Interplay Between Notch Signaling and Epigenetic Silencers in Cancer

Review

Interplay between Notch Signaling and Epigenetic Silencers in Cancer

Maria Dominguez

Instituto de Neurociencias, Consejo Superior de Investigaciones Cientificas-Universidad Miguel Herna´ndez, Campus de Sant Joan, Alicante, Spain

Abstract epigenetic silencing elements and transforming networks can be Given its role in the development and self-renewal of many defined in vivo. tissues, it is not surprising that a prominent role has recently Despite the tradition of studying cancer in mouse models, the been proposed for the Notch signal transduction pathway in fruit fly, Drosophila melanogaster has recently emerged as a tumor development. However, exactly how Notch hyper- powerful model system for these purposes (8). Indeed, a genetic activation promotes oncogenesis is poorly understood. Recent screen in the Drosophila eye revealed an unexpected collaboration findings in Drosophila melanogaster have linked the Notch between epigenetic silencing pathways and the Notch signaling pathway to epigenetic silencing and the tumor suppressor pathway during tumorigenesis (9). The Notch signaling pathway is gene Rb during tumorigenesis. Because aberrant epigenetic important in many biological processes as it governs binary cell fate decisions, patterning, cell proliferation and growth, apoptosis, gene silencing contributes to the pathogenesis of most human cancers, these findings may provide a new focal point to differentiation, and migration. This developmental pathway was understand how Notch is associated with cancers, and to help first identified in Drosophila before mammalian Notch receptors (NOTCH1-4) and ligands of the Delta (DLL-1, DLL-3, and DLL-4) develop better selective cancer therapies. (Cancer Res 2006; and Serrate (JAGGED-1, and JAGGED-2) family were shown to act 66(18): 8931-4) in mammalian development, organ self-renewal, and diseases such as cancer (10). Background Aberrant activation of the NOTCH1 receptor is estimated to Cancer is a highly varied and complex disease considered until participate in 50% of all pediatric and in almost all of the most recently to be underpinned by genetic abnormalities. However, common subtypes of T cell acute lymphoblastic leukemias (11). epigenetic alterations of gene expression now also seem to Moreover, inappropriate activation of the NOTCH transduction contribute to the pathogenesis of cancer. Thus, epigenetic pathways has also been associated with a wide range of solid processes are responsible for the inactivation of tumor suppressors tumors (12) and, in some contexts, Notch has been shown to and tumor-related genes in many cancer cells, particularly through function as a tumor suppressor gene; but how the deregulation of abnormal histone modifications and aberrant DNA methylation. Notch pathways contributes to the pathogenesis of cancer in vivo These epigenetic modifications permit heritable gene silencing remains unclear. Accordingly, it is imperative to identify the that could, in turn, contribute to the initiation and progression of oncogenes and tumor suppressors that cooperate with Notch cancer, and to metastasis (1, 2). In contrast to genetic changes (i.e., pathways to promote tumorigenesis. The recent demonstration mutations), the reversibility of epigenetic changes has led to an that Notch and epigenetic silencing pathways cooperate to expansion of epigenetic therapies targeting histone deacetylases promote tumorigenesis is a first step in this direction (9). If this (HDAC) and DNA-demethylating agents (for reviews, see refs. 3, 4). holds true for human cancers, these discoveries open the In normal cells, the epigenetic inheritance of transcription patterns possibility to treat Notch-associated cancers with epigenetic (i.e., cellular memory) helps to maintain cell identity, as well as therapies. regulating senescence and stem cell renewal. For example, proteins of the PcG form a part of the memory machinery and they maintain transcriptional repression of genes and have been Key Findings implicated in the maintenance of stem cell pluripotency and The early growth and patterning of the Drosophila eye depends plasticity (see for example, ref. 5). In humans, the up-regulation of on the autonomous and nonautonomous activity of the Notch signal PcG genes has been associated with many types of invasive transduction pathway (13). The Delta and Serrate ligands locally carcinomas, lymphomas, and leukemia (6, 7). To understand how activate the Notch receptor along a dorsal/ventral organizer. cancer initiates, and to develop specific therapies, we must Inappropriate inactivation of the Notch pathway or the expression determine how these damaging epigenetic alterations arise in of dominant-negative Notch receptors impairs eye growth, whereas the precursor cells of cancer, and why specific genes are targeted constitutive activation of this pathway or ectopic ligand expression for epigenetic silencing in such cells. To address these questions induces eye overgrowth. Given the increasing relevance of the Notch directly, animal models are required in which the role of different pathway in human cancers, we established a high-throughput forward genetic screen in the fly eye to search for ‘‘gain-of- expression’’ mutations that interact with the Notch pathway and

