Expression of lymphoid enhancer factor/T-cell factor proteins in colon cancer

Expression of lymphoid enhancer factor/T-cell factor proteins in colon cancer Marian L. Waterman, PhD Molecular genetic analysis of colon cancers has established that the Wnt signaling pathway is involved in early tumor development. Mutation of midstream components can activate the pathway, making it independent of Wnt ligands and maintaining constant pressure to change target gene expression. The transcription factors that connect the pathway to target genes are members of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family. The genes for two members of this family, TCF 7 and LEF 1, produce full-length forms that mediate Wnt signals and truncated dominant negative forms that limit Wnt signals and may function as growth suppressors. Results from studies of their expression in colon cancer suggests that because Wnt-linked cancers progress to malignancy, there may be a strengthening of the Wnt signal by selective expression of the activating forms of LEF/TCFs and a bias against suppressing, truncated forms. Curr Opin Gastroenterol 2002, 18:53–59 © 2002 Lippincott Williams & Wilkins, Inc. Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, Irvine, California, USA. Correspondence to Marian L. Waterman, PhD, Room C154 Med Sci I, Department of Microbiology and Molecular Genetics, 19182 Jamboree Blvd., University of California, Irvine, Irvine, CA 92697-4025, USA; e-mail: mlwaterm@uci.edu Current Opinion in Gastroenterology 2002, 18:53–59 Abbreviations HMG LEF/TCF high-mobility group lymphoid enhancer factor/T-cell factor ISSN 0267–1379 © 2002 Lippincott Williams & Wilkins, Inc. Wnt signal transduction Wnt proteins are secreted morphogens that trigger receptive cells to adopt specific fates or polarities or to grow and divide. The steps in the signaling pathway and the role of this pathway in cancer are already the subjects of several excellent, in-depth reviews [1–6]; therefore, only a brief overview is presented here. By binding to cell surface receptors called Frizzleds, Wnt ligands initiate a signaling cascade that reaches the nucleus via rapid movement of cytoplasmic ␤-catenin through nuclear pores (Fig. 1). To effect this nuclear localization, the Wnt signal inhibits the activity of the serine/threonine kinase GSK-3␤ [7–9], a ubiquitously expressed and constitutively active kinase. In the absence of Wnt signals, GSK3␤ cooperates with the proteins adenomatous polyposis coli and axin/conductin and other components to promote the degradation of newly translated ␤-catenin via the ubiquitin-proteasome pathway [10–12]. This activity is very efficient, and, in the absence of any incoming Wnt signal, the cytoplasm and nucleus are devoid of ␤-catenin protein. (The exception is a stable pool of ␤-catenin in cell adherens junctions where it functions as an essential adaptor protein between the cytoplasmic tail of E cadherin receptors and the cytoskeleton binding protein ␣-catenin.) Wnt signals inhibit GSK-3␤ kinase activity to stop degradation and force a rapid increase in the levels of free, cytosolic ␤-catenin and soon thereafter nuclear ␤-catenin. Nuclear ␤-catenin binds tightly to LEF/TCF transcription factors and together they alter transcription of gene targets [13–16]. ␤-Catenin does not have a DNA binding domain but has a potent transcription activation domain. LEF/TCF transcription factors do not have a strong transcription activation domain but have a good DNA binding/bending domain. Thus, when ␤-catenin binds to a LEF/TCF protein, it forms a potent transcription regulatory complex. Although the functions of this complex vary in different cell types, genes involved in cell cycling are likely to be common targets [17•,18•,19–21,22•]. Wnt-independent activation: normal mechanisms and genetic mutations The Wnt pathway can be activated at steps that lie downstream of a Wnt•Frizzled interaction. For example, GSK-3␤ can be inhibited by other signal cascades such as integrin-linked kinase and insulin [9,23]; such inhibition occurs transiently within a normal context. Genetic mutation of midstream components can also activate the pathway, but, in this case, the pathway is turned on con53 54 Large intestine Figure 1. Wnt signal transduction