[Frontiers in Bioscience 16, 759-769, January 1, 2011]
Chemoprevention of hepatocellular carcinoma by acyclic retinoid
Masahito Shimizu, Hiroyasu Sakai, Hisataka Moriwaki
Department of Medicine, Gifu University Graduate School of Medicine, Gifu, Japan 501-1194
TABLE OF CONTENTS
1. ABSTRACT
The prognosis for patients with hepatocellular carcinoma (HCC) is poor and effective prevention strategies are urgently required. Here, we review abnormalities in the expression and function of retinoids and their receptors, and how they play a critical role in the development of HCC. In particular, a malfunction of RXRalpha due to phosphorylation by Ras-MAPK signaling pathway is profoundly associated with liver carcinogenesis and thus may be a promising target for HCC chemoprevention. Acyclic retinoid (ACR), a synthetic retinoid, inhibits Ras-MAPK activation and RXRalpha phosphorylation, thereby suppressing growth in HCC-derived cells. In clinical trials, ACR has been shown to improve patient survival by preventing viral HCC development, a possible manifestation of the concept of "clonal deletion" therapy. "Combination chemoprevention" with ACR as the key drug has great potential to become an effective strategy for the prevention of liver carcinogenesis. In summary, both basic and clinical research strongly suggest that ACR plays a critical role in preventing the development of HCC and that "clonal deletion" therapy is one of the most practical approaches for this purpose.
2. INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, accounting for 500,000 to 600,000 deaths per year. The development of HCC is frequently associated with chronic inflammation and subsequent cirrhosis of the liver induced by persistent infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). This fact indicates that HCC is a major health problem in Eastern as well as Western countries where hepatitis viral infection is endemic, and the incidence is increasing (1-3). However, in spite of strenuous efforts to develop effective methods of diagnosis and treatment, there has been limited improvement in the prognosis for this malignancy. A major obstacle for HCC therapy is the high frequency of tumor recurrence after curative treatment; the recurrence rate at 5 years after definitive therapy may exceed 70% (4, 5). At present, there are no effective chemotherapeutic agents for this malignancy. Therefore, there is a critical need to develop more effective strategies for the chemoprevention and chemotherapy of HCC to improve the prognosis for patients with this malignancy; for this purpose, we must elucidate the molecular mechanisms underlying hepatocarcinogenesis. Among the several causal factors for the development of HCC, phosphorylation of retinoid X receptor- a (RXR a) by the Ras-MAPK signaling pathway is considered to play a key role (6-9).
Because of the high incidence of recurrence and the development of secondary tumors (4, 5), the curative treatment for HCC is difficult once this malignancy has developed. The high risk group, including patients infected with hepatitis, are easily identified, however. Therefore, cancer chemoprevention, an approach wherein a natural or synthetic chemical compound works to arrest or reverse premalignancies via physiological pathways (10), is one of the most promising strategies for the treatment of HCC, particularly hepatitis virus-positive patients. We previously reported that, in clinical trials, the administration of acyclic retinoid (ACR), a novel synthetic retinoid which targets phosphorylated RXR a (11-13), reduced the incidence of post-therapeutic HCC recurrence and improved patient survival (14-17). In this article, we review evidence that a malfunction of RXR a due to phosphorylation is closely involved in liver carcinogenesis. We also show the pleiotropic effects of ACR in the inhibition of HCC and suppression of cancer growth, especially focusing on the inhibition of RXR a phosphorylation and induction of RAR b and p21CIP1 expression. In addition, the possibility of "combination chemoprevention", which uses ACR as a key drug, and the concept of "clonal deletion" therapy, a practical approach to preventing HCC development, are also discussed.
3. Retinoids and their receptors
Vitamin A and its functional analogues, collectively termed retinoids, exert fundamental effects on the regulation of epithelial cell growth, differentiation, and development (18, 19). Retinoids consist of several molecular species, including retinoic acid (RA, an active metabolite that binds to its nuclear receptor), retinol (a transport form in the plasma), and retinylesters (a storage form in the tissues). In addition, large numbers of synthetic retinoids, including ACR, have been developed (Figure 1). Retinoids exert their biological functions primarily by regulating gene expression through 2 distinct nuclear receptors, the retinoic acid receptors (RARs) and RXRs, which are both composed of 3 subtypes ( a, b, and g) that are characterized by a modular domain structure. Nuclear retinoid receptors are ligand-dependent transcription factors; after ligand binding, RXRs form a homodimers, as well as heterodimers with RARs, which interact with the retinoid X response element (RXRE) or the retinoic acid receptor responsive element (RARE) located in the promoter region of target genes, thereby modulating gene expression (18, 19). In addition to RARs, RXRs also form heterodimers with other nuclear receptors including peroxisome proliferator-activated receptors (PPARs), which control energy homeostasis by modulating glucose and lipid metabolism and transport (20). Therefore, RXRs play a fundamental role in controlling normal cell proliferation and metabolism, and act as master regulators of nuclear receptors (19). These facts suggest that retinoid receptors, especially RXRs, are exciting pharmacological targets for the therapies of various human diseases, including cancer and metabolic disease (21, 22).
