Vitamin D and K signaling pathways in hepatocellular carcinoma

Accepted Manuscript Vitamin D and K signaling pathways in hepatocellular carcinoma

Manal L. Louka, Ahmed M. Fawzy, Abdelrahman M. Naiem, Mustafa F. Elseknedy, Ahmed E. Abdelhalim, Mohamed A. Abdelghany PII: DOI: Reference:

S0378-1119(17)30608-X doi: 10.1016/j.gene.2017.07.074 GENE 42098

To appear in:

Gene

Received date: Revised date: Accepted date:

20 June 2017 25 July 2017 27 July 2017

Please cite this article as: Manal L. Louka, Ahmed M. Fawzy, Abdelrahman M. Naiem, Mustafa F. Elseknedy, Ahmed E. Abdelhalim, Mohamed A. Abdelghany , Vitamin D and K signaling pathways in hepatocellular carcinoma, Gene (2017), doi: 10.1016/ j.gene.2017.07.074

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ACCEPTED MANUSCRIPT Vitamin D and K signaling pathways in hepatocellular carcinoma *Manal L. Loukaa, Ahmed M. Fawzy b, Abdelrahman M. Naiemb, Mustafa F. Elseknedyb, Ahmed E. Abdelhalimb, Mohamed A. Abdelghanyb a

Medical Biochemistry Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt

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Undergraduate, Armed Forces College of Medicine (AFCM), Cairo, Egypt

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Corresponding Author: *[email protected], +201222191007, Medical Biochemistry Department, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt, 11381

ACCEPTED MANUSCRIPT Abstract Hepatocellular carcinoma (HCC) is a primary liver malignancy, and is now the six most common in between malignancies. Early diagnosis of HCC with prompt treatment increases the opportunity of patients to survive. With the advances in understanding the molecular biology of HCC, new

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therapeutic strategies to treat HCC have emerged. There is a growing consensus that vitamins are important for the control of various cancers. Biochemical evidence clearly indicates that HCC cells

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are responsive to the inhibitory effect of vitamin D, vitamin D analogues and vitamin K. In this

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review, we summarize the mechanisms used by vitamin D and K to influence the development of HCC and the latest development of vitamin analogues for potential HCC therapy.

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Keywords: hepatocellular, carcinoma, vitamin, signaling, analogues

ACCEPTED MANUSCRIPT 1. Introduction Hepatocellular carcinoma (HCC) is a primary liver malignancy, which originates mainly from the hepatic stem cells almost in patients with chronic liver disease (Alison et al., 2005). It is now the six most common between malignancies (Bravi et al., 2013). It is the fifth most common cancer in

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men, worldwide, and seventh among women, with over half a million new cases diagnosed annually worldwide. It is the second leading cause of cancer related mortality in the world (World

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Health Organization Mortality Database, 2008).

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HCC represents a public health problem in Egypt. It constitutes 70.48% of all liver tumors among Egyptians, representing the second most common malignancy after bladder cancer in men and

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breast cancer in women and the second most common cause of death in men (Curado et al., 2007;

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Baghdady et al., 2014). It has a prevalence of 14.8% among causes of cancer mortality. It is

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expected that by 2050, the incidence will increase up to three folds in comparison with the incidence in 2013 (Ibrahim et al., 2014).

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Early diagnosis of HCC with prompt treatment increases the opportunity of patients to survive so

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this necessitates the surveillance for it especially in individuals with high risk (Kim et al., 2011).

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The Barcelona-Clınic Liver Cancer (BCLC) staging system is the gold standard system in grading and staging of HCC (El-Serag, 2011). Diagnosis of HCC can be confirmed not only by imaging finding but also by the clinical picture of patient as liver cirrhosis or chronic hepatitis. Also lately, it was proved that the alpha feto protein has no adequate sensitivity and specificity for effective diagnosis (Singal, 2009). The current treatment of HCC include surgical resection, thermal or chemical ablation, medical treatment and liver transplantation. With the advances in understanding the molecular biology of HCC, new therapeutic strategies to treat HCC have emerged (Villanueva

ACCEPTED MANUSCRIPT et al., 2007). While there is a growing consensus that vitamins are important for the control of various cancers, the mechanistic foundation for this protection is still being determined. In this review, we summarize the current thinking on the mechanisms used by vitamin D and K to influence the development of HCC and the latest development of vitamin analogues for potential

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HCC therapy.

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2. Vitamin D

Vitamin D, identified as a hormone to maintain blood calcium homeostasis and promote skeletal

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mineralization, has been demonstrated to exert additional functions, including anti-proliferative,

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pro-differentiation, pro-apoptotic, anti-angiogenesis and anti-invasive characteristics in many

clinical trials (Diaz et al., 2015).

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cancer cell systems during the past two decades. These antitumor activities have led to several

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Epidemiological evidence indicated that decreased concentrations of vitamin D were associated

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with an enhanced risk of various types of cancer (Giovannucci et al., 2009) as colorectal (Garland et al., 1989), breast (Bertone- Johnson et al., 2005) and prostate cancer (Ahonen et al., 2000).

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The role of vitamin D in the pathogenesis of hepatic diseases is actually of great interest. Previous

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studies have indicated that 1,25(OH)2D3 inhibited the proliferation of HCC in a dose dependent manner (Pourgholami et al., 2000). It was suggested that low vitamin D levels might be a risk factor for hepatocellular carcinoma. This was supported by the finding of each 10-nmol/L decline of serum vitamin D being associated with a 20% increase in HCC risk in the EPIC (The European Prospective Investigation into Cancer and Nutrition) (Colombo et al., 2014). Recently, it was reported that serum vitamin D acted as a prognostic parameter in 200 patients with HCC. The overall survival rates in patients with lower serum 25 (OH) D3 concentrations

ACCEPTED MANUSCRIPT (≤10ng/ml) were significantly lower than those in patients with higher serum 25 (OH) D3 concentrations (>10ng/ml) (Finkelmeier et al., 2014). Vitamin D deficiency was associated with low rate of sustained virological response (SVR) in patients affected by hepatitis C virus (HCV) under interferon-alfa therapy. Furthermore, an

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intervention trial showed that vitamin D supplementation improved the probability of achieving a

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SVR following antiviral treatment in patients with recurrent hepatitis (Gascon-Barré et al., 2014). Vitamin D status is recommended to be assessed in all patients with Chronic Liver Disease and, if

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deficiency is present (<50 nmol/L or 20 ng/ml), supplementation with 1000–4000 IU/day of vitamin D3 should be initiated, with the initial dose dependent upon baseline 25(OH) D3 levels.

