Disruption of the TGF-β pathway and modeling human cancer in mice

Mutation Research 576 (2005) 120–131

Review

Disruption of the TGF-␤ pathway and modeling human cancer in mice John J. Letterio ∗ Laboratary of Cell Regulation and Carcinogenesis, The Center for Cancer Research, NCI, NIH, Bethesda, MD 20892-5055, U.S.A. Received 27 December 2004; received in revised form 28 February 2005; accepted 1 March 2005 Available online 1 June 2005

Abstract There is considerable complexity underlying the mechanisms through which the TGF-␤ signaling pathway regulates the initiation and progression of cancer. Analysis of this pathway and the role that it plays in human malignancy continues to elucidate novel mechanisms through which various genetic and epigenetic events subvert the controls that TGF-␤ exerts over cell growth, differentiation, and malignant transformation. Modeling these events in the mouse represents an important goal, as the relevant preclinical models are essential not only for improving our understanding of the role of the TGF-␤ pathway in the molecular pathogenesis of cancer, but also as tools for evaluating the impact of novel therapeutics on TGF-␤ signaling and the role they may play in the prevention and treatment of malignancies. Here, we consider highlights from a number of in vivo murine model systems and relate a few of the significant observations to what we know about TGF-␤ signaling in human cancer. © 2005 Published by Elsevier B.V. Keywords: TGF-␤; Cancer; Smad; Signaling; Inflammation

Contents 1. 2. 3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Complex roles of TGF-␤ in carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disruption of TGF-␤ signaling in models of intestinal neoplasia in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Gastrointestinal tumorigenesis in the Tgf-␤1−/− mouse model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Tissue-specific expression of TGF-␤ signaling: dominant-negative TGF-␤ receptors and conditional deletion of the type II TGF-␤ receptor gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Gastrointestinal tumorigenesis associated with disruption of SMAD genes in mice . . . . . . . . . . . . . . . . . . . . . . .

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0027-5107/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.mrfmmm.2005.03.004

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TGF-␤ signaling in models of hematopoietic malignancy in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Targeting the TGF-␤ receptors and lymphoid neoplasia in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. A specific role for SMAD3 in leukemogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Our understanding of the role of the transforming growth factor-␤ pathway in the neoplastic process has been advanced by the application of transgenic and gene targeting technology in mice. Alterations in TGF␤ signaling have been frequently implicated in human cancer, including malignancies of both hematopoietic and epithelial origin. For many of these human cancers, a tumor-suppressor function for TGF-␤ has ultimately been most clearly defined through the analysis of genetically engineered mouse models in which disruption of TGF-␤ signaling either initiates or promotes the neoplastic process. In this chapter, we will concentrate on lessons learned from several in vivo murine model systems. These include those characterized by a disruption of TGF-␤ signaling through the expression of dominant negative-acting TGF-␤ receptors, by targeted disruption of genes encoding the type 1 isoform of TGF-␤, and by disruption of genes encoding components of the signaling pathway, specifically the SMAD genes. These new models represent a valuable asset in the exploration of novel chemopreventive and therapeutic strategies for cancer.

2. Complex roles of TGF-␤ in carcinogenesis The transforming growth factors-␤ (TGF-␤) were first identified in culture supernatants of Moloney MuSV-transformed mouse fibroblasts and named for their ability to support the anchorage-independent growth of a responder cell line in a standard transformation assay [1]. Our initial view that this ‘growth factor’ was an effector of transformation has given way to the understanding that this family of the ligands signal through an important tumor suppressor pathway. Moreover, extensive in vivo studies in murine model systems have also demonstrated that this cytokine serves a dual role in oncogenesis that, in part, may have been

