overexpression and inactivity of SIRT1 in diseased state. In addition, when a SIRT1-activating molecules were given to the organisms, profound therapeutic benefits and neuroprotective effects were recorded against age-dependent neurodegenerative diseases (89).

### SIRT1 AND CARDIOVASCULAR DISEASE

Nowadays, the sirtuin protein family is thought as one of the important target for cardiovascular diseases (CVD). Therefore, the role of SIRT1 protein and its downstream molecules also gains importance in the experimental studies related with CVD development. In cardiomyocytes, during prenatal period, SIRT1 is found in the nucleus, however, it is mostly located in the cytoplasm of myocytes of adult heart of rodents (90). Previously, we found that the level of the SIRT1 expression was significantly higher in the CVD patients compared to the levels of SIRT1 in healthy subjects pointing the crosstalk between SIRT1 protein expression and reactive oxygen species (61). In this previous study, we also found a significant increase in the oxidative stress parameters which may be an inducer for SIRT1 expression. In cardiomyocytes, myoblast gains resistance against to oxidative stress by increasing expression of nuclear SIRT1 protein. To do produce this antioxidative activity, SIRT1 protein enhances the level of MnSOD expression through p53 deacetylation (90). In addition, activation of FoxO1 dependent oxidative pathway by overexpression of SIRT1 protein is another regulatory way of protection of cardiomyocytes from oxidative stress (91). By the help of this pathway, cardiac infarct volume is reduced to ameliorate and recover cardiac function after ischemia/reperfusion in mice (92). It is also thought that transcriptional activity of NF-κB protein, a preconditioner in cardiac ischemia, is inhibited by SIRT1 protein to promote cell protection (93, 94). In literature, it was reported that the activity of SIRT1 protein is directly or indirectly controlled via the JNK1- SIRT1 link by accumulated oxidative stress which is caused by an increase in the ROS level due to aging or age-related diseases enzyme (1). It was demonstrated that ROS inhibited JNK phosphatases which activated the JNK1 to phosphorylate SIRT1 (95, 96). Furthermore, this phosphorylation increased the activity of SIRT1 resulting its translocation into the nuclei (97). Alcendor et al. (91) noted that the rate of SIRT1 overexpression had two-sided action in the cardiovascular system. For example, 2.5- to 7.5-fold increase in the SIRT1 expression attenuated apoptosis, the symptoms of cardiac dysfunction, age-related cardiac hypertrophy and expression of senescence markers. On the other side, 12.5-fold increase in the expression of SIRT1 resulted in increased cardiac hypertrophy due to oxidative stress and apoptosis. This study explained clearly the relation between oxidative stress and overexpression of SIRT1 to the pathological levels in CVD patients. In one of our previous studies (61), we found a positive correlation between total antioxidant level and SIRT1 level in CVD patients. Therefore, we can conclude that the increase in the SIRT1 level may be a compensatory mechanism to increase the antioxidants against oxidative stress in CVD patients.

Contrary to some previous studies (98, 99), the SIRT1 level significantly decreased approaching to control values in the CVD patients receiving statin therapy (100). The decline in the SIRT1 level by statins can be explained by the statins' inducement effect on PPARγ activity to protect patients against the progression of atherosclerosis (101). On the other hand, PPARγ inhibits SIRT1 expression at the transcriptional level which interrupting compensatory action of increased SIRT1 expression (102).

### CONCLUSION

Recent studies have shown that age-related diseases or endocrine system dysfunctions are associated with an increase in SIRT1 expression levels, but with a decrease in their activity. The oxidative stress produced during these processes may lead to compensatory or protective increase in the SIRT1 expression to deal with the decline of the SIRT1 activity.

### AUTHOR CONTRIBUTIONS

BE wrote the draft of the manuscript and UK finalized the manuscript.

#### REFERENCES

implications for chronic inflammation and aging. Am J Physiol Lung Cell Mol Physiol. (2007) 292:L567–76. doi: 10.1152/ajplung.00308.2006

in obesity-resistant alphaMUPA mice. Int J Obes (Lond) (2011) 35:226–35. doi: 10.1038/ijo.2010.125

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Elibol and Kilic. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# SIRT1 and Estrogen Signaling Cooperation for Breast Cancer Onset and Progression

#### Sergio Liarte1 \*, José Luis Alonso-Romero2 and Francisco José Nicolás 1 \*

1 Laboratorio de Oncología Molecular y TGFβ, Instituto Murciano de Investigaciones Biosanitarias Arrixaca, Murcia, Spain, 2 Servicio de Oncología, Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain

Breast cancer remains a significant female mortality cause. It constitutes a multifactorial disease for which research on environmental factors offers little help in predicting onset or progression. The pursuit for its foundations by analyzing hormonal changes as a motive for disease development, indicates that increased exposure to estrogens associates with increased risk. A prevalent number of breast cancer cases show dependence on the increased activity of the classic nuclear estrogen receptor (ER) for cell proliferation and survival. SIRT1 is a Type III histone deacetylase which is receiving increasing attention due to its ability to perform activities over relevant non-histone proteins and transcription factors. Interestingly, concomitant SIRT1 overexpression is commonly found in ER-positive breast cancer cases. Both proteins had been shown to directly interact, in a process related to altered intracellular signaling and aberrant transcription, then promoting tumor progression. Moreover, SIRT1 activities had been also linked to estrogenic effects through interaction with the G-protein coupled membrane bound estrogen receptor (GPER). This work aims to summarize present knowledge on the interplay between SIRT1 and ER/GPER for breast cancer onset and progression. Lastly, evidences on the ability of SIRT1 to interact with TGFß signaling, a concurrent pathway significantly involved in breast cancer progression, are reported. The potential of this research field for the development of innovative strategies in the assessment of orphan breast cancer subtypes, such as triple negative breast cancer (TNBC), is discussed.

 It constitutes a multifactorial disease for which epidemiologic studies over environmental determinants offer little help in predicting disease onset or progression, thus, gender, aging, diagnosed first-degree relatives or previous history of BC remain dominant risk factors (2, 3). The pursuit for BC causes over molecular biology techniques led to the establishment of few genetic markers, such as BRCA1, BRCA2, or DBC-1, which driver mutation predispose for disease while also explaining cases of familiar clustering; still, the majority of mutations detected account for genes of low penetrance and frequently altered across genomes (4). Whilst, concurrent research efforts tried to find a predictable BC marker or cause based on hormonal changes.Although for most of hormonal hypothesis proposed data is still inconclusive (2, 5), it is accepted

#### Edited by:

Yang Yang, Northwest University, China

#### Reviewed by:

Xianyong Lan, Northwest A&F University, China Yang Zhi, Fourth Military Medical University, China Sylvie Babajko, INSERM U1138 Centre de Recherche des Cordeliers, France

#### \*Correspondence:

Sergio Liarte<EMAIL_ADDRESS>Francisco José Nicolás<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology

Received: 05 June 2018 Accepted: 30 August 2018 Published: 27 September 2018

#### Citation:

Liarte S, Alonso-Romero JL and Nicolás FJ (2018) SIRT1 and Estrogen Signaling Cooperation for Breast Cancer Onset and Progression. Front. Endocrinol. 9:552. doi: 10.3389/fendo.2018.00552

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that increased exposure to estrogens associates with higher risk, while reducing exposure is believed to result in protection (6). Therefore, factors such as early menarche, nulliparity or late menopause are associated with enhanced BC likelihood.

