From Gut Homeostasis to Cancer - Ingenta Connect

Current Molecular Medicine 2006, 6, 275-289

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From Gut Homeostasis to Cancer Freddy Radtke1,*, Hans Clevers2 and Orbicia Riccio1 1Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Chemin des

Boveresses 155, 1066 Epalinges, Switzerland 2Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584CT

Utrecht, The Netherlands Abstract: The mammalian intestine has one of the highest turnover rates in the body. The intestinal epithelium is completely renewed in less than a week. It is divided into spatially distinct compartments in the form of finger-like projections and invaginations that are dedicated to specific functions. Intestinal cells are constantly produced from a stem cell reservoir that gives rise to proliferating transient amplifying cells, which subsequently differentiate and migrate to the correct compartment before dying after having fulfilled their physiological function. In recent years, a substantial body of evidence has accumulated to support the concept that signaling pathways known to be crucial for embryonic development of multiple organisms play a critical role in tightly regulating and controlling the self-renewing process of the intestine. Moreover, the same pathways appear to be deregulated in several hereditary and sporadic colorectal cancer syndromes due to activating and/or inactivating mutations of key components of such pathways. In this review we discuss recent findings demonstrating that differentiation and homeostasis of the intestine are controlled by developmental pathways such as Wnt, Notch, TGF-β and Hedgehog, and illustrate how their deregulation contributes to intestinal neoplasia.

DEVELOPMENT OF THE GUTANDGENERAL ORGANIZATION The gut can be viewed as a tube composed of three germ layers: the endoderm forming the epithelial cell layer of the intestine, the mesoderm contributing smooth muscle and connective tissues, and the ectoderm which provides the enteric nervous system. Formation of the gut during embryogenesis is initiated simultaneously in the anterior and posterior portions of the developing embryo (around E7.5 and E9.5 during mouse embryogenesis) by a series of invaginations of the endodermal layer. These invaginations elongate and fuse at the midline of the embryo, leading to formation of a gut tube. This gut tube comprises stratified epithelium composed of highly proliferating cubiodal endodermal cells surrounded by splanchic mesodermal cells “Fig. (1)”. Around E14.5, this multistratified epithelium commences conversion into a single layer characterized by invaginations of the endoderm. The surrounding splanchic mesodermal cells differentiate into smooth muscle cells and connective tissue. All of these morphogenetic changes take place in the form of an anterior-toposterior wave-like process that is completed around E18.5, and already permits the distinction between the small and large intestine. The fetal small intestine is characterized by finger-like projections called villi, which mainly consists of differentiated cells, while *Address correspondence to this author at the Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland; E-mail: [email protected] 1566-5240/06 $50.00+.00

undifferentiated proliferating cells are located in the epithelium between the villi. The large intestine does not contain villi, but forms crypt-like structures instead. Proliferative cells reside at the bottom of the crypts while more differentiated cells locate to the surface epithelium. Morphological changes and reshaping of the gut continues during the first three weeks after birth. In the small intestine the proliferative regions located between the villi invaginate and form crypts, also known as ‘crypts of Lieberkühn’. The crypts of the large intestine continue elongation and grow deeper into the intestinal submucosa “Fig. (1)”. For a more comprehensive overview on embryonic development of the gut we refer the interested reader to reviews by Wells and Melton [1] and de Santa Barbara and colleagues [2]. The small intestine can be further subdivided anatomically from anterior to posterior into duodenum, jejunum and ileum, whereas the large intestine is subdivided into colon and rectum. The predominant function of the small intestine is absorption of nutrients whereas the large intestine compacts the stools. These functional differences are also represented by anatomical differences. The small intestine is much longer in length than the large intestine, and contains villi that dramatically increase the cell surface area to more efficiently absorb nutrients. The cellular composition of the small and the large intestine is also different. The epithelium of both the small and large intestine is composed of four different cell types, absorptive, enteroendocrine, mucus-secreting Goblet and Paneth cells. The most abundant cells within the © 2006 Bentham Science Publishers Ltd.

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Fig. (1). Developmental stages of the intestinal epithelium. During embryonic life, mesodermal outgrowth towards the lumen of the multilayered stratified epithelium results in the formation of small villi, and in conversion to a unistratified epithelium. Long, thin villi of the small intestine are separated by proliferating cells in the intervillus region. After birth, invagination of the intervillus region within the small intestine leads to the formation of the crypt compartment. In contrast, the large intestine forms flat, wide crypt-like structures during embryonic development, that elongate deeper into the different mesodermal layers after birth to form colonic crypts.

small intestine are enterocytes, which absorb nutrients from the food and secrete hydrolases into the lumen. Enteroendocrine cells represent less then 1% of all cell types and secrete hormones such as serotonin, substance P and secretin. Goblet cells are mucus-producing cells, and are most abundant in the large intestine in order to protect the intestinal epithelium as compaction of stools increases [3,4]. Paneth cells, which reside at the bottom of the crypts of the small intestine, can be seen as the “innate immune system” of the intestine as they secrete antimicrobial peptides and enzymes such as cryptidins and lysozyme in order to control the microbial flora of the intestine [5,6].