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predicted by the simple transformation assay used to identify TGF-␤ activity. A principal reason that the TGF-␤ ligands play such a complex role in the pathogenesis of cancer is that their effects on stroma, vasculature, and immune cells, all contribute to the ultimate development, growth, and metastasis of a tumor cell. The existence of a ‘prooncogenic’ activity for TGF-␤ is largely a consequence of the effects of this cytokine on cells in the ‘environment’ in which a cancer cell grows. This point can be highlighted by the observation that subcutaneous injection of TGF-␤1 enhances skin tumor promotion, which suggests distinct roles for stroma-derived paracrine and autocrine epithelial TGF-␤1 in the maintenance of epithelial homeostasis [2]. The production and secretion of TGF-␤ by tumor cells can enhance their survival by stimulating the production of matrix and stroma necessary for tumor cell attachment, growth, and ultimately invasion through basement membrane barriers. Studies have shown that TGF-␤ can enhance the metastatic potential of mammary tumors by contolling their ability to destroy and invade basement membrane barriers [3]. Paracrine effects of tumor-derived TGF-␤ on surrounding normal cells are also known to decrease the production of collagenase, stromelysin, and other enzymes that degrade extracellular matrix, while providing a chemotactic and inductive signal to fibroblasts and promoting the deposition of fibronectin, collagen, and the expression of integrins. These pro-metastatic properties clearly co-exist with the tumor-suppressive effects of TGF-␤, and each is undoubtedly influenced by factors in the microenvironment and by additional genetic alterations within the tumor cells. This point has been illustrated by in vivo studies in which the Neu (ErbB2) proto-oncogene was co-expressed with TGF␤1, each driven by the MMTV promoter. The histologic grade, local invasiveness, and metastatic potential of the Neu-induced tumors were all increased by the coexpression of TGF-␤1. The MMTV-Neu tumor cells exhibited increased activation of AKT and MAPK that

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were abrogated by an inhibitor of TGF-␤ signaling, clearly indicating an intact autocrine TGF-␤ loop [4]. A second and important mechanism underlying the pro-oncogenic nature of TGF-␤ is the suppression of the host anti-tumor immune response. Early studies demonstrated that TGF-␤1 suppressed the macrophage tumor cytocidal activity in vitro [5], and later in vivo studies confirmed these findings [6–8]. Studies of xenotransplants in athymic mice have shown that production of TGF-␤ by both colonic and breast carcinomas suppresses cytotoxicity of activated monocytes and NK cells [9]. This effect is reversed by systemic administration of blocking antibodies to TGF-␤ which inhibits development of primary tumors and distant metastases [10]. Similarly, a newly established preclinical model shows that chronic exposure to a TGF-␤ soluble receptor has a protective effect against the development of metastases [11]. In this study, transgenic expression of the human FC/T␤RII chimeric protein is not associated with any adverse effect, but the mechanisms mediating suppression of metastasis remain to be determined and may involve the suppression of cytotoxic T cell development and function. T cell-mediated immunity is generally impaired by TGF-␤ in tumor-bearing hosts. The suppression of CD4+ T cell function associated with the growth of the MH134 hepatoma in vivo can be reversed by systemic administration of anti-TGF␤ antibodies [12]. It is noteworthy that the induction of TGF-␤ by immunosuppressive agents such as rapamycin and cyclosporine may not only underlie effects on lymphocyte function but also promote tumor progression by a cell-autonomous mechanism involving the induction of autocrine TGF-␤ activity in tumor cells [13]. This effect of TGF-␤ on T cell-mediated tumor immunity has also been explored more extensively through the use of genetically engineered mouse models. In one example, mice expressing a dominant negative-acting type II TGF-␤ receptor (dn-T␤RII) in T cells are able to spontaneously develop immunity to tumors that produce TGF-␤ and are typically nonimmunogenic [14]. This model is complicated by the onset of autoimmunity in mice that survive beyond 5 months of age. T lymphocytes in older CD4-dn-T␤RII mice spontaneously differentiate into type 1 or type 2 cytokine secreting cells, with all CD8+ T cells capable of producing IFN-␥ and CD4+ T cells secreting IFN-␥ and/or IL-4 in vitro [15]. Evidence implicating CD8+ T

