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The Department of Pharmacology and Toxicology, Dartmouth Medical School, and the Norris Cotton Cancer Center, Dartmouth Hitchcock Medical Center, Hanover, NH 03755
3To whom correspondence should be addressed. E-mail: michael.spinella{at}dartmouth.edu.
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ABSTRACT |
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 TOP ABSTRACT Retinoids as chemopreventive...Human embryonal carcinomaRetinoic acid activates the...Gene expression profiling of...SummaryLITERATURE CITED |
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KEY WORDS: • retinoic acid • embryonal carcinoma • chemoprevention • microarray • differentiation
The concept of chemoprevention was first coined by Sporn et al. (1
) as a framework for therapeutic interventions at early stages of carcinogenesis.
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Retinoids as chemopreventive agents |
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TOPABSTRACTRetinoids as chemopreventive...Human embryonal carcinomaRetinoic acid activates the...Gene expression profiling of...SummaryLITERATURE CITED |
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). Thus differentiation-based cancer therapy holds promise as a relatively nontoxic alternative to the cytotoxic therapies currently available. This concept has been with us for many years and has been demonstrated for a variety of agents in cell culture and, in some cases, in animal models (2
). However, the only approved anticancer regimen with demonstrated in vivo differentiation activity is the use of the retinoid, all-trans–retinoic acid (RA)4 (3
), for treatment of acute promyelocytic leukemia. Induction of complete remission in this disease is associated with a conversion of promyeloid blasts to mature granulocytes (3
,4
).
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–5
)]. Preclinical studies, first reported by Wolbach and Howe in 1925 (6
), indicated that vitamin A–deficient rodents developed squamous metaplasia. These metaplastic lesions were reminiscent of those found in smokers and were reversed by vitamin A repletion. Data derived from other experimental animal models revealed retinoid chemopreventive effects on the epithelium of various tissues after exposure to chemical mutagens (7
). Epidemiological evidence indicated an inverse relationship between cancer incidence at specific sites and serum vitamin A or ß-carotene levels (3
). Taken together, these findings provided a basis for the use of retinoids in clinical cancer prevention. During the past decade, associations between nuclear retinoid receptor abnormalities and specific cancers [e.g., the translocation of a portion of the retinoic acid receptor (RAR)
gene in acute promyelocytic leukemia] suggest that the dietary vitamin A deficiency reported by Wolbach and Howe may be mimicked in specific tissues, perhaps precipitating early carcinogenic events. Findings indicate that at least some of these events are reversible with pharmacologic retinoid doses. For example, early trials using isotretinoin (13-cis-RA) and other retinoids led to reductions in second cancers of the head and neck, lung and liver after retinoid treatments (3
–5
). However, one limitation of retinoid-based therapy is systemic vitamin A–associated toxicities. Clinically, this has led to the necessity of reducing retinoid doses and to difficulties in administering potent retinoids on a chronic basis in prevention trials. One strategy for designing more effective chemopreventive agents is to uncover new pharmacological targets based on a mechanistic understanding of the beneficial and harmful downstream targets of existing pleiotropic chemopreventive agents such as the retinoids.
Nuclear retinoid receptors are members of the steroid receptor superfamily (8
,9
). Nuclear retinoid receptors function as ligand (hormone)-dependent transcription factors. Two subtypes of retinoid receptors exist, the RARs and the retinoid X receptors (RXRs). RARs are activated by all-trans-RA, whereas 9-cis-RA activates both RARs and RXRs. Retinoid-mediated changes in gene expression occur by the binding of RAR/RXR heterodimers or RXR/RXR homodimers to defined RA-responsive elements (RAREs) within target genes. Three RARs (, ß and 
) and three RXRs (, ß and ) exist and have cell context-dependent expression patterns and interact with coregulators. The identity of target genes, which mediate the differentiation, growth suppressive and antitumorigenic effects of retinoid-activated RARs, are largely unknown.
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Human embryonal carcinoma |
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TOPABSTRACTRetinoids as chemopreventive...Human embryonal carcinomaRetinoic acid activates the...Gene expression profiling of...SummaryLITERATURE CITED |
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). TGCTs are the most common carcinomas of men between the ages of 15 and 35 (11
). Despite recent progress, the molecular basis for germ cell tumorigenesis is poorly understood (12
). In our studies the human TGCT-derived EC cell line NT2/D1 is used. This line was developed from a metastatic lesion of a testicular cancer patient (13
). Human EC cells, including NT2/D1 cells, appear distinct from murine EC in terms of genetic, phenotypic and biologic markers (14
,15
). Based on these differences, it has been proposed that NT2/D1 cells may represent an earlier stage of differentiation compared to most murine EC cells available (14
,15
).
