Strategy and mechanism for the prevention of hepatocellular carcinoma: phosphorylated retinoid X receptor alpha is a critical target for hepatocellular carcinoma chemoprevention

Abstract

Hepatocellular carcinoma (HCC) is a major health care problem worldwide. The prognosis of patients with HCC is poor because even in the early stages when surgical treatment might be expected to be curative, the incidence of recurrence in patients with underlying cirrhosis is very high due to multicentric carcinogenesis. Therefore, strategies to prevent recurrence and second primary HCC are required to improve the prognosis. One of the most practical approaches to prevent the multicentric development of HCC is 'clonal deletion' therapy, which is defined as the removal of latent (i.e. invisible) (pre)malignant clones from the liver in a hypercarcinogenic state. Retinoids, a group of structural and functional analogs of vitamin A, exert their biological function primarily through two distinct nuclear receptors, retinoic acid receptors and retinoid X receptors (RXR), and abnormalities in the expression and function of these receptors are highly associated with the development of various cancers, including HCC. In particular, a malfunction of RXRalpha due to phosphorylation by the Ras-mitogen-activated protein kinase signaling pathway is profoundly associated with the development of HCC and thus may be a critical target for HCC chemoprevention. Acyclic retinoid, which has been clinically shown to reduce the incidence of a post-therapeutic recurrence of HCC, can inhibit Ras activity and phosphorylation of the extracellular signal-regulated kinase and RXRalpha proteins. In conclusion, the inhibition of RXRalpha phosphorylation and the restoration of its physiological function as a master regulator for nuclear receptors may be a potentially effective strategy for HCC chemoprevention and clonal deletion. Acyclic retinoid, which targets phosphorylated RXRalpha, may thus play a critical role in preventing the development of multicentric HCC.

Figure 1

Chemical structures of natural and representative synthetic retinoids. Retinyl esters (mainly retinyl palmitate; R, fatty acid), stored in the liver stellate cells, are hydrolyzed to retinol, which is then transported to target cells through the circulation after binding to retinol‐binding protein. Retinoic acid (RA) is biosynthesized from retinol via the intermediate metabolite retinal by oxidization in the cells of peripheral tissues. Three well‐known isomers of RA, all‐trans RA, 9‐cis RA, and 13‐cis RA activate the retinoid receptor retinoic acid receptor (RAR), whereas only 9‐cis RA activates the other receptor, retinoid X receptor (RXR). A number of synthetic retinoids have been developed to carry out their pharmacological applications including cancer chemoprevention. Acyclic retinoid and N‐(4‐hydroxyphenyl) retinamide (4HPR) successfully prevented the development of hepatocellular carcinoma and breast cancer, respectively, in clinical trials (see review reference^{( 27 )}).

Figure 2

Retinoid refractoriness due to phosphorylation of retinoid X receptor (RXR) α and its restoration by acyclic retinoid (ACR) in hepatocellular carcinoma (HCC) cells. In normal hepatocytes, when the ligand binds to and activates RXRα, the receptor becomes able to heterodimerize with other nuclear receptors, such as retinoic acid receptor (RAR), and then activates the expression of target genes, which may regulate normal cell proliferation and differentiation, by binding to the specific response elements. Thereafter, RXRα dissociates from the dimer, is ubiquitinated (Ub), and is degraded by the proteasome. The whole process from ligand binding to proteasomal breakdown of RXRα is estimated to take approximately 6 h (A).^{( 14 )} In HCC cells, the Ras–mitogen‐activated protein kinase (MAPK) pathway is highly activated and phosphorylates RXRα at serine residues, thus impairing dimer formation and the subsequent transactivation functions of the receptor. Furthermore, phosphorylated RXRα (pi‐RXRα) escapes from ubiquitination and proteasomal degradation (B). Therefore, pi‐RXRα accumulates and interferes with the physiological function of the remaining unphosphorylated RXRα, presumably, in a dominant‐negative manner, thereby playing a critical role in the development of HCC (C). ACR is not only a ligand for RXRα but also suppresses the Ras–MAPK signaling pathway, inhibiting phosphorylation of RXRα, restoring the function of the receptor, and thus subsequently activating the transcriptional activity of the responsive element (D). ACR also directly or indirectly inhibits the ligand (i.e. specific growth factor)‐dependent receptor tyrosine kinase (RTK) activities in cancer cells (E). These effects may contribute to the inhibition of extracellular signal‐regulated kinase (Erk) and RXRα phosphorylation, thus causing inhibition of the growth of HCC cells.

Figure 3

The concept of ‘clonal deletion’. Persistent inflammation caused by hepatitis B virus (HBV) or hepatitis C virus (HCV) infection transforms the liver into a ‘precancerous field’ (A). Therefore, the high incidence of hepatocellular carcinoma (HCC) as well as its recurrence in cirrhotic patients strongly suggests the presence of latent malignant clones that arise through multicentric carcinogenesis and are undetectable clinically by image analysis (invisible) (B). These multiple clones demonstrate different grades of malignancy (atypia) in the cirrhotic liver and, at some point, turn into clinical HCC (visible) (C). Even when primary HCC could be found in an early stage and surgical treatment might be expected to be curative, other clones still survive in the remaining liver and thus grow into secondary HCC again (D). Therefore, the eradication of such transformed clones, referred to as ‘clonal deletion’, may be one of the most effective strategies to prevent secondary HCC (E). Clinical experience suggests that acyclic retinoid (ACR), which inhibits phosphorylation (Pi) of retinoid X receptor (RXR) α (F), reduces the recurrence of HCC on the basis of this concept because this agent causes a decrease in the serum levels of lectin‐reactive α‐fetoprotein isoform 3 (AFP‐L3) and protein induced by vitamin K absence or antagonist‐II (PIVKA‐II), which are produced by latent malignant clones, by eradicating or inhibiting these clones. Once such clones are deleted, the preventive effect on HCC lasts several years without any continuous administration of ACR. In fact, 1‐year administration of ACR inhibited secondary HCC for the next 3 years.^{( 11 )} Therefore, this agent can significantly improve the survival rate of such patients.