cells as the principal target of TGF-␤-induced immunosuppression was provided by adoptive transfer studies involving the co-transfer of either CD4+ or CD8+ T cells of the dn-T␤RII mice into lymphocyte-deficient RAG2−/− mice. Rejection of TGF-␤-secreting tumors could be established in reconstituted mice but only if their CD8+ T cells were derived from dn-T␤RII mice, and regardless of whether the transferred CD4+ T cells were responsive to TGF-␤. While TGF-␤ can modulate CD8+ T cell-activity and effector function, it is likely that the predominant effects of TGF-␤ may be exerted during differentiation of na¨ıve CD8+ T cells, at which point TGF-␤ primarily opposes many of the inductive effects of co-stimulatory molecules and interleukins [16]. Until recently, most of the emphasis has been on the effects of TGF-␤ produced by tumor cells. For example, introduction of TGF-␤ antisense expression vectors into the TGF-␤-producing 9 L gliosarcoma converted the tumor phenotype to highly immunogenic, and rendered these cells as effective as CTLinducing whole tumor cell vaccines [17–18]. Remarkably similar results have been obtained in a rat model of prostatic carcinoma [19]. However more recent evidence suggests that TGF-␤, derived from other sources, has an equally significant role in the suppression of host T cell function. For example, in a murine model of fibrosarcoma, BALB/c mice was injected subcutaneously with the fibrosarcoma 15-12RM-developed tumors that exhibit a period of spontaneous regression followed by tumor recurrence. The spontaneous regression in this model is immune-mediated and dependent on CD8+ CTLs [20]. In a recent report by Terabe et al., it has been demonstrated that the recurrence of tumor growth is the direct result of immune suppression by TGF-␤ [21]. Importantly, the source of TGF-␤ is not the fibrosarcoma, but rather a myeloid population defined by the expression of GR-1 and CD11b. In this system, a population of cells defined as CD4+ - and CD1d-restricted, most likely NKT cells [22], were also necessary to downregulate this CTL-mediated tumor immunosurveillance, and were found to induce TGF-␤ in the myeloid population via a mechanism involving IL-13. Overall, the results of these and similar studies imply that blockade of TGF-␤ signaling in vivo would have an overall effect of suppressing tumor progression by enhancing tumor immunity. However, as discussed below, the underlying mechanisms leading to tumor

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formation will ultimately dictate whether interference with TGF-␤ signaling in T cells will have the effect of inducing tumor suppression.

3. Disruption of TGF-␤ signaling in models of intestinal neoplasia in mice 3.1. Gastrointestinal tumorigenesis in the Tgf-β1−/− mouse model Since the original publication of the phenotype of the TGF-␤1 null mouse in 1992, this model has been one of the most extensively studied, and has been responsible for providing significant insight into the major biological functions of this isoform. The embryonic phenotypes and extensive immune pathologies associated with deletion of the Tgf-β1 gene have been extensively reviewed elsewhere [23,24] and will not be presented here. Recent studies focused on this model have aimed to define the role of this ligand in the suppression of tumorigenesis. The embryonic and inflammatory phenotypes that accompany global disruption of Tgf-β1 gene expression are problematic when trying to discern the contribution of this deletion to tumor susceptibility. However, enhanced survival of the Tgf-β1−/− mouse in the absence of a competent immune system has allowed for longitudinal assessment of tumor susceptibility by obviating the associated immune pathology in Tgf-β1−/− progeny. In addition, careful analyses of the Tgf-β1 heterozygote in models of chemically induced carcinogenesis have revealed TGF-␤1 to be haploinsufficient as a tumor suppressor protein [25]. It is noteworthy that enhanced susceptibility to spontaneous tumor formation in humans has so far not been associated with inactivating mutations in any of the genes encoding the TGF-␤ ligands. As noted above, this may reflect the rather complex role that these ligands play in the processes of initiation, progression, and metastasis of the tumor cell. Evaluation of the Tgf-β1−/− mouse in the immune-deficient SCID background has revealed an increased susceptibility to invasive tumors in the region of the cecum in the large bowel [26]. In this study, all Tgf-β1+/+ and Tgf-β1−/− mice in the SCID background developed a proliferative epithelial phenotype and inflammation in the intestinal mucosa, but invasive lesions, typical of adenocarcinoma,