THE GUT: THE PROTOTYPE OF A SELFRENEWING ORGAN The intestinal epithelium is a classical selfrenewing tissue like the hematopoietic system and the skin. It follows the basic principles of self-renewal by being compartmentalized into stem cells, transient

amplifying cells and terminally differentiated cells. Stem cells and undifferentiated transient amplifying cells constitute the majority of cells found in the crypts. Paneth cells are the only terminally differentiated cells found at the bottom of crypts. The turnover of the gut epithelium is very high as the average life span of an epithelial cell is less then a week, with the exception of Paneth cells, which survive approximately 20 days. Undifferentiated crypt progenitors divide every 12-16 hrs, giving rise to approximately 200 cells per crypt per day. Since the mouse intestine is estimated to contain approximately one million crypts the daily production of epithelial cells mounts to 200 x 106 cells per day [7]. This enormous production capacity requires mechanisms that tightly regulate cell production, proliferation, migration and cell death in order to maintain gut homeostasis. Cells produced in the crypts constantly migrate up towards the tip of the villi of the small intestine (with the exception of Paneth cells), or to the surface epithelium of the colon [8] where they undergo apoptosis and are

From Gut Homeostasis to Cancer

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Fig. (2). Structure and cell fate specification of the intestinal epithelium. A. Schematic representation of the crypt/villus within the small intestine. Stem cells (in yellow) and proliferating Transient Amplifying (TA) cells (in green) localize to the crypt compartment, which is maintained by both Notch and Wnt signaling. All differentiating TA cells (with the exception of Paneth cells) migrate upwards and withdraw from the cell cycle at the crypt/villus boundary. Migration of non-proliferating differentiated cells continues towards the tip of the villus where they are shed into the lumen. BMP (Bone morphogenic protein) and TGF-β signaling occur in the villi. In contrast, Paneth cells migrate downwards and are found at the bottom of the crypt. B. Immunostaining for Ki67 (in brown) shows proliferating cells within the crypt compartment of the small intestine, whereas differentiated post-mitotic cells are found in the villi. C. Schematic representation of the large intestine. Stem cells (in yellow) within the colon reside at the bottom of the crypt. Three quarters of the colonic crypt consists of proliferating cells intermingled with mucus-secreting Goblet cells (in violet). Cells in the upper part of the crypt epithelium undergo cell cycle arrest and differentiate to form the colonic surface epithelium. Wnt, Notch and Hh signaling are indicated. D. Immunostaining for Ki67 (in brown) shows proliferating cells within the crypt compartment of the colon. E. Cell fate specification within the intestine. The intestinal epithelium consists of four differentiated cell types (Enterocytes, Enteroendocrine cells, Goblet cells and Paneth cells), which are all derived from pluripotent stem cells. The current model predicts that a stem cell can divide asymmetrically to give rise to a TA cell and to another stem cell. The undifferentiated crypt compartment is maintained through the concerted action of the Wnt and Notch signaling pathways. Loss of Wnt signaling results in loss of the crypt compartment, and simultaneous differentiation into Enterocytes. In contrast, while loss of Notch signaling also results in the loss of the crypt compartment, in this case all cells differentiate into post-mitotic Goblet cells. Genetic studies suggest that a number of intestinal cell fate specifications occur through binary cell fate choices. A first binary cell fate choice occurs within TA cells that have to choose between the adsorptive and the secretory lineages. This process seems to be regulated by Hes1 and Math1. Math1 is required for secretory cell lineages while Hes1 represses Math1, resulting in the differentiation of adsorptive cells. Once the secretory lineage is specified, cells have to choose between the Goblet and Enteroendocrine cell fates, a choice which appears to be regulated by Neurogenin-3 (Ngn-3), essential for the Enteroendocrine lineage. So far it is unclear how the Paneth cell lineage is specified, however maturation as well as positioning is under control of the Wnt pathway.


shed into the lumen of the intestine, a process called exfoliation “Fig. (2A)”. In order to maintain epithelial cell numbers in the steady state intestine, the rate of apoptosis must equal the rate of cell production within the crypts. Proliferation of crypt progenitors is a cell non-autonomous process [9] mediated by proliferative permissive signals in the crypt niche. In the small intestine, proliferating cells are only found within the crypt compartment because as soon as migrating cells reach the villi they cease proliferation and express specific terminal differentiation markers “Fig. (2B)”. Thus, the transition from crypt to villi during migration compartmentalizes undifferentiated proliferating cells from post-mitotic terminally differentiated cells within the gut [10] “Fig. (2A)”. Similarly, in the large intestine as soon as cells reach the upper third of the colorectal crypt compartment where proliferative signals are absent, the migrating epithelial cells undergo cell cycle arrest and subsequent differentiation “Fig. (2C and D)”.