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However, increasing evidence suggests that supplementation should be considered for a 25(OH)

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D3 level<75 nmol/L, especially in those considering interferon-based antiviral therapy for CHC

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(Kitson et al., 2012).

Several studies have shown high levels of CYP24A1 expression in HCC. An increase in CYP24A1

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can lead to lower levels of vitamin D thereby allowing for tumor growth. Treatment with vitamin

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D3 decreased the proliferative rate in numerous HCC cell lines (Kennedy et al., 2013).

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Vitamin D has 2 types: vit D2 and vit D3, both of them are rarely found in food. They originate from different sources; vit. D2 (ergocalciferol) is synthesized from ergosterol of yeast, while vit. D3 (cholecalciferol) is produced from 7- dehydrocholesterol (7-DHC) of lanolin. Exposure to sunlight provides about 90% of the human requirement of vitamin D (Holick, 2003). Biologically, vitamin D is inactive, Vitamin D (cholecalciferol) is first hydroxylated in the liver by vitamin D25-hydroxylase (25-OHase) to be (25-OH cholecalciferol) (Schuster, 2011). The level of

ACCEPTED MANUSCRIPT circulating vitamin D is measured by the level of 25-OH cholecalciferol. It circulates in blood bound to vitamin D-binding protein. Once it enters the kidney, it is hydroxylated again mainly in the PCT (Proximal Convoluted Tubule) by the enzyme 25(OH) D-1α -hydroxylase or CYP27B1. Finally, this is the most active

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form of vitamin D and may be hydroxylated again by the action of 24-hydroxylase to terminate its action (Schuster, 2011). These enzymes may be found in some other extra renal tissue (Townsend

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et al., 2005) and act and degrade locally in an autocrine or paracrine mechanism that is regulated

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in a tissue-dependent mechanism and has no relation in calcium homeostasis. Once 25(OH) D3 is internalized in the cell its fate depend on the relative expression of 24-OHase to 1α-OHase with

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relative dominancy of 1α-OHase. 25(OH) D3 is converted into 1α,25(OH)2D3 which perform its

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non-calcemic action and increase the expression of 24-OHase which degrades the locally

2.1. Functions of vitamin D

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generated 1 α,25(OH)2D3 (Townsend et al., 2005).

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The action of vitamin is mediated through vitamin D receptor (VDR) which is a member of nuclear

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receptor family (Haussler et al., 1997). Vitamin D-VDR complex can modulate gene expression in a-cell and tissue specific manner (Norman, 2006). There are 2776 VDR binding sites being

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identified by a chip sequencing method located within 229 vitamin-D-regulated genes (Ramagopalan et al., 2010). Since the presence of VDR in tissues wasn't associated with calcium and bone metabolism (Stumpf et al., 1979), the 1α,25(OH)2D3 induced anti-proliferation, anti-inflammatory response, prodifferentiation,

pro-apoptosis

and

immune

regulation

were

well

found to be tissue- and cell-specific (Chiang et al., 2009; Adams et al., 2010).

established

and

ACCEPTED MANUSCRIPT VDR requires further dimerization with retinoid X receptor (RXR) to form a heterodimer to bind to vitamin D response element in the promoter region of vitamin D responsive gene to perform its genomic action (Tsai et al., 1994). The VDR-mediated gene expression is further modulated by a multiple of co-factors (Haussler et al., 1998).

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2.1.1. Anti -inflammatory and anti-fibrotic effect

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Proinflammatory signals in monocytes and macrophages may regulate the local metabolism of vitamin D, auto-inducing the expression of CYP27B1 and the local production of 1α,25(OH)2D3,

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and thus controlling the excessive inflammatory response ( Liu et al., 2006). Almost 90% of the tissue macrophages are in the liver (Bilzer et al., 2006) which suggests that the liver production of

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active vitamin D is affected during inflammatory diseases of the liver. Furthermore, VDR is

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expressed in both macrophages and other non-parenchymal cells and biliary epithelial cells

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(Gascon-Barré et al., 2003). In the liver, vitamin D acts as an “immune-modulator” suppressing fibroblast proliferation and collagen production. Hammad et al. (2013) indicated that vitamin D,

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as well as IL-6 and IL-17, were potential biomarkers for the development of HCC in patients with

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hepatitis C.

Importantly, vitamin D was inversely proportional to IL-6 (Hammad et al., 2013) which had an

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effect on secretion of thrombopoietin (TPO) p that stimulated hepG2 and hep3B cells up to 1.5 fold in incubation periods 24-h (Wolber and Jelkmann, 2000). So, the use of 1α,25(OH)2D3 inhibited the proliferation of HepG2 cells after 5 days of treatment so there was decrease in cell numbers at 10 nmol/L level (Wolber and Jelkmann, 2000; Akhter et al., 2001; Hammad et al., 2013). Any decrease in vitamin D reflected the grade of hepatocellular injury (El Husseiny et al., 2012).

ACCEPTED MANUSCRIPT Chronic inflammation and fibrosis are hallmarks of HCC. Vitamin D has an anti-fibrotic effect in the liver by suppressing the production of collagen in stromal hepatic stellate cells (HSCs), thus dietary vitamin D supplementation may be a therapeutic strategy to combat HCC progression or the associated loss of liver function (Duran et al., 2016).