A simplified schematic of Wnt signaling in a mammalian cell shows that signaling begins when Wnt ligands bind to transmembrane receptors (Frizzled) on the cell surface. Binding causes phosphorylation of disheveled and subsequent interaction and recruitment of GBP (GSK3-␤ binding protein, also known as FRAT) to GSK3-␤ for potent inhibition of its kinase activity. In the absence of Wnt signals, GSK3-␤ phosphorylates ␤-catenin and adenomatous polyposis coli within a large multiprotein complex brought together by axin (but not ␤-catenin proteins that are bound to the cytoplasmic tail of E-cadherin in cell adhesion junctions). Phosphorylation allows Slimb/TrcP proteins (not shown) to lead ␤-catenin through the ubiquitin-dependent proteolysis pathway. Inhibition of GSK3-␤ activity prevents degradation and allows ␤-catenin to accumulate in the nucleus where it can bind to any one of four lymphoid enhancer factor/T-cell factor proteins. Together these proteins bind and regulate expression of Wnt target genes. stitutively, and this problem leads to cancer. Loss-offunction mutations in the tumor suppressor adenomatous polyposis coli or axin, or mutations that make ␤-catenin unable to be degraded (mutation of GSK-3␤ serine substrates in the N-terminus), allow ␤-catenin protein to accumulate to very high levels. Abiding, high levels of ␤-catenin translate into constitutive Wnt signaling—a permanent turning on of the pathway [24–26]. This constitutive activity has been implicated as a root cause of several different kinds of cancers, including colon, prostate, melanoma, hepatocellular carcinomas, and many others [1–6]. Presumably, high levels of ␤-catenin•LEF or ␤-catenin•TCF complexes form in the nucleus, and the target genes of this complex are thus misregulated. This is observed in normal chicken embryo fibroblasts that are transformed when a LEF-1•␤-catenin fusion protein is expressed [27]. Likewise, the highly related ␥-catenin (plakoglobin) protein, which forms active complexes with LEF/TCFs, greatly upregulates c-MYC expression to transform epithelial cells [28••]. However, target gene regulation by the catenins and LEF/TCFs is not very well understood. A common assumption is that there is a preexisting pool of LEF/TCF proteins that are able to recruit the huge excess of ␤-catenin to gene targets. However, as we now know, there are inhibitory pathways that limit the activity of ␤-catenin•LEF/TCF complexes in the nucleus (ie, the TAK/NLK pathway [29], I-mfa [30], ICAT [31], HBP-1 [32], and Sox3, Sox 17␣/␤ [33]), and there are also forms of LEF/TCFs that suppress Wnt signals rather than transmit them. It is the expression of suppressive forms of LEF/TCFs relative to full-length activating forms of LEF/TCFs that may differ between normal gut mucosa and colon cancer. LEF/TCF family: protein structure The LEF/TCF family of transcription factors in mammals is composed of four different proteins (Fig. 2) [34,35]. The founding family members, human and mouse LEF-1 and TCF-1, were first discovered in differentiating T and B lymphocytes [36–38]. Two other mammalian LEF/TCF proteins (TCF-3 and TCF-4) were later identified in other tissues by low-stringency cloning procedures, and their expression patterns differ [39]. LEF/TCF proteins are expressed in many tissues during embryogenesis in overlapping but distinct patterns [39,40]. Shortly after birth, their expression is shut off or downregulated, presumably when cells of these tissues become terminally differentiated. The known exceptions to this phenomenon are the thymus (LEF-1, TCF-1), bone marrow (LEF-1) [18•], the skin and dermal papillae at the base of hair follicles (LEF-1, TCF-3) [17•,41], the crypts of colon (TCF-4) [42,43••], intestinal mucosa (TCF-1, TCF-4) [42,44••], testes (LEF-1) [14], and mammary epithelia (TCF-1, TCF-4) [44••,45]—all sites of continual cell growth and differentiation from stem-cell populations. Of the functional domains that have been mapped in LEF/TCF proteins, four are described here. The first is the high-mobility group (HMG) DNA binding domain, Expression of lymphoid enhancer factor/T-cell factor proteins in colon cancer Waterman 55 Figure 2. The human lymphoid enhancer factor/T-cell factor family of transcription factors There are four members