STEM CELLS OF THE GUT Lifelong maintenance of self-renewal requires the presence of long-term stem cells within the gut. However identification of such intestinal stem cells has not been straight forward despite intensive research efforts, mostly due to the lack of reliable stem cell markers. In addition, no good experimental test systems such as those used in the hematopoietic or skin fields have been developed to quantify or functionally characterize individual gut stem cells or stem cell populations. Consequently, determination of self-renewal and multi-lineage differentiation capacity remains a challenge. Gut stem cells have mainly been identified through elegant label retaining assays in which, after successive injections of 3H-thymidine either early during post-natal development or during tissue regeneration, individual cells that have retained the label long-term are observed within the crypts. Crypt cells that symmetrically divide (and consequently differentiate) lose half their label with every division. In contrast, after asymmetric cell division, the selfrenewing daughter cell will retain the label as the old DNA template is retained, while the new DNA template segregates with the other, differentiating daughter cell. Such asymmetrically dividing cells are postulated to be gut stem cells. More recently expression of the Musahi-1 gene has been identified as a potential stem cell marker of the intestine. Musashi was originally identified as an RNA-binding protein thought to be involved in asymmetric cell division during Drosophila neural development. Antibody staining and in situ hybridization for the murine and human orthologue Musashi-1 showed expression and staining of individual cells within the intestinal crypts, reinforcing the possibility that asymmetric division might be a hallmark of ‘bone fide’ gut stem cells [11,12]. Additional studies using chimeras [13,14] combined with regeneration studies after injury have allowed estimation of stem cell

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number, location and/or stem cell properties. Using these assays, stem cell numbers have been estimated to be between 4 and 6 per crypt in the adult intestine [7,15], however their precise location within the crypts is still unclear. In the colon the consensus is that stem cells reside at the bottom of the crypts, while for the small intestine two alternative views currently exist: one puts stem cells just above the Paneth cell compartment at position 4 (with position 1 being the lowest cell found within the bottom of the crypt) (reviewed in [16,17]) “Fig. (2A)”, while the second view locates the stem cells within the Paneth cell compartment intermingled with Paneth cells [14,18,19]. Low dose irradiation experiments show that gut stem cells are extremely sensitive to irradiation, leading to the interpretation that stem cells prefer to undergo apoptosis instead of repairing the defect, even after minor DNA damage. Furthermore, these studies showed that the immediate descendents of stem cells located up to position 7 could be recruited to become new stem cells once the original stem cell has undergone apoptosis. Thus, those cells in close proximity to stem cells are uncommitted and pluripotent, and if necessary can acquire stem cell features to maintain the crypt [7,15]. In contrast, those cells above position 7 appear to have lost the ability to regenerate the stem cell pool. Whether these cells receive signals within the crypt compartment that predispose them to adopt different cell fates is currently unknown, as they do not express any specific markers and thus cannot be identified. However indirect evidence from mice with intestines chimeric for the dbl-1 locus (Dolichos lectin binding locus) supports the existence of intermediate progenitor cells with a restricted differentiation potential [14]. Similarly, gene-targeting experiments for several basic Helix-Loop-Helix (bHLH) transcription factors within the Notch signaling pathway (see below) indicate that lineage specification within the gut is the result of multiple binary cell fate decisions “Fig. (2E)”.

SIGNALING PATHWAYS INTESTINAL HOMEOSTASIS

REGULATING

As described above, the gut epithelium has a very high turnover rate, therefore processes such as proliferation, differentiation, migration and cell death must be tightly regulated in order to ensure homeostasis. Despite the diversity of cellular responses, these processes are apparently controlled by a relatively small number of signaling pathways. These include the Wnt, Notch, TGFβ/BMP and Hedgehog pathways, most of which were first identified in lower organisms where they play important roles during embryogenesis and/or organ development. These same pathways are often used in adult organisms to regulate and control multiple self-renewing organs. In recent years more and more evidence has accumulated demonstrating that signaling mediated by these pathways is often


deregulated in pathological situations [20-26]. In this section, we will first introduce the different pathways and subsequently discuss their diverse roles during gut homeostasis and cancer.