4. ABNORMALITIES IN THE RETINOID/RETINOID RECEPTOR AXIS AND HCC
Because retinoids and their receptors play an essential role in normal cell proliferation, differentiation, and death (regulation of apoptosis), abnormalities in the expression and function of these molecules, especially RXR a and RAR b, are strongly associated with the development of various human malignancies including HCC. For instance, the RAR b gene is an HBV integration site and its expression is markedly decreased in human HCC (23, 24). In the chemical-induced rat liver carcinogenesis model, both RAR b protein and mRNA levels are also decreased in HCC (25). These findings are interesting because among the retinoid receptors, RAR b is thought to be one of the most important receptors in the regulation of cell growth and apoptosis (26).
The expression of RXR a is also decreased not only in HCC and liver cell adenoma, but also in glutathione S-transferase placental form-positive foci, a precancerous HCC lesion in the chemical hepatocarcinogenesis model in rats (25). These findings suggest that the repression of RXR a occurs even in the early stage of liver carcinogenesis. Moreover, recent studies have revealed that liver carcinogenesis is accompanied by an accumulation of the phosphorylated (i.e. inactivated) form of RXR a (p-RXR a) (27). Specifically, RXR a protein is anomalously phosphorylated at serine and threonine residues, and accumulates in both human HCC tissue and HCC cell lines (9). Phosphorylation at serine 260 of RXR a, a MAPK consensus site, is closely associated with its retarded degradation, low transcriptional activity, and the promotion of cancer cell growth; the abrogation of phosphorylation by a MAPK inhibitor restores the degradation of RXR a in a ligand-dependent manner (9, 11). In addition, although RXR a is unphosphorylated and highly ubiquitinated in a normal liver, rendering it sensitive to proteasome-mediated degradation, p-RXR a is resistant to ubiquitination and proteasome-mediated degradation in both human HCC tissues and a human HCC cell line (28). Furthermore, the phosphorylation of RXR a abolishes its ability to form heterodimers with RAR b, and this is associated with uncontrolled cell growth and resistance to retinoids (29). These findings suggest that the accumulation of p-RXR a, (i.e., non-functional RXR a) may interfere with the function of normal RXR a in a dominant-negative manner, thereby playing a critical role in the development of HCC (Figure 2). There are also some reports that show the analogous effects of phosphorylated RXR a in the negative modulation of its heterodimeric binding partners (30-32). Therefore, the inhibition of RXR a phosphorylation and the restoration of its heterodimeric activity with other nuclear receptors may be an effective and important strategy for the prevention and treatment of certain types of human diseases, especially malignant disorders including HCC (6-8, 33-35).
5. ACR IN HCC CHEMOPREVENTION: EXPERIMENTAL STUDIES
ACR, which was initially developed as an agonist for both RXR and RAR (36, 37), has been demonstrated to produce several beneficial effects on the prevention of HCC development and inhibition of growth in HCC cells (ACR is the same substance as NIK-333 and Peretinoin; Kowa Pharmaceutical Co., Tokyo, Japan; See Figure 3). In rodent studies, ACR inhibits both chemical-induced hepatocarcinogenesis in rats and spontaneously occurring HCC in mice (38). ACR also inhibits growth of HCC-derived cells by inducing cell proliferation and apoptosis, which effects seem to be associated with upregulation of RAR b expression (13, 36, 39-44). In human HCC and squamous carcinoma cells, ACR causes cell cycle arrest in G0-G1, increased cellular levels of p21CIP1, and decreased levels of cyclin D1 and the phosphorylated form of retinoblastoma proteins (44-46). These findings suggest that RAR b and p21CIP1 are one of the critical targets of ACR with respect to growth inhibition and apoptotic induction in cancer cells.