Requests for reprints: Maria Dominguez, Instituto de Neurociencias, Consejo that convert tissue overgrowths into tumors. For this, we exploited Superior de Investigaciones Cientificas-Universidad Miguel Herna´ndez, Campus de the ‘‘large eye’’ phenotype caused by overexpression of the Notch Sant Joan, Apto. 18, 03550 Alicante, Spain. Phone: 34-96591-9390; Fax: 34-96591-9561; ligand Delta (14) coupled to the Gene Search (GS) transposon E-mail: [email protected]. I2006 American Association for Cancer Research. system to systematically generate gain-of-expression mutations (15). doi:10.1158/0008-5472.CAN-06-1858 This P element–based GS transposon contains five tandem Gal4 www.aacrjournals.org 8931 Cancer Res 2006; 66: (18). September 15, 2006

Figure 1. Top, the proposed mechanism by which cancer is initiated by the Notch signaling pathway and the PcG-mediated cellular memory machinery. The hyperactivation of the Notch pathway initiates the repression of target genes, such as the Rbf gene. Subsequently, Pipsqueak (Psq) and/or Lola could bind to the repressed genes and enforce gene silencing via the recruitment of HDAC and histone lysine methyltransferases. Thereafter, binding of bromodomain and chromodomain proteins, such as Pc, to the methylated H3 lysine residues initiates chromatin condensation and ‘‘permanently’’ silences the Notch target genes. This would foment tumor growth and metastasis. Bottom panels, overexpression of the Notch ligand Delta (ey-Gal4 > Dl) or deregulation of Psq and Lola (data not shown) alone is insufficient to promote tumor formation. However, the coexpression of psq, lola,andDelta in the developing eye tissue induces the development of highly invasive tumors. Macroscopically visible metastases (red) can be found throughout the body of the fly (9).