Observations on BC incidence are thought to be significantly contributed by the activities of classic nuclear hormone receptors for estrogens and progesterone (ER, PR). This is supported by tumor characterization using microarray techniques, which allowed to discriminate BC subtypes on the expression of key molecular markers including classic receptors. Applying such methods, BC is classified in ascending order indicating for aggressiveness: luminal-A; luminal-B (HER2–); luminal-B (HER2+); HER2-enriched; and basal-like. The first three subtypes share positivity for the expression of ER and/or PR along with the presence or absence of the human epidermal growth factor receptor 2 (Erb-B2; HER2), member of the epidermal growth factor receptor (HER/EGFR/ERBB) family. At last, the basal-like, which displays worsened prognosis and develops predominantly in pre-menopausal women, is characterized for the expression of specific basal-epithelium markers such as keratins (7). However, in clinical practice, cost-effective immunohistochemical methods are preferred to determine receptor presence, as such sub-typing, while providing prognostic information, also allow setting therapies to target specific oncogenic markers (**Table 1**). The anatomopathological absence of ER, PR, and HER2 led to the expression "triple negative" (TNBC), discerning cases devoid for all three markers (15). Worth noting, TNBC is somewhat regarded a surrogate for basal-like, as 70–80% overlap has been described between classifications (8, 16). In any case, while BC triage usually allows for adapted treatments improving prognosis in receptor-positive categories, that does not apply for TNBC cases, for which the lack of targeted approaches frequently restrict options to chemotherapy, with obvious consequences for the prognosis and lethality of the disease (17).

Interest on histone deacetylases (HDACs) is expanding as accumulated findings highlight their impact on regular physiology and pathological condition, staying SIRT1 the most studied (18). Sirtuins comprise a family of proteins (SIRT1– 7) described as type III HDACs relying on NAD+ availability to perform a gatekeeping role in the configuration of the cell transcriptome, function that appears highly dependent on the cellular context and has been involved in a variety of biological processes, from modulating energy metabolism to development and cellular senescence (19–21). Intriguingly, Sirtuins in general and SIRT1 in particular display a paradoxical role in cancer, with histological studies showing increased or decreased expression patterns upon cancer origin and/or stage (22–24). In that sense, numerous contributions support the notion that SIRT1 activities influence hormone receptors (HR) actions, expressly those mediating long-term estrogenic effects in the mammary gland, namely the classic ERs, for which a relevant degree of interdependence between these factors has been described with apparent importance for BC onset and development. Moreover, interaction between the anew membrane-bound G-protein coupled estrogen receptor (GPER), which ubiquitously mediates short-term estrogenic effects, and SIRT1 has been also proposed, an interplay which could help fostering BC survivability and progression. Altogether, the conjoint actions of SIRT1 and HRs pose deep implications for BC onset and progression, which turn significantly relevant for the case of drug-resistant cases and conceivably HER2-enriched and TNBC. From that scope, this review synthetizes current knowledge on this emerging field.

### KNOWN IMPLICATIONS OF SIRT1 FOR CANCER ONSET AND PROGRESSION

Initially described to deacetylate histones H1, H2, and H4 (18), SIRT1 is supposed to contribute to chromatin remodeling beneficial for tumor progression. However, despite obvious epigenetic capabilities, HDACs and SIRT1 current relevance for cancer strive on their now known ability to act on different substrates. SIRT1 is regarded an established modulator of significant non-histone nuclear proteins, such as p53, E2F1, or NF-kB (25, 26). SIRT1 overexpression has been related to tumor cell survival through the deacetylation and subsequent degradation of the p53 tumor suppressor (27, 28). Additionally, SIRT1 is known to deacetylate the FOXO family of transcription factors, resulting in repression of pro-apoptotic elements (29– 31). In this line, using both hormone-responsive and TNBC models, MCF-7 and MDA-MB-231 cell lines respectively, SIRT1 has been found to localize to the promoters of silenced tumor suppressor genes, state reverted upon SIRT1 activity inhibition (32). Additionally, there is open debate on the ability of SIRT1 to act over cytosolic targets, with proposed tumorigenic implications related to PI3K/IGF-1R signaling (33, 34).

Moreover, SIRT1 shows a convoluted role in the regulation of the epithelial-mesenchymal transition (EMT) process, with apparent relevance in the case of reproductive tumors such as prostate and BC (35, 36). Notably, SIRT1 has been found to upregulate the expression of matrix-metalloproteinases in BC cells, condition known to promote invasiveness (37, 38). Moreover, SIRT1 activities seem to coordinate cancer stem cell-EMT changeover through deacetylation of a complex circuitry of transcription factors (39). To this regard, SIRT1 has been recently found to interact with Smad proteins, TGFß signaling canonical transducers (40). Interestingly, TGFß deregulation is regarded a cornerstone for EMT and tumor dispersion, among others markers, affecting the expression of matrix-metalloproteinases (4). While having a demonstrated role on the degradation of the inhibitory-Smad Smad7, SIRT1 has been also shown to relate with receptor-regulated Smads (Smad2 and Smad3), possibly coactivator-Smad Smad4 as well, in a process linked to altered transcriptional output (36, 40–42). Consequently, such interplay prompts for wide implications on cell transformation and tumor progression/dispersion.

### ESTROGEN SIGNALING AND BREAST CANCER: THE ER PATHWAY

Among steroids, estrogens comprise a set of hormones involved in the development and maintenance of the female reproductive system, the main representative of which is 17ß-estradiol (E2),

Concurrent marker detection is achieved for establishing subtypes. Not mentioned minor special histological types may respond to targeted therapies. Erb-B2, human epidermal growth factor receptor 2 (HER2); \*overexpressed or amplified; ER, Estrogen Receptor; PR, Progesterone Receptor; Ki-67, proliferation marker; Cytokeratins, basal-like marker (8–14). \*\*highly overexpressed; ↓ low expression/risk; ↑high expression/risk; ↑↑very high expression/risk.

constituting a major hormonal input along the monthly cycle. For its synthesis, androgens are converted into estrogens on the action of CYP19A1, known as Aromatase, an enzyme being mainly expressed in the ovaries but found in other tissues including the mammary gland (43). ERα and ERß correspond with the classic HRs responsible for E2 long-term effects (44). Both receptors, along with PR, integrate into the nuclear receptor family, which include sex steroids receptors as well as receptors for corticosteroids (45). Its members mostly localize to the nucleoplasm, but a minority of isoforms which help finemodulating the overall response may also appear at alternate locations, involving a cytoplasmic-nuclear shuttling mechanism (46, 47). Although ERα and ERß share significant sequence homology, both receptors display unique expression patterns depending on either tissue or organ. ERα signaling comes crucial for the regulation of mammary gland development and function (44), also contributing to cancer onset and progression. Elevated ERα levels expressed in benign breast epithelium correlate with enhanced BC risk, whereas estrogen-dependent cancers require of E2 for cell survival and growth (44). Worth noting, PR positivity can be usually regarded a surrogate of ER positivity, as PR expression requires proper ER functioning to occur (48).

