Retinoic Acid Receptors (RARA, RARB, and RARC)
Role of RARs in Embryonic Development
Research from the 1950s implicated vitamin A deficiencies as a cause for a number of congenital malformations and defects observed in the development of animals. These results were known long before the discovery of retinoic acids as the most biologically active forms of vitamin A. Later research then investigated the role of retinoic acid in early embryonic development and in the early 1990s led to the discovery of the retinoic acid receptors (RARs). Since then much work has been done to determine the role that RARs play in embryonic development.
Genetic studies were conducted in the early 1990s to investigate the role of all three RAR isoforms in embryonic development by generating single and double RAR mutant mice. While mice with a mutation deleting only one RAR isoform contained certain developmental abnormalities, they were still viable, indicating a certain redundancy among isoforms (Mark et al. 2009). However, RAR double null mutants, containing genetic deletions of any two of the RAR isoforms, died in utero or at birth due to severe developmental deficits mainly in the vertebrae, brain, and limbs indicating the importance of the expression of the RARs on the formation of these structures. The same deficits were observed by a number of investigators where various components of retinoic acid signaling were knocked out such as retinaldehyde-synthesizing enzyme RDH10 (Sandell et al. 2007), RA-synthesizing enzyme RALDH2 (Halilagic et al. 2007), or RALDH3 (Dupe et al. 2003). In addition, treatment of wild-type animals with synthetic pan-specific RAR antagonists also produced the same defects (Kochhar et al. 1998; Wendling et al. 2001). This demonstrated that not only expression of RARs was essential for normal development but that the receptors required activation by retinoic acid ligands to mediate their important roles in developmental signaling programs.
While much has been learned about the important roles played by the RARs in embryonic development, research is now shifting toward elucidating the roles played by these receptors during the postnatal development. To do this, new strategies have emerged to allow for the selective mutation of the retinoid receptors in specific cell types so as to further understand the functions of these receptors in the postnatal animal as it develops and grows (Metzger and Chambon 2001; Metzger et al. 2003).
Role of RARs in Regulating Cell Proliferation and Cancer
In addition to the effects that retinoic acids and the RARs have on developmental pathways, a large amount of evidence has emerged implicating RARs in the control of cell-cycle pathways and cellular proliferation. In normal cells, retinoic acids generally inhibit cell-cycle progression by instituting a block in the G1 phase of the cell cycle (Mongan and Gudas 2007). Of all the RAR isoforms, these effects are mostly mediated by RARβ2 following binding and activation by retinoic acids (Faria et al. 1999). Several studies have shown in a number of cell types that activation of RARβ2 leads to the transactivation of several genes involved in cell-cycle arrest such as p21CIP1 and p27KIP1 (Li et al. 2004; Suzui et al. 2004). In addition to activating cell-cycle arrest proteins, RARs also mediate both the downregulation of mRNA expression as well as protein ubiquitination and degradation for both the Cyclin D and E families which prevents progression of the cell cycle from the G1 to S phase (Tang and Gudas 2011). Moreover, RARs induce apoptosis following binding of retinoids in a number of cell types as a guard against tumor formation. Retinoic acid binds to RARα and induces apoptosis in both acute lymphoblastic leukemia cells as well as myeloid leukemia cell lines (Chikamori et al. 2006; Luo et al. 2009). In addition, it has been reported that RARγ induces apoptosis upon binding to retinoids in both skin keratinocytes as well as pancreatic adenocarcinoma cells (Hatoum et al. 2001; Pettersson et al. 2002). Finally, RARβ2 has been implicated in the induction of apoptosis in breast cells. Taken together, these observations have provided clear evidence that RARs play key roles in the regulation of cell-cycle progression and cell growth as well as apoptosis and therefore when RAR-mediated signaling pathways are disturbed, there can be major implications for the development and progression of cancer.
The loss of retinoic acid signaling is not restricted to APL and in fact reduction in expression of both RARα and RARβ2 has been shown to occur in several types of cancer such as embryonal carcinomas, acute myeloid leukemia, and breast cancer (Mongan and Gudas 2007; Altucci et al. 2007). In contrast to APL where RARα is mutated into the PML/RARα fusion protein, most cancer cell types do not contain mutated RARs but rather have dramatically downregulated expression of these receptors. Understanding the underlying mechanisms behind the silencing of RARs in cancer cells has been a major focus of recent research and several mechanisms have been uncovered. For example, it was reported that the RARβ2 promoter in many cancer cell types is silenced by hypermethylation at CpG regions of its promoter. In addition, corepressors such as PRAEME, meningioma 1, acinus-S′, HACE1, and SMRT have all been shown to inhibit expression of either RARβ2 or RAR response genes either due to overexpression of these corepressors or a greater affinity for the RARβ2 response elements concomitant to changes caused by aberrant AKT signaling in a variety of cancer cell types (Tang and Gudas 2011). In contrast, repression of RAR coactivator expression has also been observed to downregulate expression of RARs in neuroblastoma cells. Taken together, these reports indicate that a multitude of mechanisms are at work in various cancer cell types that all result in the repression of RARs and their downstream target genes. Regardless of mechanism, the end result is an absence of RAR-mediated balances between differentiation and proliferation and demonstrates the vital roles RARs play in the progression of cancer.