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VDR knockout mice spontaneously develop hepatic fibrosis. In turn, VDR ligands inhibit TGF-βinduced liver fibrosis by binding to co-regulated genes such as Tgfb1, Mmp7 and Ctgf and reduce

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Smad3 occupancy at these sites (Ding et al. 2013). VD metabolites suppress TGF-β-mediated

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fibrosis through modulating multiple pro-fibrotic proteins, for instance, lowering collagen I and III expression and raising expression levels of MMP8, a metalloproteinase that degrades collagens

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(Artaza et al., 2009).

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VD deprivation (VDD) in in vivo models of liver tumor formation revealed striking three-fold

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increase in tumor burden in Smad3 mutant mice, with a three-fold increase in Toll-like receptor 7 (TLR7) expression compared to controls. Molecular interactions between TGF-β pathway and

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VDD were clinically validated, where an absence of VD supplementation was associated with low TGF-β pathway member expression levels and β-catenin activation in fibrotic/cirrhotic human

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liver tissues. TGF-β member expression with lower β-catenin levels were restored by

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supplementing VD. VDD promotes tumor growth in the context of Smad3 disruption, potentially through regulation of TLR7 expression and β-catenin activation. Vitamin D could therefore be a strong candidate for liver cancer prevention in the context of aberrant Smad3 signaling (Chen et al., 2016). The anti-fibrotic effect of vitamin D involves regulating activation of stellate cells and reducing Smad3 occupancy on the promoter of pro-fibrotic genes, suggesting a permissive role of vitamin

ACCEPTED MANUSCRIPT D that favors the quiescent state of non-activated stellate cells in the physiology of the liver (Ding et al., 2013). 2.1.2. Anti-proliferative and pro-apoptotic effect

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The anti-proliferative effect of 1α,25(OH)2D3 on HCC was mainly attributable to cell cycle arrest

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at G0/G1, leading to increased fraction of cells at G0/G1 phase and decreased fraction of cells at

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S phase (Caputo et al., 2003).

Cell proliferation requires fluent transition of cells through a sequence of events leading to their

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replication. Cell cycle progression is positively and cooperatively regulated by cyclins and CDKs.

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The catalytic activity of CDKs, in turn, is negatively controlled by CDK-inhibitors (CKI) such as the members of the Cip/Kip family, also known as tumor suppressor genes. Among the members

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of this family are p21 and p27, implicated in promoting the G0/G1 cell cycle arrest (Biggs et al.,

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1995). Some cell cycle regulatory factors are targets of calcitriol either directly or indirectly through VDREs in their promoter regions. A 1,25(OH)2D3 action, is through the induction of p21

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and p27, leading to suppression of cyclins (D1, E and A) and cyclin- dependent kinases 2 and 4 in

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many cancer cell lines (Wu et al., 1997; Hager et al., 2001; Sundaram et al., 2003).

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HDAC2 is usually expressed at high levels in tumors, and its downregulation leads to high expression levels of cell cycle components, including p21(WAF1/Cip1). 1, 25(OH)2D3 treatment reduced the expression of HDAC2 and increased the expression of p21(WAF1/Cip1), in a dosedependent manner, resulting in the reduction of HCC growth (Huang et al., 2016). Data from in vitro studies supported the significance of vitamin D in chronic hepatitis C, suggesting 25(OH) D as a suppressive factor of HCV replication (Matsumura et al., 2011).

ACCEPTED MANUSCRIPT There are several associated pathways with vitamin D. It has an effect on induction of thioredoxininteracting protein which is reduced in patients in many cancers (Kim et al., 2007; Zhou et al., 2011). Thioredoxin-interacting protein (TXNIP) expression suppresses the proliferation and induces the apoptosis in liver cells and it is notably that TXNIP is a priming of these cancer cells

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for oxidative injury. Significantly, TXNIP is decreased in HCC so these findings suggest that

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stimulation of TXNIP expression, by factors such as vitamin D3, may attenuate the pathogenesis

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of HCC in patients with chronic liver disease (Hamilton et al., 2014).

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Additionally, 1α,25(OH)2D3 downregulates the expression of Epidermal growth factor receptor. As a result it inhibits mitogen-activated protein kinase (MAPK) that induces apoptosis and

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differentiation of cells to stop the growth (Deeb et al., 2007).

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2.1.3. Anti-angiogenic effect

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Above all, 1α,25(OH)2D3 inhibits blood vessel formation in order to prevent the vascular endothelial growth factor (VEGF)-mediated hepato-cacinogenesis (Iseki et al.,1999; Chung et al.,

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2006).

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2.2. Vitamin D receptor (VDR), Vitamin D binding protein (DBP) and vitamin D

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metabolizing enzymes 2.2.1. Polymorphism

Vitamin D receptor (VDR) is an important mediator for effect of vitamin D on cells as it mediates its gene expression and it interacts with other pathways that affect cancer development (Walters, 1992; Deeb et al., 2007). The vitamin D receptor (VDR) gene is located on chromosome 12. It encodes a 48 kDa soluble protein that is a member of the nuclear receptor family of ligand-activated transcription factors. In

ACCEPTED MANUSCRIPT vitro VDR has a role in cell proliferation and differentiation and stimulation of apoptosis (Slattery, 2007; Gu et al., 2014). Vitamin D receptor is highly polymorphic and there are many single nucleotide polymorphisms (SNPs) in VDR gene such as polymorphism of the Bsm I (G/A) and Apa1 which they are believed

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to be a silent SNPs. They notably do not change the sequence of amino acid but affects the

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expression of the gene via regulation of mRNA stability (McClung et al., 2006; Köstner et al.,

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2009; Youssef et al., 2016; Zhou et al., 2009). SNPs is variations in gene of VDR that affect vitamin D effect against the tumors so it is associated with increasing the risk of many tumors such

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as breast (Dalessandri et al , 2012 ), prostate (Shui et al , 2012 ), and colorectal (Rasool et al ,

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2014). Importantly, Bsm Ι (G/A) has been found in epithelial origin cancers like prostate, breast, lung, ovarian, skin cancers and HCC (Raimondi et al., 2009, Yin et al , 2009; Falleti et al., 2010 ).