WNT SIGNALING IN THE GUT The Wnt pathway is certainly one of the beststudied pathways in the intestine due to its relevance to gut biology and cancer. The finding that the Drosophila segment polarity gene wingless is the orthologue of the mouse mammary oncogene int-1 [27] was the starting point for its identification (from wingless and int-1). Numerous subsequent studies using different animal models revealed that this pathway is evolutionarily conserved [28,29]. To date, 19 Wnt genes have been identified in mouse and human. Wnts are secreted glycoproteins that mediate cell-to-cell communication during development. They bind to the family of Frizzled receptors [30] (7-transmembrane molecules) in a complex with the Low-density lipoprotein receptor related proteins (LRP-5 and –6) [31-33]. Activation of these receptor complexes results in stabilization and accumulation of a protein called β-catenin [34]. In the absence of Wnt signaling, β-catenin is retained in the cytoplasm in a multi-protein complex comprising the tumor suppressor APC (adenomatous polyposis coli), the scaffold protein Axin [35,36], Caseine Kinase-1 (CK-1) [37] and Glycogen Synthase Kinase-3β (GSK-3β) [38]. In this complex β-catenin is sequentially phosphorylated by Caseine Kinase-1 and GSK3-β at conserved serine/threonine residues within its N-terminus [38], and thereby tagged for ubiquitination and proteosomal degradation [39]. Upon activation of the Wnt pathway GSK3-β is inhibited by Dishevelled (Dsh) [40-42] and β-catenin can no longer be phosphorylated at its N-terminus. Unphosphorylated β-catenin is more stable so it accumulates in the cytoplasm and translocates to the nucleus where it binds transcription factors of the TCF/LEF family thereby activating transcription of downstream target genes “Fig. (3A)”. β-catenin has an additional role as a component of adherens junctions as it binds to the cytoplasmic tail of Cadherin proteins thereby linking them to the cytoskeleton of epithelial cells. The mechanisms controlling whether β-catenin joins the signaling pool or is part of adherens junctions is currently unknown. In recent years a substantial amount of evidence has accumulated demonstrating that the Wnt pathway is essential for maintenance of the proliferating transient amplifying cells within the crypt compartment “Fig. (2E)”. Most of these in vivo studies were performed in engineered mice expressing either mutant, dominant negative or dominant active forms of Wnt-pathway components. For example, TCF4 gene-targeted mice die shortly after birth because they have lost the proliferative cell compartment within the small intestine which is exclusively populated by post-mitotic differentiated cells [43].


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Similarly, gut specific overexpression of the secreted Wnt-inhibitor Dickkopf1 (Dkk-1) (antagonizes Wnt signaling by binding to the LRP co-receptors) results in severe loss of proliferative cells in both the fetal and adult intestine coinciding with the loss of the crypt compartment [44,45]. Furthermore, the intestines of these mice lack all secretory cell lineages, while enterocyte differentiation appears to be normal suggesting that Wnt signaling has an additional function in lineage specification of secretory cells. Expression of a dominant negative form of TCF4 or knockdown of β-catenin in colorectal cancer cell lines results in cell cycle arrest [46]. These results are consistent with gain-of-function experiments expressing a dominant active form of βcatenin lacking the N-terminal phosphorylation domain. The intestinal epithelium of these mice is hyperproliferative and forms adenomatous intestinal polyps [47]. Furthermore mutations within the APC gene, a negative regulator of Wnt signaling also results in hyperproliferaion of the epithelium followed by the development of adenomas [48,49]. Mice in which the APC gene is specifically inactivated in the gut confirm these results, and in addition show that APC-deficient cells maintain a "crypt progenitor-like" phenotype due to increased β-catenin-mediated Wnt signaling [50]. Gene expression profiling of TCF4 target genes has shown that the β-catenin/TCF4 specific gene expression program in crypts cells and colorectal cancer cell lines is very similar suggesting that adenoma cells represent the transformed counterpart of crypt cells [46]. The identification of Ephrin receptors as TCF4 target genes led to the suggestion of an additional role for Wnt signaling in the gut. Ephrin receptors are a large family of tyrosine receptors that are involved in axon guidance and migration of neural crest cells (reviewed in [51]). TCF4-mediated Wnt signaling inversely controls the expression of the EphB2/EphB3 receptors and their ligands Ephrin-B1 and Ephrin B2 along the crypt/villus axis. Expression of these two receptors is in the form of a gradient, with highest expression levels at the base of the crypts, decreasing along the crypt/villus axis. The corresponding ligands are expressed in an inverse pattern, low levels at the base of the crypts and high levels towards the villi. Disruption of EphB2 and EphB3 genes in mice results in intermingling of proliferating and differentiated cell populations in the intestinal epithelium. In addition, Paneth cells in EphB3 gene targeted mice are mis-positioned and found scattered along the crypt/villus axis. These data suggest that Wnt signaling is necessary for positioning Paneth cells near the base of the crypts, and for separating proliferating and differentiated cells. Both of these processes seem to be regulated by Wnt dependent expression of specific Ephrin receptors in the gut [52]. Very recent data suggest that Wnt signaling is not only necessary for correct positioning of Paneth cells