Development of RAR Ligands for Use as Therapeutics
Given the correlation between the reduction in RAR-mediated retinoic acid signaling and the progression of a number of cancers, therapies have been developed to treat cancer patients with natural retinoids such as all-trans retinoic acid (ATRA) to induce differentiation and cell growth arrest. This strategy has been extremely successful in the treatment of APL as pharmacological doses of retinoic acid stimulate irreversible differentiation of leukemic cells into granulocytes. Moreover, it has been reported that pharmacological doses of retinoic acid also trigger growth arrest and differentiation of leukemia stem cells known as leukemia-inducing cells (LICs) (Tang and Gudas 2011). When combined with other apoptosis-inducing chemotherapeutic drugs such as anthracyclins, retinoic acid treatment reverses gene silencing and leads to induced cell death of the cancer cells curing 70–80% of APL patients (Altucci et al. 2007). This treatment is successful since the expression of the PML/RXRα fusion protein is high and the RARα portion of this protein contains a functioning ligand-binding domain and coactivator recruitment site that allows for the retinoic acid-mediated activation of a number of RARα genes that stimulate differentiation.
While cell differentiation therapies using high levels of natural retinoids such as ATRA have proven to be very successful in the treatments of some cancers, there are significant drawbacks to their therapeutic use. Retinoids are powerful teratogens that at pharmacological concentrations can induce congenital defects and toxicity in all vertebrate species. In addition, there are some cancers such as prostate cancer where ATRA and other synthetic retinoid agonists are not effective in inducing growth arrest and/or apoptosis. Moreover, a common feature of many cancers is the development of resistance to the growth inhibitory effects of retinoids limiting the utility of these therapies. Even APL, which responds well to differentiation therapy, has several variants that display retinoid resistance and does not respond to this therapy. For these reasons, efforts have been underway to develop new types of synthetic ligands for RAR that can promote the positive effects of retinoids without the detrimental side effects.
A number of synthetic retinoids have been developed as potential therapeutics for a variety of cancers. These are often referred to as atypical retinoids or retinoid-related molecules because they are based on the retinoic acid structure and have been shown to bind and transactivate RARs. Many of these compounds have been approved for the treatment of a number of diseases such as cancer, acne, and psoriasis (Altucci et al. 2007). The majority of these atypical retinoids are RAR agonists; however, there have been some RAR antagonists that have also been synthesized. In some cancers such as prostate cancer, pan-specific antagonists of RAR such as AGN194310 demonstrated much more significant anti-proliferative and pro-apoptotic effects than any RAR natural or synthetic agonist. In fact a number of synthetic molecules known as the retinoid-related molecules such as MX781, AGN 194310, and ST1926 have demonstrated potent anti-proliferative activities against large panels of human tumor cells (de Lera et al. 2007). Until recently, all of the synthetic retinoid-related molecules reported that directly bind and modulate RAR activity share structural similarities to the natural agonist retinoic acid. This means that while some have proven efficacious in the treatments of a number of important cancers, they could still be susceptible to the same limitations regarding retinoid resistance as the natural retinoids. Interestingly, a recent report has identified the first synthetic non-retinoid, non-acid RAR modulator that binds and activates all three isoforms of RAR (Busby et al. 2011). Synthetic structures such as these may provide the basis for novel chemical scaffolds of non-retinoid, non-acid RAR modulators that may be developed that are potent and efficacious toward restoring RAR signaling while at the same time overcome the challenges of toxicity and resistance seen with use of natural retinoids such as ATRA.
The retinoic acid receptors (RARs) are ligand-dependent transcription factors that belong to the NR1B subtype of the nuclear receptor (NR) superfamily. RARs are ligand-dependent transcription factors that bind to retinoids, the most potent biologically active forms of vitamin A, and heterodimerize with the rexinoid X receptor (RXR) to regulate many genes involved in the regulation of cellular growth and differentiation. RARs play significant roles in a number of developmental cascades from formation of limbs and organs to the central nervous system. In addition, all three RAR isoforms are instrumental in the control of a cellular growth through the inhibition of the cell cycle. That combined with the activation of genes involved in differentiation provides multiple pathways that RARs regulate cellular growth. Given these critical roles in cellular growth, it is not surprising that a great deal of evidence has emerged that either mutations or reductions in RAR expression are correlated with a number of cancers. This has led to the development of differentiation therapies alone or in combination with other types of drugs to restore RAR-mediated retinoic acid signaling in a number of cancers. Due to the potential toxicity and emergence of retinoid resistance in some cancers, synthetic retinoid-related molecules have been developed including one novel non-acid non-retinoid chemical scaffold that may provide safer, more efficacious ways to treat cancer by restoring normal RAR-mediated RA signaling. Further understandings of the roles of the various RAR isoforms in the progression of cancer and how to modulate the activities of RARs may provide important clues to develop novel therapies to treat cancer.
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