Title: Role of Anti-aging Gene Klotho in Oral and Gastrointestinal Cancers

Authors: Gauri Pathare, Kavita Shalia

Sir H.N. Medical Research Society, Sir H.N. Hospital and Research Centre, Mumbai 400 002, India

Corresponding Author: Gauri Pathare, Sir H.N. Medical Research Society, Sir H.N. Hospital and Research Centre, Court House, L. T. Road, Mumbai 400002, India.

Email: gauri.pathare@rfhospital.org

Phone: 022 67673885, 9869067287

Abstract:

Klotho, the anti-aging gene, has various roles, one of them being tumor suppressor. Dysregulation of insulin/Insulin Growth Factor-1 (IGF-1), Wnt and fibroblast growth factor (FGF) signalling is major contributor to cancer. The tumor suppressor effects of Klotho have been attributed to its ability to modulate these pathways. In cancer cells, Klotho gene is silenced primarily through promoter hypermethylation. The ectopic expression or the restoration of Klotho directly corresponds to reduction in cancer. This implies its role in cancer therapeutics. In this article, the role of Klotho in oral and gastrointestinal cancers has been reviewed.

Keywords: Insulin/IGF-1, FGF, Wnt signalling, promoter hypermethylation.

INTRODUCTION

In Greek mythology, the lifespan of mortals is decided by the three Fates – Clotho spins the thread of life, Lachesis measures it and Atropos decides the time of death by cutting it. Klotho gene, identified by Kuro-o et al. in 1997, is named after the Spinner. Its expression was found to increase lifespan, whereas its defect caused development of aging phenotypes and accelerated death (Kuro-o et al., 1997). Although observed in all age groups, advancing age is one of the risk factors for cancer. A global scenario indicates that majority of tumours are detected in aged populations. The biological and clinical changes that accompany aging such as cellular senescence, genomic instability, telomere biology, autophagy are also significant aspects of cancer biology (Blasco et al., 2007). Given the parallels between cancer and aging, it is expected that Klotho will be involved in cancer biology.

The Klotho gene (KL) discovered by Kuro-o et al., is located on chromosome 13q12 in humans, spans over 50kb in length and comprises of five exons and four introns. It encodes Klotho, or more precisely α-Klotho, which is a single pass transmembrane protein consisting of an intracellular, a transmembrane and an extracellular domains (Kuro-o et al., 1997). The extracellular domain has KL1 and KL2 internal repeats separated by a proteolytic cleavage site (Figure 1) (Shiraki-Iida et al., 1998). Klotho is expressed primarily in the kidney - mainly in the distal convoluted tubule, the parathyroid gland, and the choroid plexus in the brain (Kuro-o et al., 1997). The principal function of Klotho is to regulate phosphate and vitamin D metabolism by acting as a co-receptor for bone-derived fibroblast growth factor-23 (FGF-23) (Kurosu et al., 2006). Apart from the membrane form, a secreted form of Klotho is found in circulation. It is generated by alternative mRNA splicing as well as cleavage of extracellular domain of the transmembrane protein and has humoral activity (Kuro-o et al., 1997; Shiraki-Iida et al., 1998). β-Klotho and γ-Klotho or Klotho-related protein (Klrp) identified later, were found to have sequence similarity with Klotho gene. β-Klotho located on 4p14, is not found in secreted form and is predominantly expressed in liver and adipose tissue. Its main function involves metabolic regulation, glucose uptake, bile acid synthesis and fatty acid metabolism, independent of α-Klotho (Ito et al., 2000). Klrp is a transmembrane protein that binds to fibroblast growth factor receptor (FGFR) -1b, FGFR-1c and FGFR-2c but its function remains unknown (Yahata et al., 2000; Shinji Ito et al., 2002; Yaylaoglu et al., 2005; Xu and Sun, 2015).