Pluripotent human EC cells are an important model of early human development (13
). They resemble early embryonic stem cells in morphology, antigen expression patterns, biochemistry, developmental potential and gene regulation (13
). NT2/D1 cells are multipotent, differentiating into well developed, morphologic and immunophenotypic central nervous system-like neurons upon culture in RA, with associated loss of cell growth and tumorigenicity (16
–18
). It should be noted that NT2/D1 cells are particularly informative compared with other differentiation models because commitment to differentiation occurs with a single agent, RA, without additional specialized conditions such as serum starvation, application of mitotic inhibitors or cAMP or confluent growth and aggregation (16
,18
). The neuron-like cells express the major neurofilament proteins, a functional glutamate receptor and sodium ion channels. They also form functional synapses and in general possess many of the electrochemical properties of central nervous system neurons (13
,19
). Comparisons of human embryonic stem (ES), murine ES and human EC cells have demonstrated that human EC and human ES lines share many phenotypic and morphological features not possessed by murine ES cells (20
,21
). Because NT2/D1 cells have been widely characterized, can be propagated without the use of feeder lines or leukemia inhibitory factor and can be readily transfected, these cells are a valuable and practical complement to human ES cells as an in vitro model of human embryology. In addition, RA treatment of EC is an important model of antitumorigenesis because RA causes a reversal of the malignant phenotype. However, the genes and pathways regulated during neuronal differentiation, lineage commitment and repression of tumorigenic potential of pluripotent EC cells are not well defined.
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Retinoic acid activates the intrinsic transactivation function of p53 |
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TOPABSTRACTRetinoids as chemopreventive...Human embryonal carcinomaRetinoic acid activates the...Gene expression profiling of...SummaryLITERATURE CITED |
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, do not arrest in the G1 phase of the cell cycle or differentiate in response to RA (18
,22
). Importantly, overexpression of RAR will partially restore RA responses in these cells (23
,24
). The strategy was to compare the expression and activity of a panel of G1 regulators in response to RA between RA-sensitive and -resistant cells (18
). Unexpectedly, in reporter assays RA was found to activate the transactivation function of p53 up to 20-fold in NT2/D1 cells, in a retinoid receptor-dependent manner (25
). NT2/D1-R1 cells were devoid of this RA-inducible transactivation activity and were coresistant to cisplatin. This indicates that RA and cisplatin may share a common pathway involving p53 in EC. Prior clinical studies showed that aberrant p53 expression in premalignant lesions of the head and neck and oral leukoplakia predicts retinoid resistance (26
,27
). RA had no effect on p53 steady-state protein levels in either cell line. RA enhanced expression of endogenous p53 target genes such as insulin-like growth factor binding protein (IGFBP)3, p21, BTG2 and IGFBP6 in NT2/D1 but not NT2/D1-R1 cells (25
,28
). In addition, RA induces transactivation activity of a gal4-p53 fusion protein, suggesting that RA activates p53 independent of increasing p53 levels or sequence-specific DNA binding. This activity is absent in NT2/D1-R1 cells but can restored on cotransfection with specific RARs (25
). Transient transfection of dominant-negative p53 in NT2/D1 cells blocks the RA-mediated decline of a differentiation-sensitive reporter gene, fibroblast growth factor (FGF)4, and also promotes cisplatin survival (25
). Based on these findings we propose that the regulation of a subset of target genes by RA is through activation of p53, which contributes to the G1 cell cycle arrest and subsequent differentiation seen in cells treated with RA. This may be a unique activation mechanism because it was shown not to involve alterations in p53 localization or stability, mdm-2, or p53 DNA binding ability (Curtin and Spinella, unpublished observations).