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In hepatocellular carcinoma, it was found that carriage of the (GG) genotype of Bsm Ι (G/A) was

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strongly associated with the occurrence of HCC in patients with liver cirrhosis (Falleti et al., 2010). However there was no association between VDR Bsm Ι (G/A) polymorphism and HCC in patients

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with chronic HBV infection (Xing et al., 2013).

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Additionally Lange et al. (2013) aimed to determine the relationship between genetic determinants

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of vitamin D serum levels and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Associations between CYP2R1, GC, and DHCR7 genotypes that are determinants of reduced 25-hydroxyvitamin D (25[OH]D3) serum levels and the risk of HCVrelated HCC development were investigated for 1279 chronic hepatitis C patients with HCC and 4325 without HCC, respectively. The well-known associations between CYP2R1 (rs1993116, rs10741657), GC (rs2282679), and DHCR7 (rs7944926, rs12785878) genotypes and 25(OH) D3 serum levels were also apparent in patients with chronic hepatitis C. The same genotypes of these

ACCEPTED MANUSCRIPT single nucleotide polymorphisms (SNPs) that were associated with reduced 25(OH) D3 serum levels were found to be associated with HCV-related HCC. In contrast, no association between these genetic variations and liver fibrosis progression rate or outcome of standard therapy with pegylated interferon-α and ribavirin was observed, suggesting a specific influence of the genetic

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determinants of 25(OH) D3 serum levels on hepato-carcinogenesis. In hepatocellular carcinoma (HCC), complicating alcohol related cirrhosis, variation in the allele

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frequency of the BsmI, ApaI, and TaqI, but not FokI VDR polymorphisms was associated with

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HCC development when compared to cirrhotic patients without HCC. Furthermore, there was a significant difference in allele frequency of these VDR polymorphisms in alcohol-related cirrhosis

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compared to cirrhosis complicating chronic viral hepatitis (Falleti et al., 2010).

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However recently, VDR genetic polymorphisms (FOKI) were significantly associated with the occurrence of HCV related HCC especially f allele carriers. Carriage of (ff) had a significantly

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higher risk for development of HCC after adjustment with age, HCV infection, BMI and HOMA-

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IR with F allele as a reference. This could be considered as a risk factor of hepatocellular carcinoma and could be used as a molecular marker to predict the risk and to evaluate the disease severity of

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HCC in those infected with HCV (Nada et al., 2016).

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Beilfuss et al. (2014) demonstrated that the polymorphisms in the VDR gene influenced VDR expressions and production and was involved in the fibrotic processes in human hepatic stellate cells. The rs2228570 (FOKI) polymorphism is located in the 5′ end of the VDR gene. This polymorphism resulted in an alternative transcription initiation site, leading to a protein variant with three additional amino acids at the amino terminus (Uitterlinden et al., 2004).

ACCEPTED MANUSCRIPT The homozygous FokI (TT) and heterozygous (CT) polymorphism and vitamin D levels had independent effect on cancer development and were not synergistic in their actions (Targher et al., 2007). Moreover, expression of VDR and CYP27B1 were significantly lower in hepatocellular carcinoma

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compared with non-tumorous liver in a study done by Horváth et al. (2016). This indicated decreased bioavailability of 1,25-Dihydroxy vitamin D3, providing an escape mechanism from the

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anti-tumor effect. CYP27B1-1260 promoter polymorphism resulted in reduced 1,25OHD levels

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and were associated with failure to achieve sustained virological response (SVR) in patients with hepatitis C virus (HCV) genotypes 1, 2, and 3 (Lange et al., 2011). The change in the VDR protein

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structure led to altered biological functions of vitamin D and was involved in carcinogenesis (Arai

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et al., 1997).

vitamin

D

synthetic

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Thanapirom et al. (2017) reported that when patients were genotyped for functional variants on pathway

including GC (rs4588,

rs7041,

rs22020,

rs2282679),

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CYP2R1 (rs2060793,rs12794714), CYP27B1 (rs10877012),and DHCR7 (rs12785878),the DHCR

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7 polymorphism could be a pre-treatment predictive marker for response to PEG-IFN-based therapy in chronic HCV genotype 1 infection.

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Vitamin D binding protein (DBP) is the primary transport protein of 25(OH) D3 in circulation, with approximately 88% of 25(OH) D3 being bound to DBP (Powe et al., 2011). DBP is predominantly produced in the liver, but also in kidney, gonads, fat, and neutrophils. DBP determines how much free 25(OH) D3 substrate is available for 1 α-hydroxylase as well as the amount of free 1α,25(OH)2D3 ligand available to activate the VDR and influences downstream gene transcription (Kitson and Roberts, 2012).

ACCEPTED MANUSCRIPT In addition to being a carrier for vitamin D metabolites, DBP also has anti-inflammatory and immune regulatory functions, and has been identified playing a role in several chronic disease including cancers (Anic et al., 2014). The gene for the DBP is highly polymorphic (Pibiri et al., 2014). SNPs in the DBP locus associated with low 25(OH) D3 levels are rs2282679, rs7041,

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rs3755967, rs17467825, rs2298850, and rs1155563. The DBP rs7041 polymorphism contributed

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to an increased HBV-related HCC risk in Chinese population (Peng et al., 2014).

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Moreover, a proteomic study had shown vitamin DBP to be one of three metaproteins associated

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with sustained virological response (SVR) (Patel et al., 2011). DBP levels were significantly lower in subjects with significant or advanced fibrosis (METAVIR F2-4) compared with those with

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absent or minimal fibrosis (F0/1) and healthy controls (Gressner et al., 2009, Ho et al., 2010).

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Thus, vitamin D deficiency appears to be common in CHC and may be associated with adverse outcomes such as lower treatment response; more advanced fibrosis stage and increased severity

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of necroinflammation. It remains, however, uncertain as to whether vitamin D supplementation

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improves the SVR rate in patients receiving combination anti-viral therapy with PEGIFN and

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RBV.