1. MECHANISM OF ACTION OF KLOTHO AS A TUMOR SUPPRESSOR

1.1 Via inhibition of Insulin/ Insulin Growth Factor-1 (IGF-1) Signalling Pathway

IGF-1 functions via binding to the IGF-1 receptor (IGF1R), a tyrosine kinase protein that is widely distributed in all tissues. This binding results in receptor autophosphorylation which leads to activation of phosphatidylinositol-3-kinase (PI3K)-Akt and RAS/ Rapidly Accelerated Fibrosarcoma (RAF)/mitogen-activated protein kinase (MAPK) signalling pathways (Brahmkhatri et al., 2015). Via these pathways, IGF-1 regulates metabolic and cell growth responses. The metabolic effects are induced by the activation of enzymes involved in gluconeogenesis, glucose uptake, protein synthesis, and lipogenesis, whereas the cell growth responses are mainly induced by the mechanistic target of rapamycin (mTOR) pathway (Kumar et al., 2017). Thus, it effects balanced growth among multiple tissues and organs.

Clinical and animal studies indicate that the insulin/IGF-1 signalling pathways have a significant role in tumorigenesis. It play a major role in cell survival, proliferation, differentiation and metastasis (LeRoith and Roberts, 2003, Laviola et al., 2007). Studies have reported elevated IGF-1R (key signal transduction receptor of IGF-1 pathway) activity to be associated with multiple aspects of cancer progression including enhanced carcinogenesis, tumorigenesis, metastasis as well as resistance to chemotherapeutics and other molecularly targeted drugs (Rosenzweig and Atreya, 2010). Inhibition of the IGF-1R signalling pathway significantly induces apoptosis in vitro and in vivo, restricts cellular growth, and tumor metastasis (Yakar et al., 2005). Dysregulation of IGF-1 signalling pathway has been implicated in various cancers including brain (Mangiola et al., 2015), breast (Christopoulos et al., 2015), ovarian (Lojkin et al., 2015), prostrate (Roberts, 2004), gastrointestinal (Golan and Javle, 2011), bone (Hiraga et al., 2012) and lung cancer (Kumar et al., 2017).

The tumor suppressor activities of Klotho, associated with inhibiting activation of insulin/IGF-1 pathway, were first identified by Wolf et al. (2005) in breast cancer cells. In another study, Wolf et al. (2008) have reported that the forced expression of Klotho in breast cancer cells or treatment with soluble Klotho inhibited the activation of insulin/IGF-1 pathway. They further demonstrated that it induced up-regulation of the transcription factor CCAAT/enhancer-binding protein b (a breast cancer growth inhibitor) that is negatively regulated by the IGF-1-Akt axis. Further, Xie et al. (2013) have proposed that Klotho inhibited the phosphorylation of IGF-1R, which subsequently inhibited insulin receptor substrate -1 (IRS-1) phosphorylation and PI3K-Akt-mTOR signalling. This inhibition induces cell cycle to arrest at G0 and G1 phase in tumor cells.

1.2 Via Inhibition of Wnt Signalling

Wnt signalling plays principal regulatory roles in many developmental and biological processes, including embryonic development, cell proliferation, differentiation and tissue homeostasis. The Wnt signalling, activated by the binding of Wnt ligands to their cell surface receptors, is commonly divided into β-catenin dependent (canonical) and independent (non-canonical) pathways. The hallmark of the canonical Wnt pathway is the accumulation and translocation of β-catenin into the nucleus. Without Wnt signalling, cytoplasmic β-catenin is degraded. In the nucleus, β-catenin forms an active complex with lymphoid enhancer factor (LEF) and T-cell factor (TCF) proteins (the LEF/TCF DNA-binding transcription factors). This complex binds to the promoter of target genes which are required during embryogenesis as well as oncogenesis (Komiya and Habas, 2008; Behrens et al., 1996).

Aberrant increase in Wnt signalling is found in many human diseases, including cancer. A hallmark feature of stem cells, simulated by cancer cells, is their ability to maintain long telomeres by function of the TERT gene, which is directly enhanced by binding of β-catenin to its promoter region (Park et al., 2009). Thus, cancer stemness- the self-renewal potential of cancer cells, is linked to increased Wnt activity and is observed in colorectal, lung, breast and hepatocellular carcinoma (Zhan et al., 2016). Canonical Wnt ligands and receptors are often overexpressed in cancer cells whereas secreted antagonists are silenced (Zhan et al., 2016). Klotho is one of the endogenous antagonist of the Wnt/β-catenin signalling pathway. Secreted Klotho can bind to several Wnt proteins. This prevents Wnt binding to respective cell surface receptors and disrupts Wnt signalling (Voloshanenko et al., 2013).