of the family in mammalian cells. Four functional domains are shown; percentages refer to amino-acid-sequence identity relative to LEF-1. Accession numbers for sequences used in the alignment: hLEF-1, AF288571; hTCF-1, Z47362, AF163776; hTCF-3, AB031046; hTCF-4, Y11306, CAA72166. C-termini are generated by splicing; only the most common tails are shown. an 88-amino-acid region near the carboxy terminus containing a HMG box motif and nuclear localization signal. HMG boxes were first recognized in the nonhistone, chromatin-associated HMG 1/2 proteins [46], and a hallmark of these motifs is that they bend DNA (90 to 130 degrees) [47,48]. The HMG DNA binding domain is the most highly conserved feature of the LEF/TCF family (between 93% and 99% amino-acid-sequence identity), which means that LEF/TCFs bind and bend identical DNA sequences. The second most highly conserved feature of the LEF/TCF family is a region at the extreme N-terminus. The function of this region was discovered when Behrens et al. [49] and Molenaar et al. [15] identified it as a ␤-catenin binding domain. Genetic experiments in Drosophila and Xenopus confirmed that this interaction is necessary for Wnt signaling and normal development, establishing that LEF/TCF proteins are bona fide components of the pathway [15,50,51]. Since then, all known members of the LEF/TCF family have been shown to bind to ␤-catenin or one of its orthologs. A third feature is the alternative C-termini generated from alternative splicing (designated by alphanumeric nomenclature). Different termini were first discovered for TCF1, but they have since been identified in all family members [52–54]. The function of these alternative C-termini has not been very well studied, although a protein motif in the E tail of TCF-3 and TCF-4 that binds to the transcription corepressor CtBP has been identified [55]. CtBP binding is not conserved in TCF-1E, and the LEF1 locus cannot generate an E tail. Therefore, the alternative splicing feature of the family may distinguish one member from another by the unique array of C-terminal activities. Finally, a transcription repression domain is located in the central region; the overall function of this domain appears to be conserved for each LEF/TCF, even though the primary sequences are not. LEF/TCF proteins do not carry any strong independent activating or repressing domain, rather they are considered architectural proteins that bend DNA to maximize productive interactions between DNA-bound proteins in a larger enhancesosome or repressing complex [56,57]. In addition to this structural function, LEF/TCFs directly recruit coactivators or corepressors. Although the most well known role of LEF/TCFs is to recruit the strong transcription activator ␤-catenin through direct binding to the N-terminus, the central domain of LEF/TCFs can recruit the corepressors of the Groucho family [58•]. In addition, LEF-1 can recruit HDAC1, a repressive histone deacetylase [59•], but whether this occurs directly through binding the central domain or via an indirect interaction is not yet known. The central domain in LEF-1 can also engage in protein•protein contacts with the protein ALY [60] or with activating factors that bind to neighboring sites on the T-cell receptor ␣ chain enhancer [61–63]. Nevertheless, for many if not most target genes, it is thought that LEF/TCFs function as transcription repressors in the absence of ␤-catenin binding and that this activity is carried out by corepressor recruitment via the central domain. Dominant negative LEF/TCFs Productive Wnt signaling relies on the expression of fulllength LEF/TCF proteins from the N-terminal ␤-catenin binding domain to the HMG DNA binding domain near the C-terminus, and indeed most tissues with normal LEF/TCF expression show abundant levels of full-length protein. However, both the TCF7 (encod- 56 Large intestine ing TCF-1 protein) and LEF1 genes contain two different promoters for transcription (Fig. 3) [52,54]. The first promoter in each gene produces mRNA-encoding fulllength protein with an intact ␤-catenin binding domain at the N-terminus. The second promoter is located in the second intron and produces a truncated protein missing the ␤-catenin binding domain. Truncated LEF-1 and TCF-1 proteins