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Fig. (3). Schematic overview of signaling pathways involved in regulating intestinal homeostasis. A. The canonical Wnt pathway. The central protein in the canonical Wnt pathway is β-catenin. In the absence of secreted Wnt proteins, β-catenin is bound in a multi-protein destruction complex consisting of adenomatous polyposis coli (APC) protein, the scaffold protein Axin, Casein kinase1 (Ck1), and the glycogen-synthese-kinase-3β (GSK-3β). GSK-3β phosphorylates the N-terminus of βcatenin thereby targeting it for proteosomal degradation. Signaling initiated by binding of Wnt proteins to the Frizzled (Fz) receptors and the Low-density lipopotein related proteins (LRP5/6), results in the activation of Dishevelled (Dsh), which inhibits the function of the multi-protein destruction complex. Consequently β-catenin is no longer phosphorylated, avoids destruction and enters the nucleus where it heterodimerizes with members of the TCF transcription factor family to drive transcription by converting them into transcriptional activator through the displacement of co-repressors such as Groucho. B. The Notch pathway. Notch signaling is initiated through ligand-receptor interactions between two neighboring cells. This initiates two proteolytic cleavages (mediated by TACE and γ-secretase activity of Presenilins) liberating the cytoplasmic domain (NICD) of Notch receptors. NICD translocates to the nucleus and binds the transcription factor CSL, converting it from a transcriptional repressor into a transcriptional activator by displacing a co-repressor complex (CoR) and recruiting co-activators. C. The TGF-β/BMP pathway. Heterodimerization of type I and type II serine kinase receptors is initiated after binding of either TGF-β or BMPs to their corresponding type II receptors. During this heterodimerization process type II receptors phosphorylate type I receptors. These activated receptor complexes phosphorylate receptor regulated (R-) SMADs (SMAD1, 2, 3, 5 and 8) that subsequently bind to SMAD4 (the common SMAD), and translocate to the nucleus where they regulate gene expression. D. The Hedgehog pathway. In the absence of Hh ligands, the Patched receptor (Ptch) constitutively inhibits Smoothend (Smo), another transmembrane protein. This promotes the processing of the Gli transcription factors by a multi-protein complex. Gli transcription factors are thereby inactivated or turned into a repressive form. Ligand binding inhibits Ptch, which results in the de-repression of Smo and the release of unprocessed Gli transcription factors, now able to activate Hh target genes.

but also for their maturation. Comparative gene expression profiling of fetal TCF4 deficient and TCF4 heterozygote small intestines has identified a substantial number of TCF4 target genes, which are typical Paneth cell markers such as multiple

cryptidins and peptidoglycan recognition proteins. Conditional inactivation of the Wnt receptor Frizzled5, which is expressed in the (developing) crypts of neonatal and adult mice, abrogates expression of these genes in Paneth cells [53].


In summary, Wnt signaling plays multiple roles during gut homeostasis. It is considered the gatekeeper for crypt progenitor cells by controlling the progenitor gene expression program. It is necessary for the development and or differentiation of the secretory cell lineages, positioning and maturation of Paneth cells as well as for the separation of proliferating undifferentiated and postmitotic differentiated cells.

NOTCH SIGNALING DURING GUT HOMEOSTASIS The Notch pathway is another well-known developmental pathway that plays a central role in intestinal homeostasis. The Notch signaling pathway is highly conserved in evolution and is found in organisms as diverse as worms and humans. At the beginning of the 20th century Thomas Hunt Morgan and colleagues described notches at the margin of wing blades of fruit flies (Drosophila melanogaster) [54]. These notches were later found to be the result of a partial loss of function (haplo-insufficiency) of the Notch gene which encodes a single transmembrane receptor [55,56]. Notch proteins and their corresponding ligands regulate many cell fate decisions and differentiation processes during both fetal and postnatal development [57]. Mammals such as mice and humans possess four receptors (Notch1-4), and five ligands: Jagged1 and Jagged2 (homologues of Serrate) and Delta-like1, 3 and 4 (homologues of Delta). Notch signaling is initiated by ligand binding to the extracellular domain of Notch receptors, triggering two proteolytic cleavages within the receptor. The first is mediated by the ADAM protease TACE (tumor-necrosis factor α-converting enzyme), which cleaves the receptors close to the transmembrane domain. The extracellular Notch domain is ‘transendocytosed’ by the ligand expressing neighbouring cell [58]. A second cleavage, mediated by the γ -secretase activity of the multi-protein complex of presenilins, occurs within the transmembrane domain. The liberated cytoplasmic domain (NICD, for Notch intracellular domain) translocates to the nucleus and binds the transcription factor CSL (CBF1 in humans, RBP-J in mice, Suppressor of hairless in Drosophila, Lag1 in C. elegans), converting it from a transcriptional repressor into a transcriptional activator by displacing corepressor complexes [59-61] and recruiting coactivators [60,62] “Fig. (3B)”. To date, only few Notch target genes have been identified, some of which are utilized in multiple tissues while others seem to be tissue specific. The bHLH transcription factors of the Hairy enhancer of split (Hes) family such as Hes1 and Hes5 are among the best-studied Notch target genes [63]. Notch receptors, the corresponding ligands and the downstream gene Hes1 exhibit overlapping expression patterns in the intestinal crypt compartments. In addition, some Notch gene family members are expressed in the lamina propria of the large intestine as well as in