The principal ER activation mechanism requires E2 binding, which allows conformational changes promoting receptor dimerization and nuclear translocation to interact with estrogen response elements (ERE) present in the DNA to regulate transcription (44). Additionally, alternate mechanisms for ER activation and modulation had been described through crosslinking with signaling pathways like EGFR/PI3K/ERK, based on changes of ER phosphorylation status (49, 50). Upon landing on EREs, E2-ER complexes further recruit several co-activator proteins such as p300, PPARγ, and PGCα, which leads to histone acetylation and chromatin remodeling necessary for the regulation of transcription (51). Moreover, active ERα also recruit members of the FOXO family into transcriptomic complexes, interplay that has been characterized to greatly influence transcriptomic outcomes, with implications for mammary morphogenesis, BC onset and progression to drug resistant states (52–56).

At the clinic, hormone-responsive BC is usually managed in a straightforward manner due to the multiplicity of pharmacological approaches based in the use of Aromatase inhibitors along with selective ER modulators and downregulators, like Tamoxifen or Fulvestrant, which diminish ER-mediated E2-responses at the breast (57). Consequently, these cases are mostly associated with favorable prognosis. Still, hormone-responsive tumors frequently transform over time into an estrogen-independence status, gaining the ability to proliferate in the absence of hormonal input. This conversion usually becomes a critical step for BC clinical progression, as it is related to increased aggressiveness. In that sense, ER-negative cases at diagnosis are believed to have lost ERα expression over time due to gene silencing, thus reducing therapeutic options targeting HRs and thus associating with less favorable prognosis (58).

#### ESTROGEN SIGNALING AND BREAST CANCER: THE GPER PATHWAY

Despite the fact that classical HRs may be missing for some BC cases, however, it is now acknowledged that estrogen stimulation would still play a powerful role for the evolution of the disease through the actions of membrane-coupled ERs. Turning into the XXI century, the existence of a membrane-coupled ER was revealed, that would be responsible for most of E2 rapid physiological responses which at the time lacked of proper molecular explanation. This marker corresponded with a G-protein coupled receptor which could be located both at the endoplasmic reticulum and the plasmatic membranes (59, 60). Initial reports indicated that through its binding to E2, GPER actions resulted in cAMP and inositol triphosphate production, triggering intracellular calcium mobilization. However, it was rapidly established that its actions also resulted in the transactivation of diverse intracellular signaling cascades including MAP-Kinases, PI3K, or eNOS (61– 63). In this line, further research also found GPER able to attune E2-mediated transcription regulation. This was established on co-expression experiments showing the existence of a functional cooperation between canonical-ERα-mediated and short-time-GPER-mediated signaling through a mechanism relying on the activation MAP-Kinases, capable of promoting post-translational modifications such as altering ERα phosphorylation status (64). Moreover, additional studies established GPER to be ubiquitously expressed through both reproductive and nonreproductive tissues (65), thus posing new challenges for the understanding of estrogen physiology. In that sense, it was not long before GPER was proposed to contribute for cancer development (61). Still, GPER's specific role for both neoplastic transformation and cancer progression remains unclear, as its actions seem to be highly depend on tissue origin (66). Yet, although anatomopathological studies on GPER expression are not included in routine clinical practices, in the case of estrogendependent tumors and especially BC, several works show that its altered expression can be associated with cancer progression, supporting a potential prognostic value (67–69).

At the molecular level, diverse studies advocate for GPER's ability to affect cancer cell survivability and proliferation, by influencing a myriad of signaling pathways responsible for cell cycle regulation or apoptosis control. They also play a role in the regulation of angiogenesis required for tumor nourishment and development [reviewed at (66)]. Moreover, GPER signaling has been proposed to distinctly affect cell migration and cancer invasiveness. Calpains are non-lysosomal proteases that are implicated in the regulation of cell adherence to the extracellular matrix and motility (70). Interestingly, it was reported that incubation of ER-negative BC cells with GPER agonists G-1 promoted Calpain-1 activity and altered adhesion to matrigel (71). Also in this line, GPER has been described to adjust EGFR/PI3K/ERK intracellular signaling, favoring expression changes of migration markers such as SNAIL or ß1-integrin, as well as altering plasticity of cellular adhesions by the activation of the focal adhesion kinase (FAK) (72). Conjointly, these abilities of GPER to promote viability and motility provide a molecular framework for estrogen stimulation of cancer cells despite ER-negativity. Notably, epidemiologic studies would deem clinical functionality to these capacities, as prevalence for GPER overexpression is found in HER2-enriched, basal-like and TNBC cases, also associated with both distant metastasis and recurrence (67, 73).

#### ESTROGEN SIGNALING AND BREAST CANCER: NOTED SIRT1 INFLUENCE

Numerous contributions support the notion that SIRT1 activities decisively impact HRs actions in relation to disease. With regard to classic ERs, in cellular models, E2 stimulation has been found to promote SIRT1 expression under direct influence of ERα transcriptomic complexes, observation matched by the prevalent detection of elevated levels for SIRT1 manifested for most of ERαpositive BC samples (74, 75). Interestingly, the ability of SIRT1 to interact with p300, PPARγ, and PGCα, ERα transcriptomic co-activators, has been proved to affect chromatin remodeling, disturbing developmental processes (76–78). Following studies found SIRT1 inhibition resulted in suppressed ERα expression, interfering with E2-dependent cell growth in healthy as well as malignant mammary epithelial cells (79, 80). In this line, inhibition of SIRT1 has been found to lessen ERα mediated repression of NRF2-dependent detoxifying enzymes in MCF-7 cells (81).

Simultaneously, significant efforts had been put to apprehend the effects of a less subtle SIRT1-ER interaction mechanism. Interestingly, ERα has been found to relate to and be a target for SIRT1. Upon E2-ERα activation, p300 stabilizes receptor complexes through acetylation in a process that can be reversed by SIRT1 (82). These findings initially suggested a SIRT1 inhibitory role over estrogen signaling. Still, changes in ERα acetylation status are considered to have limited effects (83). On the other hand, as previously mentioned, active ERα recruits FOXO family members into transcriptomic complexes, factors which also fall under SIRT1 spectrum of actions. Within the BC context, a recent report showed FOXN3 to be able to recruit SIRT1 into ERE-dependent transcriptomic complexes, promoting reduced transcriptomic output (84). Interestingly, alterations of FOXM1 and FOXO3a levels had been previously linked to SIRT1 aberrant activity, especially in TNBC (85).