1.3 Via inhibition of Fibroblast Growth Factor (FGF) signalling

FGF signalling is a highly complex growth factor signalling pathway, controlling a multitude of physiological functions. It drives cell proliferation, migration and survival during embryogenesis and later plays key roles in organogenesis. The mammalian FGF family comprises of 18 secreted proteins that interact with 4 signalling tyrosine kinase FGF receptors (FGFRs). Activated FGFRs phosphorylate specific tyrosine residues that mediate interaction with cytosolic adaptor proteins and the RAS-MAPK, PI3K-Akt, Phospholipase C-γ (PLCγ), and Signal Transducer and Activators of Transcription (STAT) intracellular signaling pathways (Ornitz and Itoh, 2015).

The FGF signalling pathway cross talks with the canonical Wnt signalling cascade to regulate transcription of target genes. FGF family members, such as FGF-18 and FGF-20, are directly up-regulated by the canonical Wnt signalling cascade as a result of transcriptional activation depending on the β-catenin-TCF/LEF complex (Shimokawa et al., 2003; Chamorro et al., 2005). FGF and canonical Wnt signals mutually regulate their transcription programs at the levels of ligands, receptors, and transcriptional regulators to coordinate cell fate and proliferation (Katoh and Nakagama, 2014).

Klotho as well as β-Klotho function as co-receptors for FGFs. While Klotho regulates phosphate and vitamin D metabolism through FGF-23, β-Klotho forms a complex with FGFRs and functions as a co-receptor for FGF-19 and FGF-21 (Kurosu and Kuro-o, 2009). Luo et al. (2010) suggested that β-Klotho is essential in FGFR4-dependent negative control of hepatic cell proliferation and hepatocarcinogenesis. β-Klotho mediated tumor suppressive effects are dependent on the FGFR4 kinase activity and result in activation of Extracellular Signal-regulated Kinase-1/2 (ERK1/2) signalling and reduction in Akt signalling. This in turn causes glycogen synthase kinase 3 beta (GSK-3b -a critical regulator of cyclinD1 expression) activation and subsequent cycling D1 degradation. This Akt/GSK-3b signaling has been reported to play an important role in liver cancer (Scharf and Braulke, 2003).

1.4 DOWN-REGULATION OF KLOTHO - EPIGENETIC SILENCING

Klotho down-regulation has been observed in various cancers while it's over-expression has shown opposite effects. Epigenetic alterations, namely, DNA methylation and histone modifications play an important part in silencing of tumor suppressor genes. Rubinek et al. (2012) have reported that methylation of KL promoter and deacetylation of its histones in breast cancer cells, decreased Klotho expression in the early stages of breast tumorigenesis. Further, decreased Klotho levels contributed to drug resistance in lung cancer cells (Chen et al., 2016). This strengthens its role as a therapeutic agent in cancer.

Thus in summary, Klotho has a preeminent role in cancer biology through insulin/IGF-1, Wnt and FGF pathways. However, it is beyond the scope of this review to cover all types of cancers in detail. In the last decade, role of Klotho in Gastrointestinal (GI) cancers has been evaluated in detail. GI cancers are leading contributor to cancer-related morbidity and mortality worldwide. In the year 2012, 27% of cancer related deaths in India were due to GI cancers (Sharma, 2016). In this review, we have focussed on the role played by Klotho in oral and GI cancers.