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were only noted in Tgf-β1−/− mice. Further studies in this model have linked this tumor susceptibility to the presence of the intestinal pathogen, Helicobacter hepaticus, as the rederivation of the Tgf-β1−/− SCID model into an H. hepaticus-free environment eliminates the proliferative colitis and intestinal tumorigenesis [27]. More recent work by Fox et al. clearly defined the role of H. hepaticus in the induction of intestinal neoplasia in mice [28]. While the latter studies elegantly show that tumor susceptibility is clearly influenced by the immune status and strain in mice, the data suggest that TGF-␤1 plays a key role in suppressing malignant transformation of intestinal epithelial, potentially by controlling the innate immune response in the intestinal microenvironment. Indeed, recent studies clearly demonstrate the ability of enteric bacteria to induce TGF-␤ signaling in intestinal epithelial cells, which then acts to oppose many NF␬B-dependent proinflammatory signals, and to suppress the expression of cytokines, such as interleukin 6 (IL-6) [29]. We have made similar observations by evaluating the Tgf-β1−/− mouse on a background that lacks the gene for the inhibitor of cyclin-dependent kinases, p21Cip1 . This cdk inhibitor is highly expressed in activated immune cells, and the absence of p21Cip1 expression has allowed for an extended survival of the Tgf-β1−/− mouse [30]. We first proposed the mechanism underlying this enhanced survival which was based on an increased susceptibility of the p21Cip1−/− T cells to activation-induced cell death. A recently published study in an unrelated model of autoimmunity also demonstrated a role for p21Cip1 in controlling the survival of memory T cells, and that the loss of p21Cip1 expression improved survival by ameliorating the manifestations and complications of autoimmune disease. In our model, spontaneous tumorigenesis is observed in Tgf-β1−/− mice only in the presence of homozygous inactivation of both alleles of the p21Cip1 gene. In this instance, mice are immune-competent and documented to be free of H. hepaticus. By 6 months of age, the Tgf-β1−/− /p21Cip1−/− mice exhibit invasive lesions at several sites within the gastrointestinal tract that are not associated with any chronic underlying inflammatory process. Although the deletion of the p21Cip1 gene in mice does not result in spontaneous tumor formation, our model not only supports a role for p21Cip1 in suppression of intestinal neoplasia, but also suggests that TGF-␤1 and p21Cip1 act through distinct pathways and

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cooperate to suppress malignant transformation of gut epithelia. Interestingly, while deletion of specific intracellular signaling intermediates of the TGF-␤ pathway has been associated with a metastatic colon cancer phenotype in mice [31–34], the tumors of Tgf-β1−/− mice are not metastatic. This disparity serves to highlight the complex role of TGF-␤1 in the progression of neoplasia, and it suggests that the expression TGF-␤1 may be involved in tumor dissemination beyond the intestinal wall. In total, these studies support the importance of the type 1 isoform of TGF-␤ in the homeostasis of intestinal epithelia and of local immune function, and implicate this pathway in general as a suppressor of colon cancer formation. 3.2. Tissue-specific expression of TGF-β signaling: dominant-negative TGF-β receptors and conditional deletion of the type II TGF-β receptor gene Targeted expression of a protein can be achieved in different cell types in a controlled fashion by expressing the corresponding gene either ectopically, ubiquitously, or in a tissue-specific manner through the use of its own promoter. When evaluating the function of the TGF-␤ signaling pathway, this approach has been used to interfere with signaling in a target tissue by overexpression of a ‘mutant’ or non-functional cell surface receptor. The latter are capable of acting in a dominant negative fashion to the endogenously expressed receptor proteins, and thereby preclude signaling via any of the isoforms. Each TGF-␤ isoform signals through the same heteromeric receptor complex of type I and type II serine/threonine kinases. The type II receptor (T␤RII) is necessary for ligand binding and subsequent growth suppression by TGF-␤ [35–37]. The expression of receptors carrying deletions in their cytoplasmic domains interferes with signaling via intact receptor proteins, effectively creating kinase-dead complexes. This strategy has successfully been applied in transgenic models to disrupt all TGF-␤ signaling in a tissue-specific manner. For example, expression of a dominant negative T␤RII in mouse keratinocytes leads to disruption of TGF-␤ signaling and is followed by rapid changes in cell ploidy, implicating a major role for this pathway in maintaining genomic stability [38–40]. In vivo, overexpression of a dominant negative T␤RII in the epider-