Regarding the interaction between SIRT1 and GPER, knowledge is scarce. Nonetheless, using ER-negative HER2 enriched BC cells, it was recently described that E2 actions via GPER can result in SIRT1 overexpression through activation of the EGFR/ERK/c-fos/AP-1 transduction pathway (86). Interestingly, such induction contributed specifically to enhanced cancer cell survival and proliferation, as it was reversed by the use of SIRT1 inhibitors or GPER silencing as well. Notably, these effects were also observed in cancer associated fibroblast obtained from BC patients (86), posing challenging questions for this interplay in BC progression.

### PRESENT AND FUTURE CLINICAL VALUE OF THE ESTROGEN SIGNALING/SIRT1 INTERPLAY

Finding ways to better screen and characterize BC, especially drug-resistant cases and TNBC, constitutes a standing challenge of cancer research. Efforts to establish a SIRT1 prognostic value for BC are increasing, as its overexpression can be commonly detected (9). Recent meta-analysis gathering non-connected data sources, while finding association with higher tumor stage, failed to detect correlation between SIRT1 expression levels and BC overall survival; however, these works addressed statistics letting out BC subtyping (87). Notwithstanding, a single retrospective study incorporating 822 BC patients found SIRT1 expression to correlate with tumor aggressiveness and reduced diseasefree-survival (DFS) (88). Interestingly, when BC subtyping is considered, SIRT1 overexpression associates with ER-positivity and likely shortened DFS (9). Moreover, while not associated with HER2-enriched or TNBC status, concomitant SIRT1 overexpression successfully dictates for lymph-node metastasis likelihood (9), observation which has been linked to the ability of SIRT1 to promote an altered expression of key EMT markers such as E-Cadherin, Vimentin, and SNAIL-1 (89). Hence, although data so far may be considered insufficient or poorly curated,

through either nuclear (ERα) or membrane bound G-protein coupled estrogen receptor (GPER) signaling. Total activity is also modulated by phosphorylation status. Increased Sirt1 activities and interaction with diverse factors result in pleiotropic effects supporting cell survival and transformation for cancer onset. Continued activity contributes to cell de-differentiation and epithelium to mesenchyme transition (EMT).

the accumulated evidence allows to consider SIRT1 histological detection as a valuable marker for assessing BC status, also providing hints on metastasis and relapse odds.

Per BC tumor progression, it is speculated whether SIRT1 has a role in determining tumor conversion to a drugrefractory phenotype. It has been reported that SIRT1 activity helps increasing expression of drug-resistance genes (90), in a process that involves FOXO1 deacetylation and reverts upon SIRT1 suppression (91, 92). Noteworthy, SIRT1 activity is mainly regulated by upholding absolute protein levels with little variation overtime (35), although modulation of its actions depending on MAPK-mediated phosphorylation has been also described (93). To this extent, two considerations should be made. Firstly, it ought to be reminded that estrogen signaling via either HR can augment SIRT1 levels (74, 86). Interestingly, elevated SIRT1 levels has been reported to promote increased Aromatase activity both in ER-positive and TNBC cell lines (94), thereby allowing for local E2 production. Secondly, it should be noted that MAP-kinases signaling can be activated by overexpressed HER2 subunits dimerizing with EGFR and triggering the PI3K/ERK pathway (95). Consequently, a SIRT1 potential role assisting evolution to ER-independence turns reasonable, as deleterious abilities, including aberrant modulation of p53 and FOXO or expression of drug-resistance genes, could be maintained via SIRT1 overexpression depending on GPER signaling, perhaps also through MAP-kinases dependent activation. In that sense, recent reports showing linkage between SIRT1 overexpression and enhanced SRC and AKT activities in different TNBC cell lines would support the latter notion (96, 97). Worth considering as well, classic ERs and GPER display dissimilar behavior upon exposure to pharmaceutical modulators like Tamoxifen and Fulvestrant, which in the case of GPER appear to have an agonistic function (66). Moreover, in cellular models, GPER activation has been described to negatively affect ERα protein levels (71), perhaps fostering the transition. Hence, considering that solid tumors are composed by a clones plethora subject to selective pressure, it is tempting to propose the monitoring of SIRT1 expression/activity as source of information on the efficiency of treatments related to tumor evolution at the cellular scale. To this regard, procedures based on liquid-biopsy techniques would offer an appropriate framework for such approach (98).

On the aspect of treatments, many efforts had tried to assess the potential of suppressing SIRT1 activities. However, after several years, effects described on the many inhibitors discovered and interference studies do not agree with each other. This is regarded as the result of dissimilar mechanistic involved in knocking-out or knocking-down the enzymatic activity and also on the specificity of inhibitors used (99). Yet, modulators of SIRT1 activity may still retain potential as co-adjuvant treatments, due to sensitizing capabilities useful at specific BC environments (100). In this sense, sirtuins in general, and SIRT1 in particular, have been shown to interact with numerous signaling pathways which decisively affect different aspects of the cell physiology. That is the case with the recently described interaction of SIRT1 with TGFß canonical-signaling-transducers, the Smad proteins. SIRT1 has been found incorporated into Smad-mediated transcriptomic complexes, its activity linked to reduced Smad acetylation and decreased nuclear half-life (36, 40–42). Lack of proper TGFß response is considered a major mechanism for EMT through regulating the expression of key cell adherence and migration markers (4). Interestingly, this TGFß dependent regulation involves the participation of FOXO factors (101, 102). Hence, the overexpression of SIRT1 in BC cells has the potential to facilitate aberrant regulation through these system, thus contributing for the EMT process and cancer progression. Moreover, as signaling via GPER and HER2 are known to trigger MAP-Kinases activation, and activated MAP-Kinases had been related to SIRT1 augmented activity, this interplay provides an additional framework for the promotion of both tumor ER-independence and EMT progression, potentially offering opportunity to develop tailored strategies, which would come particularly useful for the case of TNBC. Perhaps, nextgeneration inhibitors, with better specificity and increased potency, may provide advancements in this field.

Finally, a brief but necessary mention should be made on non-coding RNAs. Increasing efforts attempt to discriminate the relevance of both long-non-coding and micro-RNAs in BC pathogenesis and progression, due to their dual role as an additional prognostic information source and potential therapeutic targets (103, 104). Not surprisingly, distinct signatures could be found in BC depending ER status (105). In that sense, exposure to either estrogens or selective ER modulators had been shown extensively affect through classic ERs the microRNA profile of different mammary cell lines (106). Interestingly, within the BC context, SIRT1 activity appears to be highly conditioned by the non-coding-RNA environment, with several long- and microRNA species affected by endocrine signaling being able to promote or downregulate its expression (107, 108). Further research in this area may again probe helpful for the development of strategies targeting intrinsic BC subtypes.