2. KLOTHO IN ORAL AND GI CANCERS

2.1 ORAL CANCER

Oral cancer, a disease of multifactorial origin, refers to malignant neoplasm arising from the lining mucosae of the lips and mouth (oral cavity), including the anterior two thirds of the tongue. It is traditionally defined as a squamous cell carcinoma, because in the dental area, 90% of cancers are histologically originated in the squamous cells (Rivera, 2015). Genetic mutations and aberrant epigenetic modifications have been reported in oral squamous cell carcinomas (OSCC). Silencing of tumor suppressor genes by promoter region hypermethylation is a notable process in cancer and leads to growth advantages similar to deletions and mutations (Herceg and Hainaut, 2007). DNA methyltransferase (DNMT) is the key enzyme that brings about promoter region methylation. The DNMT family includes DNMT1, TRDMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L (Jin and Robertson, 2013). Studies have reported higher level of DNMT3a immune-positivity in squamous cell carcinoma groups (Daniel et al., 2010; Adhikari et al., 2017). A significantly decreased expression of Klotho in OSCC when compared with non-tumour cells as well as oral dysplastic lesions (ODL) has been reported by Adhikari et al. (2017). In their study, the expression levels of DNMT3a and Klotho were found to be inversely related. They suggested that the down-regulation in Klotho expression may be due to DNA hypermethylation, which may have been induced by the overexpression of DNMT3a. Thus, Klotho may be a reliable gene for early detection of methylation changes in oral tissues, and can be used as a target for therapeutic modification in oral cancer during the early stages.

2.2 GASTROINTESTINAL CANCERS

2.2.1Esophageal Cancer

Evidence suggests a role for β-catenin in the carcinogenesis of esophageal squamous cell carcinoma (ESCC). Several studies report that its accumulation in cytoplasm frequently occurs in ESCC (Kimura et al,. 1999; Zhou et al., 2002). Tang et al. (2016) found that Klotho expression was much lower in ESCC than in adjacent noncancerous tissues. It was inversely related to clinical staging, histological grade, lymph node metastasis, and invasion depth as well as with cytoplasmic accumulation of β-catenin. Thus it was proposed that the loss of Klotho expression in the tumorigenesis of ESCC allows binding of Wnt to its receptor, leading to the accumulation of β-catenin in cytoplasm. This study further supports the hypothesis that Klotho suppresses the development of cancer by binding to Wnt and inhibiting the Wnt/β-catenin signalling. However, further study is warranted to prove this association (Tang et al., 2016).

2.2.2Gastric Cancer

Aberrant DNA methylation of promoter region of gene, which causes inactivation of tumor suppressor and other cancer-related genes, is the most explicit epigenetic hallmark in gastric cancer (Qu et al., 2013). Epigenetic silencing of Klotho gene by methylation of its promoter was detected in gastric cancer cells (Xie et al., 2013; Wang et al., 2011). In vitro studies by He et al. (2014) suggest that miR-199a-5p (an oncogene in gastric cancer) functions by targeting Klotho. Demethylation of the promoter region of KL increases Klotho expression, induces apoptosis and autophagy through down-regulation of IGF-1R, IRS-1, PI3K, Akt and mTOR phosphorylation as well as inhibits growth of gastric cancer cells (Xie et al., 2013; Wang et al., 2011).

2.2.3Colorectal Cancer

The canonical (β-catenin-dependent) Wnt signalling pathway plays major role in intestinal pathology. Studies have established a direct link between Wnt signalling and colorectal cancer wherein its aberrant activation by different mutations of adenomatous polyposis coli (APC) is documented (Buchert et al., 2010; Zhan et al., 2016). The loss of APC, a negative regulator of Wnt pathway, is the classic feature of this cancer (Aoki and Taketo, 2007). Wnt3a, an important ligand in the colon is overexpressed in colorectal cancer (Voloshanenko et al., 2013). Klotho can bind to this ligand and inhibit Wnt signalling activity. It has been well documented that Klotho expression is decreased in colorectal cancer (Gan et al.,2011; Pan et al., 2011; Yang et al., 2014; Perveez et al., 2015). Thus, there is a strong basis for the role of Klotho in regulation of Wnt signalling pathway in colonic tumorigenesis. Additionally, a study by Li et al. (2014) reported that overexpression of Klotho inhibited cell proliferation and invasive potential in colon cancer cells through inhibition of IGFR mediated PI3K/Akt pathway.