have an intact HMG DNA binding domain and nuclear localization signal and can therefore reside in the nucleus and bind as tightly to Wnt response elements as the full-length forms. They cannot recruit ␤-catenin because they are missing the N-terminus and therefore cannot mediate Wnt signals. They are, however, fully capable of recruiting corepressors such as Groucho through their intact central domain. Thus, if the levels of these truncated forms exceed the levels of fulllength, ␤-catenin binding forms, they could effectively suppress Wnt signaling by occupying regulatory sites and actively inhibiting transcription. These truncated forms are called natural dominant negatives because they are able to prevent ␤-catenin•TCF complexes from activating reporter genes [15,43••]. Truncated forms are expressed in normal tissues such as thymus, where the TCF7 locus expresses mostly dominant negative forms, and the LEF1 locus appears to express equivalent amounts of both (at least at the mRNA level) [36,37,52,64]. Expression of LEF/TCFs in the colon What are the expression patterns of LEF/TCFs in normal colon tissue and colon cancer? Are the dominant negative forms expressed, and, if so, is expression altered in cancer? Recent studies over the past two years have begun to address these questions. Two members of the family, TCF-4 and TCF-1, are expressed in normal gut mucosa, and three members, TCF-4, TCF-1, and LEF1, are expressed in colon cancer (Table 1). Normal colon tissue requires TCF-4 expression for development and maintenance of its stem-cell compartment [42]. Mice missing the TCF7L2 gene (encodes TCF-4 protein) die shortly after birth with the unusual phenotype of an intestine with a few differentiated villi and no proliferating crypt stem-cell compartment. Analysis of TCF-4 expression in adult human colon shows that mRNA and protein are present in all cells of the gut epithelium from the crypt to the tips of the villi [24,43••,45]. TCF-4 continues to be expressed in colon cancer and analysis of TCF-4 protein in human colon cancer cell lines suggests that only a full-length TCF-4 polypeptide is expressed [43••]. Thus, expression of TCF-4 correlates most closely with maintenance of mitotically active cells in the intestine. TCF-1 function differs dramatically. TCF-1 is expressed in fetal and adult intestine [44••], and its expression has also been noted in mouse small intestine and human colon cancer cell lines [65]. Surprisingly, mice missing the TCF7 gene (TCF-1 protein) develop intestinal and mammary adenomas [44••]. There are dramatic increases in the number of adenomas when the APC gene is inactivated in these mice. In this respect, TCF-1 appears to function as a tumor suppressor rather than a Wnt-linked tumor promoter. Although the relative levels of full-length and truncated TCF-1 isoforms are not Figure 3. Genomic structure and protein products from the TCF7 and LEF1 genes Promoters P1 and P2 in the LEF1 and TCF7 (for TCF-1) genes produce proteins that differ at the N-terminus. The first four exons (Ia⬘, Ib⬘, I, and II) and three exons (1 to 3) are shown for the TCF7 and LEF1 genes, respectively. For both genes, the first exon encodes the ␤-catenin binding domain. A second promoter in intron 2 of both loci produces mRNA that does not have the coding sequences for ␤-catenin binding. Instead, an internal ATG translation start codon is used in exons II (TCF7) and 3 (LEF1). The truncated polypeptides from these second mRNAs contain all the functional domains necessary to localize to the nucleus and bind to DNA and to recruit transcription corepressors. Because of this latter activity, these truncated forms are referred to as dominant negative isoforms. Expression of lymphoid enhancer factor/T-cell factor proteins in colon cancer Waterman 57 Table 1. Expression and knock-out phenotypes of LEF/TCFs in colon Normal colon* Colon cancer† TCF-4 [24,42] + + TCF-1 [44••] LEF-1 [43••] + − ND + LEF/TCF Knock-out phenotype‡ No proliferative stem cells in crypts, few differentiated villi Adenomas NR +, present; −, absent; LEF/TCFs, lymphoid enhancer factors/T-cell factors. ND, not determined for primary human colon cancer; NR, none reported; *Human colon tissue. †primary human colon cancer; ‡phenotype of small intestine in