## CONCLUDING REMARKS

The accumulated evidence prompts to consider SIRT1 as an integrated player into the transduction network activated by estrogens, both through ERs and GPER, in the mammary tissue (**Figure 1**). This tight cooperation conceptualizes and supports a SIRT1 promoting role in mammary tumorigenesis, with meaning for both disease onset and progression. These implications are to be considered both in the case of signaling triggered upon endogenous estrogens exposure, also in the case of exposure to environmental pollutants like xenoestrogens, phytoestrogens, and other synthetic compounds; let aside for the response to BC treatments based on estrogen modulators. Consequently, SIRT1 detection has potential to become a powerful prognostic indicator for tumor evolution and response to chemotherapeutics. Moreover, a better understanding at the molecular level of its cooperation and impact in the signaling through connected pathways may provide opportunity for the development of innovative therapy approaches in the assessment of BC and particularly TNBC cases.

### AUTHOR CONTRIBUTIONS

SL conceptualized the work, performed searches in literature databases and prepared, reviewed and edited the original draft of the manuscript. JA-R contributed with formal analysis and the incorporation of clinical aspects, also reviewed the original draft. FN supervised the project, was responsible for funding acquisition and reviewed and edited the original draft. All authors read and approved the final manuscript.

## FUNDING

This work was supported by grants (SAF2006–09482; SAF2003– 06029) from the Spanish Ministry of Education and Science. Also, this work was supported by a grant (PI13/00794) from the Instituto de Salud Carlos III. And the Fondos FEDER Una manera de hacer Europa (ERDF, A way to build Europe). SL received a fellowship from Roche. FN was supported the Fundación para la Formación e Investigación Sanitarias de la Región de Murcia.

### ACKNOWLEDGMENTS

We are indebted to the Hospital Clínico Universitario Virgen de la Arrixaca that strongly supported this research.

## REFERENCES

a special focus on 17beta-hydroxysteroid dehydrogenase type 1, 2, and breast cancer. Oncotarget (2017) 8:30552–62. doi: 10.18632/oncotarget.15547

development of mammary gland in mice.  Onco Targets Ther. (2018) 11:2051–61. doi: 10.2147/OTT.S1 62503

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Liarte, Alonso-Romero and Nicolás. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# SIRT1 in Secretory Organ Cancer

#### Raffaele Frazzi\*

Laboratory of Translational Research, Azienda Unità Sanitaria Locale - IRCCS di Reggio Emilia, Reggio Emilia, Italy

Mammalian silent information regulator 1 (SIRT1) is reported to play a role in cancers of the secretory organs, including thyroid, pancreatic endocrine, and ovarian tumors (1–4). A recent meta-analysis conducted on 37 selected studies of human cancers analyzed the correlations of overall survival (OS), disease-free survival (DFS) and relapse-free survival (RFS) with SIRT1 expression (5). This study reported that SIRT1 overexpression was associated with a worse OS in liver and lung cancers, while it was not correlated with OS in breast cancer, colorectal cancer, or gastric carcinoma. Collectively, the meta-analysis revealed that an unfavorable OS was associated with SIRT1 expression for solid malignancies. Given the growing importance of this class of lysine/histone deacetylases in human endocrine malignancies, a rational and focused literature assessment is desirable in light of future clinical translations.

Edited by:

Yang Yang, Northwest University, China

#### Reviewed by:

Zhenxing Liang, First Affiliated Hospital of Zhengzhou University, China Daniel Christian Hoessli, University of Karachi, Pakistan Shashwat Sharad, Center for Prostate Disease Research (CPDR), United States

> \*Correspondence: Raffaele Frazzi<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology

Received: 10 May 2018 Accepted: 05 September 2018 Published: 24 September 2018

#### Citation:

Frazzi R (2018) SIRT1 in Secretory Organ Cancer. Front. Endocrinol. 9:569. doi: 10.3389/fendo.2018.00569

## INTRODUCTION

Keywords: SIRT1, cancer, secretory organs, acetylation, epigenetic modulation

The epigenetic regulation of chromatin structure and gene expression represents a major field of study and intervention for researchers focused on cancer. The class III lysine/histone deacetylases are known as Sirtuins and are represented, in humans, by 7 members (6, 7). Silent information regulator 1 (SIRT1) is the most studied and well characterized among the class III deacetylases and its targets include p53, Ku70, peroxisome proliferator-activated receptor gamma (PPARγ), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), Beclin 1 and β-catenin, among others (8–13). A relevant group of SIRT1 targets is the Forkhead box O (FOXO) family of transcription factors. FOXO3a hyperacetylation mediated by SIRT1 accompanies apoptosis while FOXO4 deacetylation activates this target enhancing its transcriptional and biological activity in the nucleus. FOXO4 activation enhances the cellular defenses against oxidative stress and leads to apoptosis resistance (8, 14, 15). Fibrosis is another aging-related disease involving FOXO1/3, well known modulators of aging and longevity playing a clear inhibitory effects on fibrogenic effector cells and extracellular matrix production (16).

Furthermore, the epigenetic regulation and dysregulation of the hypermethylated in cancer1 (HIC1)/SIRT1/p53 axis is relevant for the development of malignancies (17). SIRT1 is also a target for several miRNAs, small non-coding RNA molecules known to be deregulated in various cancers, whose expression is involved in tumorigenesis (18).

SIRT1, due to its NAD+ dependency, is also a metabolic sensor and its deacetylating activity toward regulatory target proteins affects cell metabolism (19).

The focus of this review is the role and involvement of SIRT1 in secretory organ cancers since the information recently gathered on this topic should bear new translational benefits. Correlations between SIRT1 and nuclear receptors, transcription factors, master regulators of gene expression, miRNAs and lncRNAs have been reported. The main glands in the body and the most recent literature are taken into consideration.

**196**

#### OVARIAN CANCER

Ovarian cancer is the fifth leading cause of female cancer mortality in the United States (20). This type of cancer is currently treated with a platinum and taxane-based chemotherapy after surgical cytoreduction. However, the rates of progression free survival (PFS) and OS for these patients remain dismal and no benefits seem to arise by the introduction of additional cytotoxic agents (21). Ovarian cancer is usually regarded as a single entity and current treatment protocols for the disease are not subtype specific. Actually, ovarian cancer is a heterogeneous disease that is classified by histopathological examination, including serous, clear cell, endometrioid, and mucinous subtypes. These subtypes develop differently and respond differently to chemotherapy (22).

SIRT1 overexpression is reported by many authors as associated with poor outcome and chemoresistance in ovarian cancer of epithelial origin (23–25). Ovarian cells (both normal and tumoral) express two kinds of estrogen receptors (ER), ERα and ERβ which play different roles in cell proliferation and aggressiveness (4). Specifically, ERα is associated to a poor outcome, while ERβ expression corresponds, on the contrary, to a favorable outcome. ERα levels are closely associated with estrogen-dependent growth and the increased metastatic potential of ovarian epithelial carcinoma (OEC) through the promotion of the epithelial to mesenchymal transition (EMT) (26). ERβ, in contrast, inhibits EMT in presence of 17β-estradiol (E2), thus mediating the opposite effect to ERα (26). SIRT1 is inversely correlated to ERβ mRNA and protein levels, and the specific ERβ activator KB9520 strongly inhibits SIRT1 mRNA expression. These data collectively support the role of SIRT1 as a tumor promoter in OEC (4).