mis blocks TGF-␤ signaling and results in a significant increase in the DNA labeling index in the skin [41]. Expression of a dominant negative T␤RII in the epidermis on a truncated loricrin promoter increases susceptibility to the tumor promoter, 12-O-tetradecanoly phorbol-13-acetate (TPA) [42]. These mice develop papillomas at twice the rate of control mice, and the papillomas progress to carcinomas despite withdrawal of TPA. This strategy of tissue-specific suppression of TGF-␤ signaling in mice has also demonstrated the tumor-suppressor activity of this pathway in the mammary gland [43] and prostate [44]. In a similar fashion, TGF-␤ signaling has been disrupted in the mouse intestine through the expression of a dominant negative mutant form of the TGF-␤ type II receptor under the control of the mouse intestinal trefoil peptide (ITF)/TFF3 promoter [45]. The ITF-dnT␤RII transgenic mice develop spontaneous colitis unless they are maintained under specific pathogen-free (SPF) conditions. Under SPF conditions, colitis could also be induced by the administration of dextran sodium sulphate (DSS), with diarrhea and rectal prolapse as the clinical indicators of disease. All the transgenic animals develop manifestations consistent with an autoimmune type process, including increased expression of antigens of the major histocompatibility complex class II and autoantibodies against intestinal goblet cells. As in the studies described previously, these results demonstrate the importance of TGF-␤ signaling in regulating immune homeostasis in the intestine, and suggest that this may be a critical factor controlling the epithelial cell response to normal intestinal microbial flora. Although these observations suggest that TGF-␤ signaling in the intestinal epithelial cells may be essential to maintain local immune homeostasis, they did not support the hypothesis that a disruption of TGF␤ signaling at the receptor level would lead to spontaneous tumor formation in the ITF-dnRII transgenic mice. However, the authors have demonstrated an increased susceptibility to azoxymethane-induced colon carcinoma and to H. pylori-associated carcinogenesis in the stomach in this model [45,46]. So, why would a disruption of the gene encoding the type 1 isoform of TGF-␤ lead to spontaneous tumors in the gastrointestinal tract, while loss of signaling specifically in the epithelial cells does not? One likely possibility is that the dominant negative receptor may function more as a gene “knock-down” in this model. If the ITF-dnT␤RII

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is not expressed at a level high enough to successfully compete out all ligand binding, then there may remain a minimal level of TGF-␤ signaling, and perhaps enough to maintain genomic stability and suppress epithelial cell transformation. Data supporting this conclusion have been provided by a more recent report in which TGF-␤ signaling is disrupted in epithelial cells using the approach of conditional gene targeting. In this model, Biswas et al. crossed a Tgfbr2flx/flx line with the Fabpl4xat-132 Cre driver line, thereby creating an in vivo model system in which the loss of the Tgfbr2 gene is complete and can be directly evaluated in the context of chemical carcinogenesis [47]. Nearly 50% of these mice spontaneously develop gastrointestinal intraepithelial neoplasia by 10 weeks of age and progress to multifocal colon carcinoma. As in the ITF-dnT␤RII model, exposure to azoxymethane also accelerates the initiation and increases the frequency of carcinoma in the Tgfbr2flx/flx × Fabpl4xat-132 Cre model. These models suggest that it is ultimately the disruption of TGF-␤ signaling in the epithelial cell which is a primary event leading to the induction and/or progression of colon carcinoma. However, several observations point to the importance of TGF-␤ signaling in the resident immune cells within the intimal layers of the intestinal mucosa. In the Tgf-β1−/− SCID model, the neoplastic transformation in the epithelial lineage is coupled to the inflammatory response to resident bacteria. While TGF-␤ can suppress bacterial-triggered NF␬B-dependent gene expression within the epithelia [29], resistance to TGF-␤ in the resident leukocytes could potentiate the bacterial driven inflammatory process [48], disrupt the integrity of epithelial/basement membrane interactions, and lead to the production of factors that promote neoplastic tranformation in the epithelia. This hypothesis is now supported by a study in which T cell-specific expression of a dnT␤RII (driven by a CD2 promoter) leads to accelerated tumorigenesis following induction with azoxymethane [49]. In this model, TGF-␤-resistant T cells secrete increased levels of IL-6, which then activates the JAK-STAT pathway in epithelial cells, thereby promoting the progression of epithelial tumors. The concept that epithelial neoplasia can be initiated by altered TGF-␤ signaling in a neighboring lineage within the supporting stroma has also been demonstrated by the targeted disruption of TGF-␤ signaling in fibroblasts in vivo. Conditional deletion of T␤RII in fibroblasts leads to intraepithe-