2.2.4Liver Cancer

Tang et al. (2016) found that hepatocarcinoma patients with Klotho-expressing tumors had longer periods of survival as compared to those without Klotho. Experimental data showed that Klotho expression inhibited the proliferation of hepatocellular carcinoma (HCC) cells and induced apoptosis in HepG2 and SMMC-7721 cell lines (Sun et al., 2015). Overexpression or recombinant administration of Klotho decreased the expression levels of β-catenin, C-myc, and Cyclin D1 in HepG2 cells. This inhibiton of hepatocellular tumour was mediated via inhibition of Wnt/ β-catenin signalling (Tang et al., 2016; Sun et al., 2015). Epigenetic silencing of KL via promoter hypermethylation was correlated to poor prognosis and was found in 81% of HCC cases (Xie et al., 2013). Whereas, restoration of Klotho expression induced cell apoptosis and autophagy through regulation of IGF-1R phosphorylation. (Xie et al., 2013; Shu et al., 2013).

Apart from Klotho, its homologue β-Klotho, which is predominantly expressed in the liver, pancreas and white adipose tissue has recently been implicated in inhibiting cell proliferation and tumorigenesis (Ito et al., 2000; Luo et al., 2010). Of the four FGFR tyrosine kinases, FGFR4 is dominant in mature hepatocytes and in association with β-Klotho mediates endocrine control of hepatic metabolism, plays a role in cellular homeostasis and hepatoma suppression (Luo et al., 2010). Recent studies have reported that β-Klotho expression is down-regulated in human hepatocellular carcinoma tissues compared with that in normal hepatocytes, while reintroduction of β-Klotho into hepatoma cells inhibited their proliferation (Luo et al., 2010; Ye et al., 2013). The β-Klotho-FGFR4 interaction causes a depression of Akt signalling (Luo et al., 2010). The anti-proliferative effect of β-Klotho might be linked with G1 to S phase arrest, mediated by Akt/GSK-3β/cyclin D1 signalling (Ye et al., 2013). These studies suggest that β-Klotho suppresses tumor growth in hepatocellular carcinoma, however the extent to which β-Klotho-dependent FGFR4 signalling pathways are responsible for control of hepatocyte proliferation and hepatoma suppression is a subject for future investigation.

Intrestingly, there are contradictory reports about the role of Klotho in HCC. A study by Poh et al. (2012) found elevation of β-Klotho expression in HCC tumors compared to normal liver tissues and this up-regulation correlated with multiple tumor formation. They showed that silencing of β-Klotho gene in Huh7 cells decreased cell proliferation and suppressed FGFR4 downstream signaling, resulting in decreased protein expression of alpha-fetoprotein (AFP), a HCC diagnostic marker. Thus, they suggested that β-Klotho is instrumental in driving elevated FGFR4 activity in HCC progression. Similar report by Chen et al. (2013) noted an oncogenic function of Klotho in promoting anoikis resistance via activating vascular endothelial growth factor receptor-2/p21-activated kinase-1 (VEGFR2/PAK1) signalling, and thereby facilitating tumor migration and invasion during hepatoma progression.

These contradictory reports compel further investigation of the role of β-Klotho in hepatocarcinoma.

2.2.5Pancreatic Cancer

In Panc1, MiaPaCa2, and Colo357 - pancreatic adenocarcinoma cell lines, it was demonstrated that Klotho overexpression reduced phosphorylation of the IGF-1R and its downstream targets IRS-1, Akt-1, and ERK-1/2. Furthermore, IGF-1R activation in Panc1 cells was also inhibited by soluble Klotho (Abramovitz et al., 2011). The inhibition of the IGF-1 and FGF pathways suppressed tumorgenicity and increased sensitivity of pancreatic tumors to radiation and chemotherapy (Min et al., 2005; Vickers et al., 2002). Thus, Klotho, regulator of both the pathways, plays an important role in pancreatic cancer. Recent in vitro and animal studies have revealed that KL1 domain of Klotho is responsible for its inhibitory effect on IGF-1 and FGF pathways in breast and pancreatic cancer cells. Overexpression of Klotho and KL1 domain, but not KL2, effectively reduced inhibited growth of cancer cells (Ligumsky et al., 2015; Abramovitz et al., 2011). Abramovitz et al. (2011) demonstrated that down-regulation of Klotho expression in pancreatic adenocarcinoma and attributed the tumor suppressive function of Klotho to the KL1 domain. Kl1 domain is common to all forms of Klotho.