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stroma of the intervillus region of the small intestine [64,65]. This wide expression pattern suggests that Notch signaling may play multiple roles in the intestine. The first direct genetic evidence showing an essential role of Notch signaling for the homeostasis of the intestinal epithelium is derived from inducible tissue specific inactivation of CSL/RBP-J which is the transcription factor mediating Notch signaling of all Notch receptors. Loss of CSL/RBP-J within the crypt compartment leads to a complete loss of proliferating transient amplifying cells followed by their conversion into postmitotic goblet cells [66]. Conversely, expression of a dominant active form of the Notch1 receptor (NICD) in the gut inhibits differentiation of crypt progenitor cells resulting in an intestine that largely consists of undifferentiated transient amplifying cells [67]. These complementary loss- and gain-of-function studies suggest that Notch signaling is a gatekeeper of the progenitor/stem cell compartment of the gut. Additional evidence supporting such an important role for Notch in the intestine is derived from toxicology studies of γ -secretase inhibitors. These small molecules are currently being developed by pharmaceutical companies to inhibit the protease (γ secretase) activity of presenilins for the treatment of Alzheimer’s disease. The primary target of these drugs is the disease causing amyloid precursor protein (APP). However Notch receptors are also substrates of these proteases, which cleave them upon ligand mediated activation, thereby liberating NICD. Mouse models for Alzheimer’s disease treated with γ -secretase inhibitors, display unwanted side effects such as a large increase in mucus secreting goblet cells [68,69] due to inhibition of Notch signaling. Taken together these results suggest that Notch signaling is essential for maintenance of undifferentiated crypt progenitors. In this respect the Notch and Wnt signaling pathways appear to work hand in hand. Loss of either signaling cascade results in loss of the crypt compartment. Thus Notch and Wnt cascades apparently synergize as gatekeepers of self-renewal in the intestinal epithelium “Figs. (2A and C)”. The fact that loss of Notch signaling results in goblet cell differentiation while enterocyte differentiation is lost suggests that Notch has an additional function in lineage specification of enterocytes. This is also supported by genetic evidence from gene-targeted mice for the downstream target gene Hes1. Hes1 -/- mice die perinatally due to severe neurological defects. The fetal intestine of Hes1 mutant mice shows increased numbers of mucus secreting and enteroendocrine cells at the expense of adsorptive cells (enterocytes) [70]. The bHLH transcription factor Math1 is transcriptionally repressed by Hes1 in multiple organs including the intestine [70,71]. In Math1 deficient mice, the intestine is only populated by enterocytes


[71]. This phenotype is largely reciprocal to that of Hes1 deficient mice, strongly suggesting that Math1 is required for the differentiation of secretory cell lineages such as mucus secreting and enteroendocrine cells [71]. In summary these results suggest that Notch-mediated Hes1 expression regulates a binary cell fate decision of intestinal progenitors that have to choose between adsorptive or secretory cell fates “Fig. (2E)”. Another binary cell fate decision that occurs within secretory cell lineages is that specifying enteroendocrine versus mucus secreting cells. This process is regulated by the bHLH transcription factor Neurogenin3 (Ngn-3) [72] since mice deficient for this molecule specifically lack enteroendocrine precursors [73] “Fig. (2E)”. Neurogenins can be negatively regulated by Notch signaling through Hes mediated repression [74] raising the possibility that this binary cell fate decision might also be controlled by Notch signaling. BETA2/neuroD, another bHLH transcription factor well known for its function during pancreas development, specifies subsets of the enteroendocrine cell fate [75]. As mentioned above, adenomas result from uncontrolled Wnt signaling, very often caused by loss-of-function mutations within the tumor suppressor gene APC. Recent gene expression profiling experiments revealed a highly conserved expression pattern between colorectal cancer cells and crypt cell progenitors [46]. Gene expression analysis of Notch signaling components performed on adenomas of APC min (multiple intestinal neoplasisa) mice show that this symmetry between crypt and intestinal neoplasia also extends to the Notch pathway. Genes such as Delta-like1, Notch2 and Hes1 are also expressed in adenomas [66], suggesting that activation of the Notch and the Wnt pathways occurs simultaneously in proliferating adenomas, as in intestinal crypts. This observation leads to the question whether proliferating adenoma cells can be differentiated and withdrawn from the cell cycle by interfering with the Notch signaling pathway, similar to what is observed with crypt cell progenitors. Indeed, γ -secretase treatment of APC min mice induces goblet cell differentiation and reduces proliferation in such adenomas, suggesting that specific inhibition of the Notch pathway can drive cells out of cycle towards a secretory cell fate despite the fact that Wnt signaling remains active [66]. This ‘proof of principle’ experiment highlights the Notch pathway as a potential drug target for the treatment of intestinal neoplasias. While the Wnt signaling pathway is aberrantly activated at the onset of the transformation process, the Notch pathway most probably remains intact at this stage. However it is possible that with tumor progression strong selective pressure might cause activating mutations within the Notch pathway. Such activating mutations are frequently found in the Notch1 gene in patients suffering from acute lymphoblastic T cell leukemias [76]. Oncogenic properties of aberrant Notch signaling have also been reported for T cell