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lial neoplasia in the prostate and invasive squamous cell carcinoma in the forestomach [50]. In this model, the paracrine effects of hepatocyte growth factor produced by the TGF-␤-resistant fibroblasts represented a potential mechanism underlying the induction of epithelial cell proliferation. Overall, if one considers the collective data from these in vivo studies, it is clear that TGF-␤ signaling in the leukocyte, mesenchymal and epithelial populations act in concert to maintain homeostasis in the gastrointestinal tract. More importantly, the effect of this cytokine on each lineage appears to be critically important in the suppression of gastrointestinal neoplasia. Further studies in these model systems will be important to establish a link between loss of TGF-␤ signaling alterations in tissue homeostasis and organ-specific events during tumorigenesis. 3.3. Gastrointestinal tumorigenesis associated with disruption of SMAD genes in mice In a manner similar to what has been observed following the targeted disruption of TGF-␤ ligand and receptor genes in mice, global disruption of the expression of genes encoding members of the Smad family has documented the critical importance of their function during embryogenesis. With the exception of the mutational inactivation of SMAD3, disruption of SMAD gene expression uniformly leads to embryonic lethality in mice, and these developmental phenotypes will not be considered here [51–57]. Three distinct models in which the SMAD3 gene is deleted have been described [57–59]. Mutations in the MADH3 gene have not been associated with human intestinal tumorigenesis, and the lack of a role for Smad3 in intestinal neoplasia is supported by the phenotypes of two distinct SMAD3 mutants produced by two different laboratories. However, while these mutations yielded identical phenotypes, a third SMAD3 mouse mutant is distinguished by the development of a metatstatic colon cancer, with 100% frequency on a pure SV129 background [59]. The results have been considered surprising [60], although the importance of strain and potential differences in enteric microbial flora might contribute to these disparities. This particular mutation has also been combined with a disruption in the gene encoding p27Kip1 , an inhibitor of cyclin-dependent kinases [61]. While the authors did observe spontaneous intestinal tumors in this study, they found no evidence

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for metastatic spread of the SMAD3−/− colon carcinomas, and failed to see any cooperativity between Smad3 and p27Kip1 in the suppression of intestinal neoplasia. These tumors continue to express APC, suggesting that inactivation of the ␤-catenin/APC pathway is not involved, and there was no evidence to suggest loss of heterozygosity for SMAD3 or tumorigenesis in the SMAD3+/− mice. Disruption of the expression of the SMAD4 gene in mice has yielded a valuable model that clearly validate the relevance of Smad4-dependent signal transduction in the suppression of colon carcinogenesis. Although embryonic lethality accompanies complete deletion of the SMAD4 gene and obviates the assessment of tumor suppressor function, SMAD4 heterozygotes are phenotypically normal and have been useful for this purpose [31,62]. Taketo and Takaku have shown that as SMAD4 heterozygote mice (SMAD4+/− ) age, they develop multiple polyps within the stomach and duodenum. These polyps having abundant stroma and eosinophilic infiltrates, features that are also true of the human juvenile polyposis syndrome, in kindreds that carry germline mutations in the SMAD4 gene. More importantly, loss of heterozygosity for the SMAD4 gene occurred in 100% of polyps of 18-month-old heterozygotes, indicating LOH for SMAD4 as an early event in polyp formation. Perhaps not surprisingly, there is now experimental evidence that a disruption in Smad4-dependent signaling in T cells might contribute to this phenotype. In a model developed in our laboratory, the conditional deletion of the SMAD4 gene in T cells leads to a progressive intestinal epithelial hyperplasia (unpublished). We observe a significant increase in T cell production of IL-6 in vivo and in vitro in this model, consistent with the transgenic model in which T cell resistance to TGF-␤ is induced by a CD2-driven dnT␤RII. More importantly, we also see a significant production of IL-5, a key factor in the induction, activation and migration of eosinophils. We are currently utilizing this model to explore the role of Smad-4-dependent signaling in T cells and the contribution that it makes to the suppression of gastrointestinal tumorigenesis. Taketo and Takaku have also created a mouse model of the human familial adenomatous polyposis syndrome by introducing the Smad4 mutation into the Apc716 knockout mouse [63]. These investigator utilized meiotic recombination to create compound heterozygotes carrying both mutations on mouse chro-

mosome 18 [33,34]. When compared to the Apc716 knockout mouse, the compound heterozygotes develop tumors that are highly malignant and invasive in phenotype. This model verifies the importance of Smad4 as a suppressor of colon tumorigenesis, and creates a valuable tool to further define the role of this pathway in the homeostasis of intestinal epithelia, and for testing new strategies for the management and prevention of the gastrointestinal cancers linked to these mutations. It also creates the opportunity for investigators in the field to identify loci that may modify disease expression, and for developing novel chemopreventive strategies.