2.2.2. CpG islands and DNA methylation

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Vitamin D signaling pathways including those encoding VDR and CYP genes have large CpG islands in their promoter regions. Vertebrate CpG islands (CGIs) are short interspersed DNA sequences that deviate significantly from the average genomic pattern by being GC-rich, and predominantly non methylated. They comprise 1–2% of the genome (Gardiner-Garden et al., 1987). Silencing of CGI promoters is achieved through dense CpG methylation. CGIs are therefore generically equipped to influence local chromatin structure and regulation of gene activity.

ACCEPTED MANUSCRIPT Excessive hyper-methylation can lead to the transcriptional silencing of genes critical to the normal anti-neoplastic process (Costello et al., 2001). However, hyper-methylation of promoter regions that bind transcriptional repressors can lead to increased transcription by interfering with the action of the repressor (Fetahu et al., 2001).

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Specific proteins that bind to CpG islands and protect them from de novo methylation including

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proteins containing a zinc (Zn) finger binding domain specific to non-methylated CpG regions and

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various histone demethylases that can actively demethylate CpG (Sceusi et al., 2011) . VDR principally targets several genes encoding histone modifiers and chromatin remodelers.

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Increased methylation levels in the promoter region of VDR and CYP genes may result in altered

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gene transcription, leading to impaired vitamin D synthesis associated with severe disease activities (Zhu et al., 2013; Zhou et al., 2014; Pilon et al., 2015).

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The study of methylation in HCC was challenging, as there were several well-known risk factors

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for HCC, such as alcohol-induced cirrhosis and chronic viral hepatitis B or C infection. HCC routinely arises in a distinctly abnormal and often cirrhotic liver. Because cirrhosis is in the causal

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pathway (from normal liver to HCC), it induces interactions resulting in carcinogenesis via

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silencing of tumor suppressor genes (APC, GSTP1, cox2, p16, E-cadherin, 14-3-3s, p15, SOCs1, RIZ1 etc) (Sceusi et al., 2011). As a consequence of inactivation of tumor suppressor genes,

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various signaling pathways were deregulated in HCC subsets, such as the Wnt/ ß-catenin pathway (Yang et al., 2011). Expression levels of VD-related genes correlated with TGF-β superfamily in human liver cancer. In specific patient populations with disruption of TGF-β signaling, low VD markers correlated with activation of the Wnt pathway and a high risk of tumorigenesis (Chen et al., 2016). However, epigenetic gene involvement of VDR in HCC pathogenesis remains to be further elucidated.

ACCEPTED MANUSCRIPT Moreover, CYP24A1 methylation (the enzyme responsible for the first step in calcitriol catabolism) tended to correlate with better prognosis in HCV-related HCC (Deng et al., 2010). Recently, Zhou et al. (2014) revealed a strong correlation of baseline DNA methylation profiles of CYP2R1 and CYP27A1 genes with response to vitamin D supplementation.

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2.2.3. Mutation

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The whole genome and transcriptomic analyses revealed that somatic mutations in genes involved

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in VD metabolism and VD-related genes occur frequently in hepatocellular cancers correlating with the TGF-β superfamily, suggesting that combined inactivating somatic mutations in VD-

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related genes and the TGF-β signaling members play a critical role in liver tumorigenesis (Chen

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et al., 2016).

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3. Vitamin D analogues

Scientists everywhere have tried their best to discover a new way for cancer's treatment, and that

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is because of its big influence on people all over the world.

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As no effective regimens are available for advanced HCC, new strategies are urgently needed.

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Morris et al. (2002) conducted a phase I clinical trial, where 1α,25(OH)2D3 dissolved in 5 mL lipiodol was injected through the hepatic artery. This could avoid the 24-OHase-mediated

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degradation of 1α,25(OH)2D3 in the liver and therefore could obtain higher concentrations of 1α,25(OH)2D3 in HCC. Eight cases of refractory HCC were administered with either 50, 75, or 100 mg 1α,25(OH)2D3 and none of the patients developed hypercalcemia over grade III, indicating this was a safe way to deliver 1α,25(OH)2D3. However, no obvious benefit on survival was observed in spite of transient stability of alpha-fetoprotein. Multiple studies have shown that calcitriol blocked the growth of cancer-derived cell lines in culture, and also in a variety of animal models (Flanagan et al., 2003; Swami et al., 2012,

ACCEPTED MANUSCRIPT Kasiappan et al., 2014; Lungchukiet et al.,2014; Chen et al., 2015; Jeong et al., 2015, Murray et al., 2015). To determine whether the VDR pathway affects in vitro proliferation rates of liver cancer cell lines, HepG2 and Hep3B human liver cancer cell lines were treated with increasing doses of VD and VD-analogues, EB1089 and QW1624F (0.01–1 μM), for 7 days. Inhibition of

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proliferation of liver cancer cell lines demonstrated the role of VDR signaling pathway (Chen et

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al., 2016).

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In order to overcome the barrier of inducing hypercalcemia, several analogues of calcitriol with

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low calcemic properties have been synthesized, some of which have undergone evaluation in clinical trials for potential anti-cancer activity (Leyssens et al., 2013; Chen et al., 2014).

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3.1. Calcipotriol

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Global or HSC-specific knockout of p62 promoted liver inflammation, fibrosis, and tumor growth

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in a carcinogen and high-fat diet–induced mouse model of HCC, and knockdown of p62 in cultured human HSCs induced the abundance of a stellate cell activation marker and the expression of the

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gene encoding collagen I (Duran et al., 2016). The vitamin D analogue calcipotriol induced a

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quiescent state in cultured HSCs, but p62 knockdown prevented this effect of vitamin D (Ferrarelli et al., 2016). p62 acted as a non-cell-autonomous tumor suppressor in liver cancer by counteracting

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the activation of hepatic stellate cells (Hu and Zender, 2016). Calcipotriol failed to increase the occupancy of the RXRα/VDR heterodimer at target promoters in p62-deficient HSCs and hence could not enable repression of the expression of fibrotic genes in HSCs. Clinical trials for vitamin D or VDR-activating agents in HCC may have better outcomes if patients were stratified based on stellate cell p62 abundance (Ferrarelli et al., 2016). 3.2. 19-nor-2a- (3-hydroxypropyl)-1α, 25(OH)2D3 (or MART-10)