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leukemias in mice and mouse mammary tumors (reviewed in [22]). However, although current data suggest that Notch might also function as an oncogene during the development of colorectal cancer, further investigation is needed to confirm this notion.

TGF- /BMP SIGNALING IN THE GUT AND MALIGNANCY The TGF-β superfamily consists of a number of secreted cytokines including different TGF-β isoforms, bone morphogenic proteins (BMPs), and activins. These proteins are known to regulate a broad spectrum of different processes such as embryonic development, differentiation, proliferation, adhesion, wound healing, and inflammation [24,77,78]. Signaling is induced by ligand binding to type II serine/threonine kinase receptors (TGFβR-II) followed by heterodimerization with, and phosphorylation of type I serine/threonine kinase receptors (TGFβR-I). Signaling of these activated receptors is transduced through SMAD proteins. Three different groups of SMAD proteins exist: receptor regulated SMADs (R-SMAD1, 2, 3, 5 and 8), a common SMAD (SMAD4), and inhibitory SMADs (ISMAD6 and I-SMAD7). Activation of type I receptors results in the phosphorylation of R-SMADs followed by heterodimerization with SMAD4 and subsequent translocation to the nucleus. SMAD heterodimers either bind DNA directly or via DNA binding proteins thereby regulating gene expression [79] “Fig. (3C)”. In contrast to signaling components of the Wnt and Notch pathways (which are localized to the undifferentiated crypt compartment), TGF-β receptor and ligands are expressed in the differentiated compartment of the gut [80,81] “Fig. (2A)”. Cell culture experiments show that TGF-β signaling can exert growth inhibitory effects [82] which are mediated through various mechanisms including repression of the transcription factor c-myc [83], inhibition of cyclin D1 expression [84,85], as well as transcriptional activation of cyclin-dependent kinase inhibitors p15INK4B [86] and p21CIP/WAF [87]. However most colon cancer derived cell lines are refractory to the TGF-β mediated growth inhibitory effect [88] due to mutations in down stream signaling components of the TGF-β pathway [89-91]. Reintroduction of wild type molecules restores the ability of these cell lines to respond to TGF-βmediated growth repression [92]. These cell culture experiments suggest that mutational loss of TGF-β signaling could lead to deregulated growth, or at least hyperproliferation of the intestinal epithelium. The generation of loss-of-function mouse models for linking TGF-β function in vivo with disease development has proven to be difficult. Conventional gene targeted mice for TGF- -1 [93-95], -2 [96], and –3 [97], TGFBRII [98] or SMAD2 [78] die during embryogenesis or shortly after birth and none of heterozygotes develop intestinal cancer. Although mutations within the TGF-β pathways have not been


shown to be causative for tumor onset to date, they have been linked to the acquisition of invasive properties in benign adenomas in both mouse and humans. For example, adenomas of APC∆716 rapidly progress to aggressive carcinomas on a SMAD4 heterozygote background, mimicking the adenomacarcinoma transition observed in humans [99]. Although we have mainly discussed the growth inhibitory properties of the TGF-β pathway it should be noted that TGF-β also has tumor promoting roles, in particular during tumor invasion. TGF-β promotes epithelial to mesenchymal transition in cells that have acquired loss-of-function mutations and therefore no longer respond to its growth inhibitory properties [20,100]. BMPs, originally identified for their ability to induce cartilage and bone formation, transduce signals specifically via SMAD1, 5 and 8 “Fig. (3C)”. In the adult intestine BMPs are expressed in the intervillus mesenchyme, activating BMP receptors on the neighboring intestinal epithelium in a paracrine fashion [101,102]. Humans with germline muations in the BMP receptor type1A or SMAD4 develop the juvenile polyposis syndrome (JPS), characterized by hematomatous polyps throughout the gastrointestinal tract in 50% of cases [103-105]. Mice heterozygous for SMAD4 develop polyps in the stomach and duodenum, thus resembling JPS [106,107]. A JPS-like phenotype is observed in transgenic mice expressing the BMP inhibitor Noggin [102] and in mice in which the BMP receptor type1A gene [108] is conditionally inactivated within the intestinal epithelium. BMP inhibition in these mouse models leads to de novo crypt formation, and to increased numbers of proliferating cells in the normally differentiated villus compartment. These data suggest that BMP signaling antagonizes the Wnt pathway within the differentiated compartment, thereby helping position transient amplifying cells in the crypt pockets. A mechanistic model for BMP mediated suppression of Wnt signaling in which BMP signaling leads to PTEN activation in stem cells, and resulting in the repression of β-catenin mediated signaling through the PI3 kinase-Akt pathway was suggested by He and coworkers [108].