Recently, another association between OEC and SIRT1 was reported (27). These observations are relevant, because OEC patients often acquire resistance to paclitaxel or cisplatin, and the increased expression in SIRT1 and TWIST1 (two genes associated with drug resistance) is observed in OVCAR-5 cells that show increased cisplatin resistance, migration and invasion potential (27).

SIRT1 is a downstream target of hypoxia inducible factor 1α (HIF1α) (28). Hypoxia is a condition associated with several types of tumors and, specifically, increased HIF1α expression predicts the poor prognosis of ovarian cancer. Notably it has been demonstrated that SIRT1 expression is induced by hypoxia and that HIF1α silencing indirectly hampers SIRT1 expression. Finally, the NF-κB signaling pathway is involved in hypoxiainduced SIRT1 up-regulation, strengthening the link between this class III lysine deacetylase and ovarian cancer (28).

#### THYROID CANCER

The incidence of thyroid cancer has increased over the past few decades worldwide. This increase is mainly driven by new cases of papillary thyroid cancer (29). SIRT1 is reported to be oncogenic in thyroid and prostate murine carcinomas initiated by PTEN deficiency (2). The mechanism unveiled by mRNA transcriptional analysis revealed that SIRT1 drives oncogenesis through c-MYC regulation (**Figure 1**). Two pathways upregulated via SIRT1 overexpression are related to translation and ribosomal biogenesis, which are both controlled by c-MYC. The protein product of the c-MYC oncogene lies at the intersection of a transcriptional network regulating cellular proliferation, replicative potential, cell–cell competition, cell size, differentiation, metabolism, and apoptosis (30).

c-MYC increases in response to mitogenic stimuli, and this is accompanied by SIRT1 upregulation. c-MYC activates SIRT1 by promoting NAMPT transcription and then the NAD-salvage pathway. Furthermore, c-MYC can bind directly to DBC1 (that is a SIRT1 inhibitor) thus preventing SIRT1 blockade (31, 32). On the other hand, SIRT1 activation leads to p53 deacetylation and consequent inactivation, decreasing the levels of PUMA and p21 and eventually counteracting the pro-apoptotic effects of p53. Finally, SIRT1 stabilizes c-MYC via lysine-specific deacetylation and increases its transcriptional activity. Altogether, these data demonstrate a positive feedback loop between c-MYC and SIRT1 (31).

The translational significance of these findings is highlighted by the consistent overexpression of SIRT1 in follicular and papillary thyroid carcinomas, positively correlates with c-MYC protein levels and stabilizes c-MYC (2).

The natural phytochemical resveratrol (RSV) is known to affect mammalian cell metabolism and aging by modulating SIRT1 (33, 34). Furthermore, SIRT1 is also a reported molecular target of RSV in cancer cells of both lymphoid and epithelial origin (35). RSV regulates thyroid stimulating hormone (TSH) secretion via SIRT1 activation. RSV also arrests follicular thyroid and papillary thyroid carcinoma growth by activating the mitogen-activated protein kinase (MAPK) signal transduction pathway and p53 phosphorylation (36, 37).

Among the few studies available, regarding the role of SIRT1 in the thyroid neoplasias, most report involvement as a direct target for miRNAs. miR-212 is a tumor suppressor miRNA that inhibits the proliferation, migration, and invasiveness of thyroid cancer cells. SIRT1 is a direct target of miR-212 and is inversely correlated to miR-212 expression in thyroid cancer tissues (42 samples of human thyroid cancer) (38).

#### PANCREATIC CANCER

Pancreatic cancer (PC) is one of the most deadly tumors in western countries and the 10th most common cancer in the United States. Pancreatic ductal adenocarcinoma is the most common subtype and is recognized as "incurable" due to its aggressive clinical course and resistance to available therapies (39, 40). miR-494 is specifically involved in PC proliferation (41). The overexpression of miR-494 inhibits PC cell proliferation in vitro and in vivo in addition to inhibiting invasion. The c-MYC/SIRT1 axis is a direct target of miR-494 and there is an inverse correlation between the two. The simultaneous interference of c-MYC and SIRT1 synergistically reduces the expression of these two targets and inhibits PC proliferation, eventually stimulating miR-494 expression (41). The interplay between c-MYC and SIRT1, therefore, plays a relevant role during PC tumorigenesis and should be taken into account when designing strategies aimed at combating this cancer.

Recent studies on the miRNA-mediated regulation of autophagy in PC have been published (42). Since it is known that hypoxia induces autophagy, PC cells were cultured under normoxic and hypoxic conditions. miR-138-5p emerged among the most down-regulated miRNAs in the hypoxia-grown cells. Targets can analysis further revealed that the SIRT1 gene contains a classical and conserved domain that binds miR-138-5p. Notably, miR-138-5p specifically binds to the 3′ -UTR of SIRT1 and its overexpression leads to SIRT1 protein downregulation (42). Finally, the authors demonstrated that miR-138-5p also counteracts the hypoxia induction in cancer cells through SIRT1 inhibition in tumor xenografts.

Another player that emerged recently is miR-601, which suppresses PC proliferation and migration (43). The target of this miRNA's activity was identified, once more, as SIRT1. This Sirtuin is downregulated by miR-601 and its overexpression reverses the effect of miR-601 on PC cells (43).

miR-217 also directly targets SIRT1, it is inversely correlated to SIRT1 expression and is down-regulated in chronic pancreatitis and PC (44).

The Ex-527 synthetic inhibitor, which inhibits pancreatic cell proliferation in vitro and in combination with the first-line drug for this pathology, gemcitabine, is considered one of the most specific SIRT1 inhibitors currently available (45). The same inhibitor was shown to promote PC xenograft tumors in SCID mice and did not synergize with gemcitabine. Therefore, caution should be used when dealing with Ex-527 inhibitor to target SIRT1 in vivo.

The role of histone deacetylases (HDACs) is poorly understood in pancreatic neuroendocrine tumors (pNETs). A comprehensive expression pattern of all the five classes (I, IIa, IIb, III, and IV) of HDACs was recently described (3). The gene expression profiles of a total of n = 57 patients revealed a significant upregulation of all the HDAC classes in pNETs over controls with increased levels ranging from 1.5- to 7-fold. The expression of several HDACs, including SIRT1, correlates to the G3 stage and, thus, to pNETs tumor grading (3).