ACCEPTED MANUSCRIPT Furthermore, another vitamin D analogue, 19-nor-2a- (3-hydroxypropyl)-1α, 25(OH)2D3 (or MART-10), was studied in HepG2 cells. It was about 100-fold more potent than 1α,25(OH)2D3 in inhibiting the proliferation of HepG2 cells through cell cycle arrest at G0/G1 phase by upregulating p21 and p27 tumor suppressor genes (Chiang et al., 2011). This higher potency than 1α, 25(OH)2

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D3 was because of being a poor substrate to CYP24A1, the main enzyme to catabolize 1α,

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25(OH)2D3 in humans, so it could act longer (Luo et al., 2016). Additionally, it had a higher VDR-

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binding affinity than 1α, 25(OH)2 D3, which was confirmed by the X-ray crystallographic structure of MART-10 complex bound with VDR (Hourai et al., 2009). Therefore, MART-10 was a

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promising candidate for further studies as a new therapeutic regimen against HCC.

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In addition, MART-10 had an ability to repress head and neck squamous cell carcinoma cells

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(HNSCC). It inhibited intracellular MMP-9 expression and extracellular MMP-9 activity of FaDu cells leading to the less-invasive phenotype. This effective drug could inhibit HNSCC cell

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3.3. EB1089 (Seocacitol)

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metastasis to a greater extent than 1α, 25(OH)2 D3 (Yang et al., 2016).

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Furthermore, a less calcemic analogue of 1α, 25(OH)2 D3, EB1089 (Seocacitol) was used. Besides an in vitro study of liver cancer cells, two animal studies using either 1α, 25(OH)2 D3 or EB1089

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have been reported by James et al. (1998). Subsequently, EB1089 was found to block tumor growth in further models of breast cancer (Flanagan et al., 2003; Sundaram et al., 2003), head and neck squamous cancers (Prudencio et al., 2001), hepatocellular cancer (Ghous et al., 2008), ovarian cancer (Zhang et al., 2005, Kasiappan et al., 2014), pancreatic cancer (Li et al., 2015) and non-small cell lung cancer (Sharma et al., 2014).

ACCEPTED MANUSCRIPT Sahpazidou et al. (2003) employed C3H/Sy virgin female mice, a strain capable of developing spontaneous HCC, to study the chemo preventive effect of EB1089 on HCC. They reported that the animals receiving 0.5 ug/kg of EB 1089 every other day for 2 months had 3.9% incidence of HCC as compared to the controls with 36.4% incidence.

Hep3B,

PLC/PRF/5

and

SKHEP-1

HCC

cells

was

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Ghous et al. (2008) reported the effect of Seocacitol in vitro and in vivo. In vitro: Proliferation of significantly

inhibited

at

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all EB1089 concentrations tested, while HTC cells only responded to 1000 nM concentration

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of EB1089. Proliferation of Novikoff cells was unaffected by the drug at all concentrations examined. In vivo: EB1089 effectively inhibited SKHEP-1 tumor growth without inducing

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hypercalcemia.

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In HCC, Seocalcitol was well tolerated. Most of the patients could tolerate 10 ug/day of EB1089

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orally. Patients with inoperable advanced HCC were included in an uncontrolled study of oral Seocalcitol treatment for up to 1 year. Out of 33 patients evaluable for tumor response, two had

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complete response (CR), 12 stable disease and 19 progressive disease (Dalhoff et al., 2003).

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The toxicity profile of Seocalcitol differs significantly from that of chemotherapy or trans-arterial chemo-embolisation (TACE). Consequently, the combination of Seocalcitol with TACE in

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comparison with TACE alone in selected patients with unresectable HCC should be studied to determine if Seocalcitol can confer any progression-free survival advantage when combined with this regional therapeutic approach. Thereby, this could overcome the drawback of early disease progression in patients treated with a putative cytostatic agent alone (Dalhoff et al., 2003). 3. 4. Others

ACCEPTED MANUSCRIPT In addition to using vitamin D analogues, it was found that when 1α, 25 (OH)2D3 was combined with fish oil, the anti-proliferative effect on HCC was greatly enhanced. Lower concentration of 1 α, 25(OH)2 D3 could be used to treat hepatocellular carcinoma in the presence of PUFAs to decrease the risk of hypercalcemia caused by high concentrations of 1 α,25(OH)2D3 (Chiang et al.,

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2009). In addition, vitamin D analogues and vitamin D receptor activators such as maxacalcitol (OCT),

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16-ene analogues, 19-nor analogues, LG190119 and C-20 cyclopropylcalcitriol have been

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developed and tested in a pre-clinical study. They may be promising adjuvants in HCC treatment in the future (Fujii et al., 2015).

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4. Vitamin K

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Vitamin K (VK) is a fat-soluble vitamin that regulates clotting factor production by acting as a

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coenzyme for a VK-dependent carboxylase that catalyzes carboxylation of glutamic acid residues into γ-carboxyglutamic acid (Furie et al., 1999). VK is involved similarly in bone metabolism by

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regulating bone matrix metabolism through γ-carboxylation (Shearer et al., 1997).

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Dietary VK is absorbed from the small intestine along with dietary fat. The latest findings have

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demonstrated that a cholesterol transporter, Niemann-Pick C1-like 1, is a key regulator of intestinal VKs absorption (Takada et al., 2015). The VK cycle plays a critical role in maintaining VK function. The cycle proceeds through the coupled carboxylation and epoxidation carried out by gamma-glutamyl carboxylase (GGCX) and VK epoxide reductase (VKOR) (Suttie, 1985; Presnell & Stafford, 2002). VK can be divided into 2 groups: 1) naturally produced VK1 (phytonadione) and VK2 (menaquinone) and 2) chemically synthesized VK3 (menadione). VK3 and its derivatives

ACCEPTED MANUSCRIPT demonstrate potent anti-proliferative effects against tumor cell lines in vitro. Whereas VK2 and its derivatives demonstrate anti-proliferative effects, although they are less potent than those of VK3, against leukemia and hepatoma cell lines (Chlebowski et al., 1985; Nishikawa et al., 1995). Beyond these effects, the novel function of VK as an anti-tumor agent has been applied to the

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prevention and treatment of HCC (Jinghe et al., 2015).