In a study on tumour suppressive role of Klotho in pancreatic ductal adenocarcinoma (PDAC), Jiang et al. (2014) have suggested that miR-504, a negative regulator of human p53, acts as an oncogene in PDACs through down-regulation of expression of KL. They reported a significantly increase in miR-504 expression and phosphorylated IGF-1R levels which were correlated with the KL promoter hypermethylation. Blocking miR-504 activity using antimiR-504 antisense inhibitor convincingly recovered Klotho gene expression and inhibited proliferation, invasion, and migration of pancreatic cancer cells.

An overview of the signalling pathways modulated by Klotho in GI cancers is provided in Table 1.

CONCLUSION

Epigenetic changes in the anti-aging gene Klotho is one of the emerging areas of concern in the study of carcinogenesis. Majority of studies have depicted the exact role of Klotho via modulation of signalling pathways in GI cancers while some have indirectly correlated cancer progression with its down regulation. Further, its therapeutic role has also been experimented. Recent studies have revealed that α-Klotho and KL1 domain, but not KL2 are responsible for cancer inhibition. Full length α-Klotho and its KL1 domain have different physiologic roles. Thus, KL1 treatment specifically may be a safe and effective therapy. Additional studies are necessary to corroborate this. In addition to its use as a therapeutic agent, its promoter methylation may be used to predict the prognosis of cancer patients. However, the therapeutic potential and its reliability as biomarker in cancer needs to be validated by further studies.

CONFLICT OF INTEREST

The authors claim no conflict of interest.

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Figure 1: Klotho gene, transcripts and proteins (Bian A et al., 2015). (1) Membrane Klotho transcript arises from the Klotho gene (5 exons) and encodes a single-pass transmembrane protein. (2) Secreted Klotho arises from an alternative RNA splicing, wherein the internal splice donor site is in exon 3. The resultant alternatively spliced transcript contains insertion of 50 bp after exon 3, with an in-frame translation stop codon at the end. The translation product is the secreted Klotho, released directly into blood circulation. (3) The extracellular domain of membrane Klotho, consisting of KL1 and KL2 repeats, is susceptible to proteolytic cleavage by α/β-secretases and released into the circulation. Thus, there are two forms of Klotho protein in the circulation; one containing Kl1 domain only and another has KL1 and KL2 domains.

Table 1: Overview of various studies reporting the signalling pathways modulated by Klotho in different GI cancers.

Studies

Cancer

Type of Klotho

Experimental Finding

Signalling pathway modulated by Klotho

Tang et al., 2016

Esophageal

Klotho

Loss of expression

Wnt/β-catenin

Wang et al., 2011,

Xie et al., 2013

Gastric

Klotho

Promoter hypermethylation

Insulin/IGF-1

He et al., 2014

Might be the downstream target of the oncogene; miR-199a-5p.

-

Gan et al.,2011; Pan et al., 2011; Yang et al., 2014; Perveez et al., 2015

Colorectal

Klotho

Expression was decreased

Wnt/β-catenin

Li et al.,2014

Over expression inhibited colon cancer

IGFR mediated PI3K/Akt Pathway

Sun et al., 2015; Tang et al., 2016

Liver

Klotho

Inhibited HCC

Wnt/β-catenin

Xie et al., 2013

Shu et al., 2013

Promoter hypermethylation

correlated to poor prognosis

Insulin/ IGF-1

Luo et al., 2010;

Ye et al., 2013

-Klotho

Overexpression inhibited cell proliferation and tumerogenesis

Akt/GSK-3β/cyclin D1 signalling through FGFR4

Poh et al., 2012

Liver

-Klotho

Elevation of expression in HCC tumors

FGFR4

Chen et al., 2013

Klotho

Facilitated tumor migration and invasion

VEGFR2/PAK1

Abramovitz et al., 2011

Pancreatic

Klotho

Down-regulated in pancreatic tumour

Insulin/ IGF-1 and FGF

Jiang et al., 2014

Klotho promoter hypermethylation

Insulin/ IGF-1