mice homozygous null for the indicated gene. known for mouse and human intestine, the expression of TCF-1 in normal thymus tissue shows that the predominant forms are truncated polypeptides missing the ␤-catenin binding domain [52,64]. If this is also true for expression of the TCF7 locus in the intestine, then an abundance of dominant negative TCF-1 could limit Wnt signal transduction by competing with ␤-catenin•TCF-4 for sites on targets genes, and this could be its normal, essential function. What is the pattern of expression TCF-1 in primary colon cancer? Again, no definitive answers are yet available, but colon cancer cell lines contain variable amounts of full-length and truncated isoforms [65]. Expression of the LEF1 gene should have nothing to do with human colon. The gene is not expressed in normal colon tissue, and no defects in the intestinal tracts of mice missing the LEF1 gene have ever been reported [43••,66]. It is surprising then to find that LEF-1 expression is detected in most primary advanced human colon cancers and many colon cancer cell lines [43••]. This means that transcription of the LEF1 locus is activated by some aberrant mechanism in these tumors. It is not known whether expression of LEF1 contributes to tumor progression, but the expression pattern is aberrant because it is skewed entirely toward producing full-length forms that bind to ␤-catenin. The promoter for fulllength LEF-1 mRNA form is activated, whereas the intronic promoter that produces truncated forms is specifically left silent. Given the tumor suppressive functions of TCF-1 and the aberrantly skewed expression pattern of LEF-1 in colon, it appears that Wnt-linked tumors might progress in part by downregulating or negating expression of dominant negative LEF/TCF proteins and upregulating the expression of full-length isoforms. Loss of expression of the truncated forms could occur by loss of the gene entirely, and this has been proposed for TCF7 [44••]. Indeed, TCF7 resides on the same arm of chromosome 5 as the APC gene (5q31.1 and 5q22.1, respectively), and loss of both genes in mice causes synergistic increases in the number of adenomas. Conversely, both TCF7 and LEF1 upstream promoters contain Wnt re- sponse elements [43••,44••]. It is possible that these promoters are targets of the Wnt pathway, and in an early cancer with abundant ␤-catenin protein, expression could be activated by ␤-catenin•TCF-4 complexes (without concomitant activation of the intronic promoters). As a result, a greater level of full-length LEF/TCF protein would be available for complex formation with ␤-catenin. Higher concentrations of ␤-catenin•LEF/TCF complexes in the nucleus could dysregulate normal target genes and perhaps target genes that should never be expressed in a colon cell (such as the LEF1 gene). Whether some or all of these possibilities actually occur in primary human colon cancer is not yet known. It is important to determine more carefully the expression patterns of each LEF/TCF locus in normal gut mucosa compared with different types and stages of colon cancer. Results over the past several years emphasize that expression of LEF/TCF loci is complicated, producing isoforms with opposing activities as well as alternatively spliced isoforms with largely unexplored functions. Current in situ hybridization and immunohistochemistry techniques cannot distinguish between these various forms and thus can give no information as to what limits are imposed to shape the signal strength of Wnt in the nucleus. Even though there is a high frequency of mutations that activate Wnt signaling early in colon cancer, Wnt-linked cancers must circumvent the opposing dominant negative LEF/TCFs and the other aforementioned forces in the nucleus that act to corral Wnt signaling. At least for LEF/TCF expression, such hurdles may be overcome by altering the patterns of expression from their loci. Identifying the mechanisms that produce these altered patterns will greatly increase our understanding of how this cancer progresses to later stages of metastasis. Acknowledgments The author thanks Dr. Randall Holcombe for comments on the manuscript and members of the Waterman laboratory for scientific discussion. 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