4. TGF-␤ signaling in models of hematopoietic malignancy in mice 4.1. Targeting the TGF-β receptors and lymphoid neoplasia in mice In human lymphoid malignancies, mutations in genes encoding the TGF-␤ receptors have been reported and loss of cell surface expression of TGF-␤ receptors has been described in both T and B lymphoid tumors (reviewed in [64]). In mice, two recently described models have demonstrated the importance of the TGF-␤s in controlling normal differentiation and function in lymphoid tissue, and also highlight how a specific promoter choice can impact on the outcome of expression of a dominant negative T␤RII transgene. Expression of a kinase-defective T␤RII in T cells under the control of the CD2 promoter results in a progressive lymphoproliferative process characterized by diffuse vasculitis and expansion of CD8+ T cells in the peripheral lymphoid tissue [65]. These T cells develop abnormal ploidy and evolve into a leukemia/lymphoma-like syndrome that is responsible for early mortality within 3–4 months of age [66]. Multiple chromosomal abnormalities were observed in these leukemias including aneuploidy, deletions, and translocations, although few of these changes occurred in more than one tumor. These data suggest a significant role for TGF-␤ in the inhibition of the proliferative expansion or growth of memory or antigen-experienced T cells. Recent data suggest that crosstalk between IL-15 and TGF-␤-signaling pathways has a significant role in maintaining homeostasis in this T cell subset [67]. Alterations in the TGF-␤ signaling pathway that impair

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the ability of TGF-␤ to oppose either pro-survival or mitogenic effects of cytokines, such as IL-7 and IL-15, would create the potential for rapid growth, and allow for the expansion of a clonal population harboring an oncogenic or growth-favoring mutation. By contrast, mice expressing a dominant negative T␤RII under the control of a CD4 transgenic construct, lacking the CD8 silencer, develop an autoimmune process without any evidence for leukemic transformation [15]. These mice survive beyond 5 months of age and display many overlapping features with the Tgf-β1−/− mice, including perivascular infiltration of mononuclear cells in many organs, circulating autoantibodies, and immune complex deposition in renal glomeruli. The authors of this study also demonstrate that most T lymphocytes in older CD4-dnT␤RII mice spontaneously differentiate into type 1 or type 2 cytokine secreting cells with all CD8+ T cells capable of producing IFN-␥ and CD4+ T cells capable of secreting IFN-␥ and/or IL-4 in vitro. The results demonstrate the importance of TGF-␤ in maintaining tolerance in T cells, and that maintenance of B cell tolerance to selfantigens is dependent on normal TGF-␤ signaling in T cells. The disparities between these two models might be explained by differences in levels of expression of the specific transgene or with the timing of expression. This point can be emphasized by comparing these models with a model in which the TGF-␤ type II receptor gene is conditionally deleted in the hematopoietic stem cell through the approach of conditional gene targeting [68]. In this model, mice develop a rapidly fatal automimmune disease that is identical in most aspects to the syndrome which evolves in Tgf-β1−/− mice. In each instance, the loss of receptor or ligand is complete, and thus one could argue that the more slowly progressive syndromes in the T cell-specific dnT␤RII mice may be due to a persistence of some level of TGF-␤ signaling, and this could conceivably involve Smad-independent MAPK, PI3K or S6 kinase-related pathways [69,70]. Overall, the collective data from these and other preclinical models support the concept that this pathway functions to suppress tumorigenesis in the T cell lineage. 4.2. A specific role for SMAD3 in leukemogenesis Until recently, the evaluation of TGF-␤-resistance in human lymphoid malignancies has mostly focused