#### GASTRIC CANCER

Gastric cancer (GC) is among the most prevalent malignancies of the upper gastrointestinal tract. It is a solid aggressive carcinoma where new therapeutic options are warranted (46). Autophagy in GC may have the dual role of promoting the activity of irradiation and anticancer drugs or, on the contrary, may function as a protective mechanism for cancer cells (47–49).

SIRT1 plays a confirmed role in autophagy (50, 51). In GC, it has been demonstrated that SIRT1 deacetylates autophagyrelated gene products (ATGs) and a series of histonic and non-histonic targets, eventually interfering with the autophagic process (**Figure 1**) (52).

GC is among the few human tumors where SIRT1 is downregulated, and in one study, the prognosis of low-SIRT1 expressing GC patients was good (52, 53). However, other authors reported a meta-analysis of SIRT1 expression in gastrointestinal cancer in which the results were heterogeneous (54). The overall results from 15 studies showed an association between worse OS and high SIRT1 expression in gastrointestinal cancer. The subgroup analysis revealed that this association was particularly strong in non-colorectal gastrointestinal cancers, especially GC and hepatocellular carcinoma (54). Therefore, SIRT1 is a promising prognostic factor in GC but not in colorectal tumors.

In contrast with the abovementioned results, a study of the clinicopathological parameters in GC patients was analyzed using the Kaplan-Meier plotter (55). Here, the data concerning the seven human Sirtuins were extrapolated from NCBI GEO databases containing mRNA profiles and the corresponding clinical data of a large number of GC patient samples. The Kaplan-Meier plotter was used to investigate the predictive value of mRNA expression of the Sirtuins for the OS of GC patients. Among a total of 631 available cases for SIRT1 expression, the survival curves suggested that a high expression of SIRT1 mRNA was favorable for OS and that different Sirtuins had diverse correlations with OS. Furthermore, the first progression (FP) was positively correlated with SIRT1 expression: in other words, a longer FP time was associated with a higher SIRT1 level (55).

Opposite evidence was reported from the immunohistochemical (IHC) staining of primary GC tissues. High SIRT1 expression, by IHC, was associated with lymphatic invasion (p = 0.028), vessel invasion (p = 0.016) and lymph node metastasis (p = 0.014) and tended to be associated with more advanced disease stages (56).

Clearly, no consistent data on the exact role of SIRT1 in GC are available at present. The evidence available thus far points to a dual role for SIRT1 (that is to say it may act as a tumor promoter as well as a tumor suppressor), possibly depending on its sub-cellular localization, p53 status, and microenvironmental conditions (57, 58).

#### ADRENAL GLANDS

The function of SIRT1 in the adrenal glands has been investigated the least. One study reports the researches performed on human adrenal NCI-H295R cells (59) and, in this setting, Sirtuins (including SIRT1) did not seem to be involved in the RSVmediated lowering of androgen production.

Moreover, steroidogenesis in H295R cells was not affected by Sirtuins, even when SIRT1/3/5 are overexpressed through transgenesis, strengthening the fact that RSV-dependent effects in this setting are SIRT-independent (59).

#### HEPATIC CANCER

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a higher frequency in East Asia than in Europe and North America (60). HCC is the predominant malignancy in the liver and recent evidence shows a role for SIRT1/PGC-1alpha/mitochondrial biosynthesis pathway in protecting the liver against lipid accumulation and mitochondrial dysfunction (61, 62). Senescence induction is a recently proposed strategy with the aim of counteracting HCC (63). Metformin, for instance, induce senescence via the activation of AMP-activated protein kinase (AMPK) when used at low doses. This effect is accompanied by the phosphorylation of SIRT1 as well as p53 acetylation (**Figure 1**). The prolonged exposure to low doses of metformin leads to human HCC senescence in murine xenografts via modulation of the AMPK-SIRT1 axis (63).

The long non-coding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was recently discovered to interfere with SIRT1 activity in hepatocellular cancer (64). MALAT1 is frequently upregulated in several types of cancers and contributes functionally to the development and malignancy of tumor cells (65). MALAT1 is, among lncRNAs, a stable and highly transcribed molecule localized in the nucleus, and it has been reported that, in breast cancer cells expressing ERα, 17β-estradiol treatment negatively regulates MALAT1 transcription, thus inhibiting proliferation, migration and invasion (66). MALAT1 is also regulated during endothelial differentiation in that hypoxia induces its transcription leading to the proliferation of the vasculature (67). In HCC specifically this lncRNA promotes aggressiveness in that releases SIRT1 by sponging miR-204 (64). SIRT1 is a direct downstream target of miR-204 and results inhibited by this interaction. MALAT1 can release the suppression on SIRT1 by base-pairing with miR-204 and leading to its post-transcriptional downregulation (64).

#### CONCLUSIONS

SIRT1 should be regarded as a multi-faceted enzyme and, as already suggested by several authors, studied in a contextdependent fashion. Secretory organs are no exception TABLE 1 | Cancer models of the secretory organs in which SIRT1 has been studied.

to this. **Table 1** summarizes the secretory organ tumors in which SIRT1 has been investigated, with the relevant experimental models. The most recent publications are taken into account.

SIRT1 plays a confirmed role in ovarian, thyroid, and pancreatic cancers, while its role in GC is yet to be definitely established. The evidence available in thyroid cancer confirms that c-MYC and SIRT1 are valuable therapeutic targets, encouraging translational researches aimed at the related pathways. The recent meta-analysis spanning 37 selected studies on human solid cancers demonstrates a correlation between higher SIRT1 expression and worse OS in liver and lung cancers (5). These clinical data are consistent with the experimental ones reported for HCC where SIRT1 activation correlates to malignancy through MALAT1, a lncRNA highly expressed in various cancers including HCC (64, 68).

Furthermore, colorectal and GC do not show the correlation between OS and SIRT1 expression (5). SIRT1 overexpression has been reported either positively associated to FP or negatively associated to OS. Contradictory results also concern SIRT1 IHC and mRNA expression and prognosis of GC, suggesting that this class of lysine-deacetylases is not a reliable biomarker for GC.

In contrast, little evidence is available in the literature concerning the role (if any) of SIRT1 in the pituitary gland. In the testis, the role of SIRT1 is reported to be associated with spermatogenesis, fertility, and differentiation, but not testicular cancer (7).

In light of the literature presented here, further basic research is warranted in order to establish possible therapeutic strategies for ovarian, thyroid and pancreatic cancer, where a protumorigenic role for SIRT1 has been established. Particular attention should be paid to the role of miRNAs in regulating SIRT1 levels (miR-212 in thyroid cancer; miR-494, miR-138-5p, miR-601, and miR-217 in PC; miR-204 in HCC). Furthermore, given the growing importance of lncRNAs in cancer pathology, the involvement of MALAT1 should be exploited. For instance, by measuring its levels in HCC biopsies and, possibly, by

#### REFERENCES

monitoring its expression during therapy administration or disease progression.

#### AUTHOR CONTRIBUTIONS

RF has been studied SIRT1 in cancer and is currently assessing a possible epigenetic regulation for this enzyme. RF conceived, wrote, and formatted the manuscript.