Cancer Res 2006; 66: (18). September 15, 2006 8932 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2006 American Association for Cancer Research. Notch and Epigenetics in Cancer binding sites (upstream activating sequences, UAS) capable of control (21), we analyzed this modification with specific antibodies overexpressing or misexpressing gene(s) laying on either side of the in mutant eye discs. We observed the loss or strong reduction of GS insertion. The GS lines were crossed with ey-Gal4 flies carrying a histone H3K4 trimethylation (H3K4me3) in the developing eye tissue Delta transgene (UAS-Delta), which drives eye-specific expression of from which the tumor arises. Interestingly, this epigenetic modifi- the UAS-linked genes throughout the proliferative phase of the cation identifies open chromatin and transcriptional activation, and imaginal eye disc (14). thus, the strong reduction in histone H3K4me3 signifies that the This screen identified a GS line, that when coupled to Delta chromatin in the mutant tissue has been condensed or silenced. overexpression, produced massive overgrowths and tumors (9). Strikingly, we found a small but significant reduction of this Some of these mutant flies display macroscopically visible epigenetic marker in the overgrown eye tissues when only the Delta secondary eye tumors within the head, thorax, and abdomen, gene is overexpressed (9). Together, these results suggest that the hence the line was named ‘‘eyeful’’ (Fig. 1). These secondary eye Notch pathway controls normal tissue growth and tumorigenesis at growths (metastases) have ragged borders and a disrupted basal least in part through epigenetic silencing. membrane, and they invade their surrounding tissue (9). The eyeful The two H3 methyl tags associated with epigenetic gene GS-element causes the deregulation of two neighboring genes, silencing and chromatin condensation in flies and mammals longitudinals lacking (lola) and pipsqueak (psq; ref. 9). The lola involve H3 methylation at K9 and K27 (20). These events are gene encodes 25 alternatively spliced mRNAs that generate 19 coordinated by the Suppressor of variegation 3-9 [Su(var)3-9] different transcription factors. All of these transcription factors (H3K9me) and Enhancer of zeste [E(z)](H3K27me) genes (19, 20). share a BTB domain (Broad complex, Tramtrack, Bric-a-brac, also Human homologues of these two fly genes are often up-regulated known as POZ for Poxviruses zinc-finger), and all but one is spliced in cancer cell lines (6, 7, 20), and thus, inhibitors of HDACs are to unique exons encoding zinc finger motifs (16). The psq gene promising drugs for patients with cancer (1–4). It was therefore encodes four different proteins, three of which contain a BTB critical for us to determine whether endogenous Rpd3/HDAC and domain in the NH2 terminus. Furthermore, two of the BTB- the histone lysine methyltransferases Su(var)3-9 and E(z) were containing isoforms and the isoform that lacks the BTB domain required for tumorigenesis in vivo. Extra Sex Comb (Esc) and its have four tandem copies of a conserved DNA-binding motif known human counterpart, EED, are essential for the E(z)/EZH2-mediated as the Psq helix-turn-helix (HTH) motif (17, 18). methyltransferase activity (20, 21). Furthermore, binding of the To determine whether the lola and psq genes contribute to the chromodomain protein Pc/HPC to methylated H3K27 is required tumor phenotype, we introduced point mutations into the lola or to stably silence gene transcription. Thus, we tested the need for À psq genes. All psq knockouts induced on the eyeful chromosome these genes in a heterozygous assay in vivo. Complete loss of prevented the eyeful GS line from producing eye tumors and function of any of these epigenetic genes confers a pleiotropic À metastases (9). In contrast, whereas all lola mutations reduced phenotype or early lethality (20), whereas the heterozygous state the size of the eye tumors, including a null allele of lola, metastases (i.e., a 50% reduction in gene dosage) enables us to develop a still developed (9). Thus, transcription of both genes is required for reliable and sensitive assay. If the Rpd3/HDAC, E(z), Su(var)3-9, Pc, tumorigenesis, although Psq seems to be more important for and Esc genes are required for Psq and Lola-mediated aberrant tumor growth and metastasis. epigenetic repression, giving half the dosage should significantly À Six of the psq mutations (missense, nonsense, small deletion, impede tumor development. Indeed, the eye phenotype in flies and splice) disrupt or completely delete the conserved BTB or the with a mutant copy of the Rpd3, Su(var)3-9, E(z), Pc,orEsc sequence-specific DNA-binding domains. Thus, protein-protein suggested a clear involvement of these epigenetic repressors in the interactions via the BTB domain as well as DNA binding are likely development of tumors. to be involved in oncogenesis. The human oncogenic proteins, In searching for tumor suppressor and tumor-related genes PLZF and BCL-6, associate with HDACs, corepressors, and PcG whose silencing might be aberrant in these tumors, we found that repressors through their BTB domain (ref. 19, and citations the retinoblastoma family protein (Rbf ) gene expression was therein). Although a direct interaction of the BTB domain of Psq down-regulated in tumors, whereas the expression of a second and/or Lola with HDACs and Polycomb repressors remains to be Rbf2 gene was unaffected (9). Interestingly, Rbf gene expression shown, Psq is present in a multiprotein complex that displays PcG was also down-regulated in the eye tissue upon overexpression of repressor and HDAC activity (reviewed in ref. 20). The Psq-type the growth-promoting Delta protein alone (9), showing that the HTH motif binds to the GAGAG sequence, which is present Rbf gene is a target of the Notch pathway. More importantly, in many Polycomb-responsive elements of homeotic box (hox) reestablishing Rbf gene expression in the mutants prevented the genes, as well as at hundreds of other chromosomal sites. Thus, tumor phenotype. In contrast, further reducing the activity of Psq and Lola could be required for sequence-specific targeting of endogenous Rbf by introducing a mutant copy of this gene notably Polycomb complexes to particular genes. Indeed, genetic studies enhanced the tumor phenotype (9). These results clearly link the have identified both Psq and Lola as members of the PcG family Notch signaling pathway to cell cycle control. RB1, together with (9, 20). We therefore speculated that the deregulation of psq and lola p53, participates in one of the most important tumor suppressor might induce tumorigenesis through aberrant epigenetic silencing of pathways in humans. RB1, like its fly counterpart, Rbf, acts as a genes that contribute to the uncontrolled growth of tumor cells. constraint on the G1-S transition and therefore its inactivation is Epigenetic processes alter the accessibility of transcriptional often considered an important step towards malignancy (22). In regulators to chromatin via the local or global modification of humans, the loss of RB1 expression can occur through epigenetic histones and DNA (20, 21). Covalent histone modifications include: mechanisms, specifically due to the aberrant DNA hypermethyla- lysine acetylation, lysine and arginine methylation, serine/threonine tion of its promoter (1). In insects, such as D. melanogaster, there is phosphorylation, ADP-ribosylation of glutamic acid, and sumolya- little DNA methylation in the genome. Nevertheless, we found that tion and ubiquitination of lysines. Because lysine (K) methylation of when compared with the wild-type, the promoter and transcrip- histone H3 is important in both fly and mammalian epigenetic tion start regions of the Rbf gene are methylated in tumors (9). www.aacrjournals.org 8933 Cancer Res 2006; 66: (18). September 15, 2006