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Abnormal, uncarboxylated prothrombin (des-γ-carboxy-prothrombin [DCP]) appears in some patients with HCC who have an HCC phenotype that appears more aggressive (Nakagawa et al.,

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1999; Koike et al., 2001). It has been shown that administration of VK2 suppresses plasma DCP concentrations in patients with HCC (Furukawa et al., 1992; Orimo et al., 1998). However, to their

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knowledge, it was not known whether the administration of VK modulated disease progression.

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The effects of VK2 on disease recurrence and survival in patients with HCC were investigated

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after they underwent curative resection or percutaneous local ablation therapy.VK2 increased cJun and c-Myc mRNA expression in hepatoma cells, suggesting the induction of apoptosis through

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VK-dependent proteins (Bouzahzah et al., 1995). Another report suggested the involvement of

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geranyl geraniol VK side chains in the induction of apoptosis (Ohizumi et al., 1995).

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Another study by Ozaki et al. found that VK2 inhibited the growth of HCC cells as well as their invasiveness via the activation of protein kinase A (PKA) and the subsequent inhibition of Rho activation. They also demonstrated the activation of the transcription factors AP-2-, USF-1- and CREB in HCC cells by showing the nuclear accumulation of Ser-phosphorylated CREB, although the roles of these factors in the VK2-induced suppression of cell growth and invasion were not known. They have revealed that VK2 inhibited the growth of human HCC cells by suppressing

ACCEPTED MANUSCRIPT cyclin D1 expression through the inhibition of NF-κB activation by suppressing IKK activity (Ozaki et al., 2007). The suppression of NF-κB activation by VK2 was also observed in lipopolysaccharide-mediated macrophage activation (Ohsaki et al., 2006) and in the VK-mediated suppression of the

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osteoclastogenesis of bone cells through the RANK/RANKL pathway (Takeuchi et al., 2000). It

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had been demonstrated that VK2 inhibited the expression of matrix metalloproteinases that contain

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NF-κB binding motifs in their promoter region, and augmented the 5-fluorouracil-induced growth inhibition of HCC cells by inhibiting NF-κB activation (Ide et al., 2009; Yamaguchi & Weitzmann,

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2011). Furthermore, they elucidated that VK2 inhibited the NF-κB activation through the

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inhibition of protein kinase C (PKC)-alpha and -epsilon kinase activities, as well as through the subsequent inhibition of PKD1 activation (Zhang et al., 2011). VK2 suppressed hypoxia inducible

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to the nucleus (Xia et al., 2012).

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factor (HIF)-1 alpha activity through the inhibition of PKC by inhibiting the translocation of HIF

Koike et al. (2001) reported the protein induced by vitamin K absence or the antagonist II (PIVKA-

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II) to be a useful predisposing factor for the development of portal venous invasion in patients with

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HCC.

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The administration of menatetrenone (a VK2 compound) was the only factor related to the recurrence rate of HCC. The cumulative survival rates for the patients who received menatetrenone were 100% at 12 months, 96.6% at 24 months, and 87.0% at 36 months; and the corresponding survival rates for patients in the control group were 96.4%, 80.9%, and 64.0% (Mizuta et al., 2006). Similarly, Kakizaki et al. (2007) reported that Vitamin K2 had a suppressive effect on the recurrence of HCC and a beneficial effect on tumor recurrence after radio-frequency ablation

ACCEPTED MANUSCRIPT therapy or curative resection. However, there was no significant difference in the survival rates. The chemo-preventive effects of vitamin K2 were not sufficient. No adverse effects (for example, elevation of hepatic enzymes or thromboembolitic events) were observed because of menatetrenone treatment, and no patients were withdrawn during the follow-

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due to differences in baseline DCP levels (Mizuta et al., 2006).

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up period. Judging from these results, the reduced rate of HCC recurrence does not appear to be

Oral administration of vitamin K2 was shown to reduce the ability of liver cancer cells to invade

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and spread via the veins in the liver (portal venous system). This could be because high doses of vitamin K2 might alter the effects of key signaling molecules which stimulate cancer cells to grow.

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Most of the studies were performed using relatively high doses of vitamin K2. Several reports have

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shown that orally administered vitamin K2 was concentrated in the liver, and the concentration in

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liver tissues was at least 10 times greater than in plasma. Thus, cancer cells situated in the liver might be exposed to higher concentrations of vitamin K2 because of more prolonged exposure and

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greater uptake. The data from this study indicated that vitamin K2 supplementation actually

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reduced the growth and invasion of liver cancer (HCC) cells (Otsuka et al., 2004). Vitamin K 2 has no known adult human toxicity, which makes orally administered vitamin K2 a

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very promising treatment for preventing cancer and for keeping tumors inactive (dormant). More clinical trials are in progress to evaluate vitamin K2 in patients with primary liver cancer. However, the beneficial effects of VK administration alone were not sufficient to prevent or treat HCC in clinical settings (Otsuka et al., 2004). Two separate studies, one experimental in vitro study and one single-arm clinical study were carried on to evaluate the safety of combination therapy using sorafenib and vitamin K in HCC. In

ACCEPTED MANUSCRIPT conclusion, the results of their clinical study combined with precedent experimental studies supported their assumption that supplementing sorafenib with vitamin K produced synergistic therapeutic effects against HCC and did not increase the adverse side-effects of sorafenib (Jung et al., 2015).

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The recent progress of novel technologies, such as a genome wide association studies and computational analysis, has been the first step to the repositioning of drugs (Jiao et al., 2015, Zhang

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et al., 2015). Thus, combination of VK with other anti-cancer reagents and the development of

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more potent novel VK derivatives are the focus of ongoing research, which seeks to achieve satisfactory therapeutic effects against HCC in the future (Jinghe et al., 2015).

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-Mechanisms used by vitamin D and K to influence the development of HCC are summarized in

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table 1.