Furthermore, these studies have contributed insight into the biology of TGCTs. TGCTs are one of the few solid tumors that can be cured (up to 90%) with standard, cisplatin-based chemotherapy, even in disseminated disease (29
). TGCTs and derived cell lines like NT2/D1 express atypically high levels of p53 protein that is rarely mutated (30
,31
). This led to the suggestion by Lutzker and Levine (32
) that wild-type p53 was in a latent form in EC, suggestive of a lack of selective pressure to mutate p53 in TGCTs. Interestingly, site-directed mutation analysis mapped p53 latency in EC to a small 27 amino acid region between the proline-rich region and DNA binding domain of p53 (Curtin and Spinella, unpublished observation). Deletion of this domain resulted in 20-fold induction of p53 transactivation activity, and the repressive activity of this domain was transferable to other transcription factors (Curtin and Spinella, unpublished observations). Interestingly this region of p53 has been relatively uncharacterized in terms of function, protein interactions or posttranslational modifications.
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Gene expression profiling of human embyronal carcinoma |
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TOPABSTRACTRetinoids as chemopreventive...Human embryonal carcinomaRetinoic acid activates the...Gene expression profiling of...SummaryLITERATURE CITED |
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). The total number of RA-responsive transcripts increased by 24 and 48 h, and their pattern of expression was more symmetrical. This may be due to a "second wave" of gene regulation mediated by the early-induced genes. Many of these gene products are associated with developmental pathways including those involving signaling by TGF-ß, homeo-domain, IGF, Notch, Hedgehog and Wnt proteins (28
). Thus it is surprising that in this cell autonomous differentiation model many developmental cues mediated by communication among diverse cell types in the embryo are regulated by RA. This suggests that RA reprograms a dynamic balance of multiple signals from diverse pathways to signal the end of stem cell renewal and the onset of lineage commitment of human EC. Deregulation of development-related genes have been associated with tumorigenesis (33
). In addition, a large set of genes predicted to mediate a major remodeling of the extracellular matrix, another developmental cue with a role in cancer, is acutely regulated by RA. Other genes induced by RA are associated with cell adhesion, cytoskeletal remodeling, tumor growth suppression and intracellular signaling cascades. In contrast, the majority of repressed genes are associated with protein/RNA processing, turnover or metabolism, consistent with cells preparing to become postmitotic. In general, the results suggest that RA alters the expression of a small dynamic set of regulatory genes at this early stage of lineage commitment. Although several of the genes are known to be direct targets of RA, the majority have not been described in this context before. The genes identified are likely candidates in controlling early steps in human development, stem cell renewal and cellular differentiation (in particular, neuronal differentiation). These genes may also be pertinent to malignant progression of TGCTs and to the clinically relevant antitumor effects of RA.
One gene identified as being induced early by RA, receptor interacting protein 140 (RIP140), was characterized in more detail (Table 1
). RIP140 is a novel nuclear receptor-corepressor that can inhibit several ligand-bound nuclear receptors including retinoid, estrogen and glucocorticoid receptors (34
,35
). RA induction of RIP140 occurs within 3 h in EC and human breast cancer cells (Fig. 2
). The RA induction of RIP140 mRNA did not require de novo protein synthesis, consistent with RIP140 being a direct transcriptional target of retinoid receptors (36
). A 4-kb promoter fragment of RIP140 contains a consensus direct repeat (DR)5 RARE and is activated by RA (36
). The finding that RIP140 is a direct transcriptional target of RA is one of the first examples of acute transcriptional regulation of a nuclear receptor coregulator and exemplifies the power of large-scale expression profiling to uncover novel RA target genes. These data are consistent with a model by which the induction of RIP140 by RA supplies a negative-feedback signal toward ligand-activated retinoid receptor. Because RIP140 is a strong repressor of both retinoid and estrogen receptors (34
,35
), RA induction of RIP140 may also be a potential mechanism of cross talk between the two receptors.
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Summary |
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TOPABSTRACTRetinoids as chemopreventive...Human embryonal carcinomaRetinoic acid activates the...Gene expression profiling of...SummaryLITERATURE CITED |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported in part by the National Cancer Institute Howard Temin Award K01-CA75154 (M. J. Spinella), by American Cancer Society Research Scholar Grant RSG-01–144-01 (M. J. Spinella) and by a predoctoral fellowship from the Department of Defense Breast Cancer Research Program BC010159 (K. A. White).
4 Abbreviations used: EC, embryonal carcinoma; ES, embryonic stem; RA, all-trans-retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid responsive element; RIP140, receptor interacting protein 140; RXR, retinoid X receptor; TGCT, testicular germ cell tumor.
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LITERATURE CITED |
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TOPABSTRACTRetinoids as chemopreventive...Human embryonal carcinomaRetinoic acid activates the...Gene expression profiling of...SummaryLITERATURE CITED |
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