Significance and Future Directions seems to be very compressed, as this study (9), and earlier studies (8), These recent findings reveal the unexpected cooperation between have affirmed. One possible way to accelerate the metastatic the Notch pathway and PcG-mediated cellular memory to promote development of tumors is if the same combination of genes which tumor initiation and metastasis in D. melanogaster (see model, initiated the tumor are also responsible for its metastasis. Hence, we Fig. 1, top). They also imply that the growth control normally exerted expect further studies of the eyeful and other fly mutants to provide by the Notch pathway involves epigenetic changes, such as depleting more insights into the genetic and epigenetic changes that activate histone H3K4 methylation and down-regulating Rbf gene expression, (or inactivate) metastasis-initiating genes, driving stationary epithe- thereby linking histone modification and cell cycle control with the lial cells to become invasive (i.e., to become motile and capable of Notch developmental pathway. Given that both Notch hyper- degrading the basal lamina), and to metastasize. Although there are activation and aberrant gene silencing have been identified in clearly vast differences in the physiology of flies and mammals, the human cancers, these discoveries in the fruit fly may provide a useful basic processes and rules that govern the transformation of a healthy new focus for research into Notch-associated cancers. cell into a cancerous cell are likely to be similar. Finally, the striking eyeful mutant may help us to understand the initial events that trigger tumor metastasis. In most types of Acknowledgments malignant tumors, it takes years for a tumor to accumulate all the genetic and epigenetic alterations required to transform it from a Received 5/22/2006; accepted 6/23/2006. Grant support: Research at the laboratory of M. Dominguez is funded by grants premalignant lesion into an invasive cancer. In flies, which have a from the Spanish Ministry of Education and Science, the Regional Government of relatively short life cycle, the time for malignant tumor development ‘‘Generalitat Valenciana,’’ and the EMBO Young Investigator Programme.

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Maria Dominguez

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