Recent in vivo and in vitro studies have indicated that ACR not only binds to RXR and RAR, but also reduces the development of HCC and inhibits cancer growth by targeting growth factors and their corresponding receptor tyrosine kinases (RTKs), which play a critical role in activation of the Ras-MAPK signaling pathway (41, 46-50). These reports are significant because the activated Ras-MAPK pathway phosphorylates RXR a, thus contributing to the development of HCC (9, 27). In addition, ACR also restores RXR a function by inactivating the Ras-MAPK signaling system, leading to the dephosphorylation of RXR a, although 9-cis RA failed to suppress ERK and RXR a phosphorylation (11). Therefore, ACR, which targets the RTK-Ras-MAPK signaling pathway and RXR a phosphorylation, is a promising agent for the chemoprevention of HCC. The role of RXR a phosphorylation in liver carcinogenesis and its inhibition by ACR are schematically represented in Figure 2.
6. ACR IN HCC CHEMOPREVENTION: CLINICAL STUDIES
An early phase randomized, controlled clinical trial tested the chemopreventive effect of ACR on secondary HCC in patients who received anti-cancer treatment for an initial HCC (14-16). In this trial, oral administration of ACR (600 mg per day) for 12 months significantly reduced the incidence of secondary HCC after a median follow-up period of 38 months (P = 0.04) (14), and improved both incidence (P = 0.002) and survival (P = 0.04) after a median follow-up period of 62 months (15). Relative risk of the development of secondary HCC and death were 0.31 (95% confidence interval, 0.12 to 0.78) and 0.33 (0.11 to 0.79), respectively (14, 15). Moreover, the preventive effects of ACR lasted up to 199 weeks after randomization or 151 weeks after completion of ACR administration (16).
A phase II/III trial of ACR confirmed its effectiveness in preventing secondary HCC in hepatitis C virus-positive patients in a multicenter, large-scale (n = 401) randomized placebo-controlled trial; oral administration of 600 mg of ACR per day was tolerated and had a strong effect on the prevention of secondary HCC with a hazard ratio of 0.27 (0.07 to 0.96) after 2 years (17). The results of these clinical trials suggest that ACR is a novel first-line therapy to reduce the development of secondary HCC.
7. "CLONAL DELETION" THERAPY FOR HCC
Liver carcinogenesis is characteristically multicentric in nature, a phenomenon which is expressed by the term "field cancerization" (51). The poor prognosis for HCC, which is associated with a high incidence of recurrence and development of secondary tumors, is particularly relevant to field cancerization. Once a liver is exposed to continuous carcinogenic insults, such as hepatitis viral infection and alcohol toxicity, the whole exposed liver is regarded as a precancerous lesion which possesses multiple as well as independent premalignant or latent malignant clones. Hence, even if the first cancer is diagnosed and removed early, the next clone essentially arises to form a secondary HCC. Therefore, the most effective strategy for HCC chemoprevention is the deletion of latent malignant clones (clonal deletion) and inhibition of the evolution of such clones (clonal inhibition) before they expand into clinically detectable tumors. We have proposed that implementation of this novel concept, "clonal deletion" therapy, which is defined as the removal of latent malignant (or premalignant) clones that are invisible by diagnostic imaging from the liver when it is in a hypercarcinogenic state, is fundamental to the chemoprevention of HCC (Figure 4) (6-8).
ACR has been used to effectively demonstrate this concept in the clinical setting. In the clinical trial, serum levels of lectin-reactive a-fetoprotein factor 3 (AFP-L3), which indicates the presence of latent (i.e., invisible) malignant clones in the remnant liver, were significantly reduced by 12-month administration of ACR (52). This observation indicates that ACR eliminates or removes the AFP-L3 producing premalignant clones from the remnant liver before they expanded into clinically detectable (i.e., visible) tumors, thereby inhibiting secondary HCC. Moreover, ACR suppressed the appearance of serum AFP-L3 in patients whose AFP-L3 levels were negative at trial enrollment, whereas the number of patients whose serum AFP-L3 appeared de novo was significantly increased in the placebo group; these patients had a significantly higher risk of secondary HCC (52). This finding suggests that, in addition to elimination, ACR actively inhibits the development of AFP-L3-producing clones, which have the potential to become HCC. This is one of the reasons why only a short-term administration (12 months) of ACR exerted a long-term preventive effect on HCC development for several years after termination of treatment (16). It takes several years for the next cancer clones to arise clinically once they are eliminated or inhibited. Therefore, the promise of clonal deletion seems to be therapeutic rather than preventive, and ACR prevents the development of HCC by this mechanism.