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5. Conclusion

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In spite of the recent advancement in HCC treatments, the prognosis of HCC is still rather poor as it does not respond well to traditional chemotherapy and radiotherapy. Searching for a new

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therapeutic strategy against HCC is urgently needed. The active form of vitamin D, 1α, 25(OH)2 D3, and vitamin K has been shown to exert an array of antitumor activities in cell culture and

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animal models. Numerous less-calcemic analogues of 1α, 25(OH)2 D3 have been synthesized and evaluated in animal models and several of them have been studied in phase I and phase II clinical trials. These promising results suggest that these analogues have a potential to be developed as a new therapeutic regimen for HCC.

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Table1: mechanisms used by vitamin D and K to influence the development of HCC

ACCEPTED MANUSCRIPT Vitamin D -“immune-modulator” suppressing fibroblast proliferation and collagen production.

Hammad et al. (2013)

-inversely proportional to IL-6 which has an effect on secretion of thrombopoietin (TPO) p. Caputo et al., 2003, Huang et al., 2016

- Suppressive factor of HCV replication.

Matsumura et al., 2011

-Induction of thioredoxin-interacting protein in HCC.

Hamilton et al., 2014

- Downregulates the expression of Epidermal growth factor receptor and so inhibits mitogen-activated protein kinase (MAPK) that induces apoptosis and differentiation of cells to stop the growth.

Deeb et al., 2007

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-cell cycle arrest at G0/G1, through the induction of p21 and p27, leading to suppression of cyclins (D1, E and A) and cyclin- dependent kinases 2 and 4 in HCC.

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-Inhibits blood vessel formation in order to prevent the vascular endothelial growth factor (VEGF)-mediated hepato-cacinogenesis.

-VD deprivation (VDD) in in vivo models of liver tumor formation revealed striking three-fold increases in tumor burden in Smad3 mutant mice, with a three-fold increase in Toll-like receptor 7 (TLR7) expression.

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VDR, DBP and vitamin D metabolizing enzymes polymorphism -Bsm Ι (G/A) has been involved in the fibrotic processes in human hepatic stellate cells.

Iseki et al.,1999; Chung et al., 2006

Chen et al., 2016

Falleti et al., 2010 Lange et al., 2013

-VDR genetic polymorphisms (FOKI) were significantly associated with the occurrence of HCV related HCC especially f allele carriers.

Nada et al., 2016

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-There were associations between CYP2R1, GC, and DHCR7 genotypes that are determinants of reduced 25hydroxyvitamin D (25[OH]D3) serum levels and the risk of HCV-related HCC development.

Lange et al., 2011

-DBP levels were significantly lower in subjects with significant or advanced fibrosis (METAVIR F2-4).

Gressner et al., 2009, Ho et al., 2010

CpG islands and DNA methylation -In specific patient populations with disruption of TGF-β signaling, low VD markers correlated with activation of the Wnt pathway and a high risk of tumorigenesis.

Chen et al., 2016

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- CYP27B1-1260 promoter polymorphism resulted in reduced 1,25OHD levels and were associated with failure to achieve sustained virological response (SVR) in patients with hepatitis C virus (HCV) genotypes 1, 2, and 3. -DBP rs7041 polymorphism contributed to an increased HBV-related HCC risk in Chinese population.

Peng et al., 2014

Deng et al., 2010

-Increased c-Jun and c-Myc mRNA expression in hepatoma cells, suggest the induction of apoptosis through VK-dependent proteins.

Bouzahzah et al., 1995

VK2 inhibited the growth of HCC cells as well as their invasiveness via: -Activation of protein kinase A (PKA) and the subsequent inhibition of Rho activation. -Activation of the transcription factors AP-2-, USF-1- and CREB in HCC cells.- Suppressing cyclin D1 expression through the inhibition of NF-κB activation by suppressing IKK activity.

Ozaki et al., 2007

-Inhibited the expression of matrix metalloproteinases that contain NF-κB binding motifs in their promoter region, and augmented the 5-fluorouracil-induced growth inhibition of HCC cells by inhibiting NF-κB activation.

Ide et al., 2009; Yamaguchi & Weitzmann, 2011

-Inhibited the NF-κB activation through the inhibition of protein kinase C (PKC)-alpha and -epsilon kinase activities, as well as through the subsequent inhibition of PKD1 activation.

Zhang et al., 2011

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-CYP24A1 methylation tended to correlate with better prognosis in HCV-related HCC. Vitamin K -Administration of VK2 suppresses plasma DCP concentrations in patients with HCC.

Furukawa et al., 1992; Orimo et al., 1998

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-VK2 suppressed hypoxia inducible factor (HIF)-1 alpha activity through the inhibition of PKC by inhibiting the translocation of HIF to the nucleus.

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1, 25 Dihydroxycholecalciferol (1,25(OH)2D3 ) 7- dehydrocholesterol (7-DHC) Barcelona-Clınic Liver Cancer (BCLC) The European Prospective Investigation into Cancer and Nutrition (EPIC) Gamma-glutamyl carboxylase (GGCX) Hepatocellular carcinoma (HCC) Histone deacetylase 2 (HDAC2) Hypoxia inducible factor (HIF)-1 Matrix metalloproteinase 9 (MMP-9) Mitogen-activated protein kinase (MAPK) Proximal convoluted tubule (PCT) Pegylated Interferone (PEGIFN) Protein kinase C (PKC) Ribavirin (RBV) Single nucleotide polymorphisms (SNPs) Sustained virological response (SVR) Thioredoxin-interacting protein (TXNIP) Trans-arterial chemo-embolisation (TACE) Transforming growth factor B (TGF-β) Vascular endothelial growth factor (VEGF) Vitamin D binding protein (DBP) Vitamin D receptor (VDR)

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Early diagnosis of HCC with prompt treatment increases the opportunity of patients to survive. Vitamins are important for the control of various cancers. Vitamin D and K influence the development of HCC. The active form of vitamin D and vitamin K exert an array of antitumor activities. Vitamin analogues have a potential role in HCC therapy.

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