#### FUNDING

This work has been funded by Azienda Unità Sanitaria Locale - IRCCS Reggio Emilia, Italy.

human gastric adenocarcinoma cells. Biochem Biophys Res Commun. (2013) 431:264–9. doi: 10.1016/j.bbrc.2012.12.120

MALAT-1 in osteosarcoma cell MG-63 in an estrogen receptorindependent manner. Biochem Biophys Res Commun. (2015) 457:500–6. doi: 10.1016/j.bbrc.2014.12.114

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Frazzi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Long-Term L-Serine Administration Reduces Food Intake and Improves Oxidative Stress and Sirt1/NFκB Signaling in the Hypothalamus of Aging Mice

Xihong Zhou1 , Haiwen Zhang2 , Liuqin He3 , Xin Wu1 \* and Yulong Yin1,3

1 Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China, 2 Key Laboratory of Tropical Animal Breeding and Epidemic Disease Research of Hainan Province, Hainan University, Haikou, China, 3 Laboratory of Animal Nutrition and Human Health, College of Life Sciences, Hunan Normal University, Changsha, China

#### Edited by:

Yang Yang, Northwest University, China

#### Reviewed by:

Sandra Helena Poliselli Farsky, Universidade de São Paulo, Brazil Yang Zhi, Fourth Military Medical University, China

> \*Correspondence: Xin Wu<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology

Received: 10 May 2018 Accepted: 01 August 2018 Published: 23 August 2018

#### Citation:

Zhou X, Zhang H, He L, Wu X and Yin Y (2018) Long-Term L-Serine Administration Reduces Food Intake and Improves Oxidative Stress and Sirt1/NFκB Signaling in the Hypothalamus of Aging Mice. Front. Endocrinol. 9:476. doi: 10.3389/fendo.2018.00476 Serine has recently been shown to reduce oxidative stress and inflammation, which, when occurring in the hypothalamus, contribute to age-related obesity. To explore whether long-term serine administration reduces oxidative stress and body weight in aging mice, various concentrations of L-serine dissolved in water were administered to 18-month-old C57BL/6J mice for 6 months. The results showed that the administration of 0.5% (w/v) L-serine significantly reduced food intake and body weight gain during the experiment. Moreover, the administration of 0.5% L-serine decreased the concentrations of leptin, malondialdehyde, interleukin-1β, and interleukin-6, while it increased those of superoxide dismutase and glutathione, in both the serum and hypothalamus. Reactive oxygen species and the activity of nicotinamide adenine dinucleotide phosphate oxidase were reduced in the hypothalamus of aging mice treated with L-serine as compared with untreated control mice. Additionally, the expression of the leptin receptor increased while the levels of neuropeptide Y and agouti-related protein decreased in mice that had been treated with 0.5% L-serine. The expression of Sirt1 and phosphorylated signal transducers and activators of transcription 3 (pSTAT3) increased, while that of phosphorylated NFκB decreased in the mice treated with 0.5% L-serine. These results indicated that long-term L-serine administration reduces body weight by decreasing orexigenic peptide expression and reduces oxidative stress and inflammation during aging in mice, possibly by modulating the Sirt1/NFκB pathway. Thus, L-serine has the potential to be used in the prevention of age-related obesity.

Keywords: aging, inflammation, oxidative stress, serine, Sirt1

## INTRODUCTION

The hypothalamus is a critical part of the central nervous system that modulates the stress response and senses nutrient-related inputs. However, in aging mice, increased levels of oxidative stress and inflammatory markers are observed in the hypothalamus (1–3). Low levels of antioxidant enzymes and high levels of lipid peroxidation make the hypothalamus vulnerable to reactive oxygen species (ROS) (4–6). These elevations of oxidative damage and inflammatory responses in the central nervous system are believed to contribute to age-related diseases including obesity and various neurodegenerative disorders (7–9).

The nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (Sirt1) regulates the response to oxidative stress, which correlates with many diseases (10–12). Sirt1 activity in the hypothalamus decreases in association with aging, and its low expression contributes to a low level of antioxidants and increased oxidative damage (10). Importantly, Sirt1 is also a nutrient sensor and plays critical roles in the energy balance in the hypothalamus. Debate is ongoing as to whether hypothalamic Sirt1 has orexigenic or anorexigenic effects, although most of the evidence shows that inhibition of Sirt1 induces a negative energy balance by regulating the activity of forkhead transcription factor FKHR (FOXO1) (13–15) and the expression of agoutirelated protein (AGRP) and neuropeptide Y (NPY) (16, 17). Additionally, the transcription factor nuclear factor-κB (NFκB) regulates numerous target genes to exert its biological functions. Importantly, NFκB functions as a central regulator of the immune response and controls the secretion of inflammatory cytokines in many tissues including the hypothalamus (18–20). Moreover, NFκB is also a central mediator of stress responses under conditions of oxidative stress and upon exposure to certain chemicals in the central nervous system (18).

L-Serine is traditionally considered a non-essential amino acid. Recently, dietary supplementation with L-serine has been shown to have antioxidant effects (21–24). L-Serine is a precursor of glycine and cysteine, which can be used for the synthesis of glutathione. In addition, L-serine exerts critical functions in the mammalian central nervous system. It serves as a precursor of the neuroactive substances L-serine and glycine, and mediates neuroprotective effects (25, 26). However, it remains unknown whether L-serine has any effects on agerelated oxidative stress in the hypothalamus. Consequently, we conducted the present study to explore the effects of long-term L-serine supplementation on hypothalamic oxidative stress and Sirt1 expression in aging mice.

### MATERIALS AND METHODS

#### Animals

Eighty 18-month-old C57BL/6J male mice were included in the study. The mice were obtained from SLAC Laboratory Animal Center (Changsha, China) and were housed in a temperaturecontrolled animal facility (lighting cycle: 12 h/d), with free access to food and water. All animals were randomly assigned into four groups: control mice, which were kept untreated, and mice administered with 0.1, 0.2, or 0.5% (w/v) L-serine dissolved in the drinking water. Eighteen-month-old adult mice were used as young controls. The experiment had the duration of 6 months. Food intake and body weight were recorded every week. Upon completion of the experiment, mice were euthanized with isoflurane and the blood was collected from the retro-orbital sinus. The hypothalamus was excised and collected according to the method described in a previous study (7). The hypothalamus samples were either immediately snap-frozen in liquid nitrogen or fixed in formaldehyde solution. This study was performed in accordance with the recommendations of the Guide for Care and Use of Laboratory Animals published by the Animal Welfare Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences. The protocol was approved by the Animal Welfare Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences.

### Biochemical Assays

Superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) biochemical assays were performed with commercially available kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Interleukin (IL)-1β, IL-6, and leptin biochemical assays were performed with kits from Cusabio Biotech (Wuhan, Hubei, China; https://www. cusabio.com/). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was assayed as previously reported (27).

### Determination of Reactive Oxygen Species