8. "COMBINATION CHEMOPREVENTION" OF HCC USING ACR AS THE KEY DRUG
Combination therapy is often advantageous because it provides the potential for synergistic effects between specific drugs; ACR is no exception in this regard. For instance, ACR acts synergistically with interferon (IFN)- b in suppressing growth and inducing apoptosis in human HCC cell lines via upregulation of type 1 IFN receptor and Stat1 expression by ACR (53). The combination of ACR plus vitamin K2 (VK2) synergistically inhibits cell growth and induces apoptosis in HCC cells without affecting the growth of normal human hepatocytes (12). These findings are significant when considering the clinical use of ACR because both IFN and VK2 are expected to exert preventive effects on the development and recurrence of HCC (54-57). Therefore, we assume that "combination chemoprevention" using ACR as the key agent may be a useful strategy to prevent the development of HCC.
The expected mechanisms of ACR-based combination chemoprevention are schematically summarized in Figure 5. Initially, specific agents that target the Ras-MAPK signaling pathway and its upstream RTKs are among the most promising partners for ACR because these agents dephosphorylate RXR a. Indeed, ACR and VK2 cooperatively inhibit activation of the Ras-MAPK signaling pathway, thus suppressing the phosphorylation of RXR a and the growth of HCC cells (12). The combination of 9-cis RA (58) or ACR (unpublished data) plus trastuzumab, a humanized anti-human epidermal growth factor receptor-2 (HER2) monoclonal antibody, synergistically inhibits growth and induces apoptosis in HCC cells via cooperative inhibition of the activation of HER2 and its downstream signaling molecules, including ERK and Akt, and subsequent dephosphorylation of RXR a. Combined treatment with ACR plus valproic acid, a histone deacetylase (HDAC) inhibitor, acts synergistically to induce apoptosis and G0-G1 cell cycle arrest in HCC cells by inhibiting phosphorylation of RXR a, ERK, Akt, and GSK-3 b proteins (13).
In addition to dephosphorylation of RXR a, induction of nuclear receptors that dimerize RXR, such as RAR and PPAR (33, 59), and recruitment of their ligands may also exert synergistic growth inhibition in cancer cells when combined with ACR. Both valproic acid (13) and OSI-461 (43), a potent derivative of sulindac sulfone, enhance the ability of ACR to raise the cellular levels of RAR b and p21CIP1, thereby markedly increasing the RARE and RXRE promoter activities and inducing apoptosis in HCC cells. Therefore, these combinations may also be an effective regimen for the chemoprevention and chemotherapy of HCC.
9. PERSPECTIVE
The prevention of HCC is an urgent task on a global scale, and one of the most practical approaches to the accomplishment of this purpose is "clonal deletion" therapy. Experimental studies strongly suggest that RXR a phosphorylation is profoundly involved in liver carcinogenesis and thus may be a critical target for HCC chemoprevention. Clinical trials reveal that ACR, which inhibits RXR a phosphorylation but induces RAR b expression, is a promising candidate for HCC chemoprevention by putting the concept of "clonal deletion" in practice. ACR-based combination chemoprevention, which is expected to exert synergism, also holds great promise as a master therapeutic for HCC chemoprevention. In conclusion, ACR may play a critical role in preventing HCC development when it is used alone or combined with other drugs and, therefore, early clinical application of this agent is greatly anticipated.
10. ACKNOWLEDGEMENTS
This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (No. 18790457 to M. S. and No. 17015016 to H. M.).
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Abbreviations: ACR, acyclic retinoid; AFP-L3, lectin-reactive a-fetoprotein factor 3; HBV, hepatitis B virus; HCC, Hepatocellular carcinoma; HCV, hepatitis C virus; HDAC, histone deacetylase; HER2, human epidermal growth factor receptor-2; IFN, interferon; MAPK, mitogen-activated protein kinase; PIVKA-II, protein induced by vitamin K absence or antagonist-II; PPAR, peroxisome proliferator-activated receptors; RA, retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid receptor responsive element; RTK, receptor tyrosine kinase; RXR, retinoid X receptor; RXRE, retinoid X response element; VK2, vitamin K2
Key Words: Retinoid, HCC, chemoprevention, phosphorylated RXR alpha, Review
Send correspondence to: Masahito Shimizu, Department of Medicine, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu, Japan 501-1194, Tel: 81-58-230-6308, Fax: 81-58-230-6310, E-mail:shimim-gif@umin.ac.jp