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on assessments of the TGF-␤ receptor genes and their expression. However, a role for Smad-dependent signaling in the suppression of leukemogenesis has now been demonstrated in both myeloid and lymphoid leukemias. While mutations in the human MADH4 gene have been described in acute myeloid leukemia (AML), mutations in genes encoding the receptoractivated Smads have not been described. However, it is now clear that epigenetic mechanisms that impair either the function or expression of Smad3 protein are important in the process of leukemogenesis. In AML, the novel protein products produced by transcripts of the fusion genes generated by chromosomal translocations have been shown to physically interact with Smad3 and suppress Smad3-dependent transcriptional responses to TGF-␤. Examples include the AML/ETO and AML/Evi oncoproteins, each of which inhibit the ability of TGF-␤ to block proliferation [71,72]. The viral oncoprotein, Tax, has also been implicated in suppression of the Smad3-dependent effects of TGF-␤. We have recently reviewed these studies elsewhere [64]. More recently, an analysis of the PML-RAR␣ oncoprotein of acute promyelocytic leukemia (APL) has revealed an important role for the tumor suppressor of acute promyelocytic leukemia (PML) as an essential mediator of TGF-␤ signaling [73,74]. Lin et al. show that the expression of cytoplasmic Pml is induced by TGF-␤. More importantly, they demonstrated for the first time that cytoplasmic PML physically interacts with Smad2/3 and SARA (Smad anchor for receptor activation) and provide evidence that this interaction is required for their assembly and trafficking with the TGF-␤ receptor in the early endosome. They also demonstrated that PML-RAR␣ oncoprotein of APL can antagonize cytoplasmic PML function and provide evidence that APL cells and PML-null cells have similar defects in TGF-␤ signaling, again implicating the receptor-activated Smad3 as a principal target of a leukemogenic oncoprotein. Our laboratory has focused on potential role of Smad3 as a suppressor of leukemogenesis in the lymphoid lineage. As noted above, it had been suggested in the literature that the Smad3 protein might be a relevant target of the Tax oncoprotein of adult T cell leukemia. Our studies in the SMAD3−/− mouse model had revealed a requirement for Smad3 in mediating the inhibitory effects of TGF-␤ on the proliferation of T-cells, so we studied the role of Smad3 in

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human T-cell leukemogenesis. We identified the specific loss of Smad3 protein as a unique feature of pediatric T-cell acute lymphoblastic leukemia (T ALL) and lymphoblastic leukemia (T-cell ALL) [75,76]. Despite normal levels of SMAD3 messenger RNA, no Smad3 protein was detected in leukemic cells from children with T-cell ALL, while Smad3 remained readily detectable in pre-B ALL specimens. Smad2 was readily detected in all extracts of leukemic cells. In mice, we found that a reduction in the level of Smad3 impairs the responses of T cells to TGF-␤, and thus the suppression by TGF-␤ of T cell production of IL-2 and of the mitogenic response to T cell receptor ligation are diminished in the Smad3 heterozygote (SMAD3+/− ). The loss of Smad3 alone was insufficient to induce leukemia. However, leukemia develops in Smad3-deficient mice when coupled with the loss of the p27Kip1 , a cyclindependent kinase inhibitor, whose gene is frequently altered in human T-cell ALL. These data suggest that TGF-␤ participates in suppression of leukemogenesis, potentially by suppressing proliferative expansion of lymphocytes through a Smad3-dependent mechanism. Loss of Smad3 expression may cooperate more generally with alterations in the retinoblastoma protein pathway, and studies aimed at identifying cooperating oncogenic events represent an important focus for future studies.

5. Summary and conclusions This review should leave the reader with the basic understanding that the tumor suppressor activity exerted by the TGF-␤ pathway is mediated not only through direct effects on the cell undergoing transformation, but also through the effects of this cytokine on neighboring cells. TGF-␤ has been frequently labeled as a ‘switch’ in that it can modulate the manner in which normal cells respond to signals in their environment. These responses, whether to normal enteric micro-organisms in the gut or to either self-antigens or environmental antigens, each must be controlled to maintain normal tissue homeostasis. There is now abundant evidence to support TGF-␤ as a key factor in fulfilling this function. In each of the murine models detailed in this review, a primary emphasis has been placed on trying to utilize mutations in genes encoding components of this

pathway in mice as a means to explore the role of TGF␤-signaling in human disease. The result is the direct demonstration of a tumor suppressor function for the genes encoding Smad3 and Smad4 in intestinal neoplasia and leukemogenesis. Finally, these models will be useful for determining the genetic alterations that complement the loss of expression and/or function of Smad3 or Smad4. Finally, these models represent significant new tools for the assessment of novel therapeutic and preventive strategies, particularly those which may activate or augment TGF-␤-signaling.

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