HEDGEHOG SIGNALING IN THE GUT The Hedgehog signaling pathway is another evolutionarily conserved pathway implicated in many developmental processes such as pattern formation during embryogenesis, cell fate specification, proliferation, and differentiation [109]. Additional roles in stem cell maintenance have been attributed to this pathway [110] and deregulated hedgehog signaling is associated with cancer [111,112]. Hedgehog was originally identified as mediator of segment polarity in the fruit fly [113]. Mammals possess three hedgehog genes: Sonic (Shh), Indian (Ihh) and Desert (Dhh), all of which encode secreted proteins. Signaling is initiated through binding of


283

Hedgehog ligands to the 12-transmembrane receptor Patched (Ptch). In the absence of ligand, Ptch suppresses the 7-transmembrane protein Smoothened (Smo). Hedgehog binding inactivates Ptch, resulting in the activation of Smo, thus triggering the stabilization and translocation of members of the GLI family of Zn-finger transcription factors from the cytoplasm to the nucleus. The mammalian genome encodes three GLI family members (GLI-1, -2 and –3), which upon translocation to the nucleus regulate gene expression [114] “Fig. (3D)”. During gut development the mesenchyme segregates into four different concentric layers of variable proximity to the epithelial layer. The innermost layer of the mesenchyme adjacent to the epithelium differentiates into the lamina propria, the next layer develops into the muscularis mucosae, the third layer differentiates into the submucosa, and the final outermost region differentiates into circular and longitudinal smooth muscle layers. Shh and Ihh are expressed by the mouse endodermal epithelium whereas Hedgehog target genes have been found to be expressed in the mesenchyme, indicating paracrine signaling [115]. Studies using gene targeted mice for Shh and Ihh have shown that both proteins are involved in radial axis patterning in the intestine by specifying the ratio of lamina propria and submucosa to smooth muscle and enteric neuronal cell precursors [116]. The smooth muscle layer in both Shh and Ihh mutant mice is significantly reduced suggesting that both proteins may regulate gut smooth muscle development in a partially redundant manner [116]. Such a function is consistent with previously identified roles for Shh in smooth muscle development in the lung [117] as well as in the pancreas of mice ectopically expressing Shh in which ectopic smooth muscle formation is induced [118]. In addition, Shh mutant mice show abnormal patterning of the enteric nervous system characterized by increased numbers of neurons, even in areas just adjacent to the epithelium. This suggests that Shh plays an inhibitory role in neural migration and/or differentiation in the gut. These findings are in agreement with transplantation experiments in chick embryos. Grafts of gut epithelium or Shh expressing cells into the mesenchyme results in inhibition of neuronal differentiation in the vicinity of the graft [119]. Furthermore, multiple malformations along the gastrointestinal tract have been observed in various animal models deficient for components within the Hedgehog pathway. Gene targeted mice for Shh exhibit gut mal-rotation, annular pancreas, imperforate anus and and persistant cloaca [116,120]. Dilated colon and abnormally thin walls reminiscent of Hirschsprung disease, as well as duodenal stenosis have been reported for Ihh deficient mice [116]. Gli-2 deficient mice show imperforated anus and recto-urethal fistula, while Gli3 knockout animals have anal stenosis and ectopic anus [120, 121].


More recently, Peppelenbosch and coworkers suggested that Hedgehog signaling restricts cycling cells in the colon to the base of the crypts. They showed that Ihh expression is restricted to the noncycling, differentiated surface epithelium of the colon, both in humans and rats “Fig. (2C)”. Treatment of rats with the naturally occurring alkaloid cyclopamine (found in the plant Veratrum californicum), an inhibitor of the Hedgehog pathway, results in defective enterocyte differentiation concomitant with increased numbers of cycling cells per crypt. In vitro experiments using colon cancer derived cell lines suggest that Ihh can repress βcatenin mediated Wnt signaling [122]. Similarly, transgenic expression of the pan-Hedgehog inhibitor HhIP (Hedgehog interacting protein) in the gut results in enhanced Wnt target gene expression, increased proliferation and the formation of crypt like structures on villi tips [123]. Taken together these studies suggest that Hedgehog signaling in the intestine may antagonize Wnt signaling. Thus loss of Hedgehog signaling should lead to a growth advantage in the gut epithelium. However, to date loss-of-function mutations in the Hedgehog pathway have not been reported for colorectal cancers. In contrast, Hedgehog associated cancers are mainly the consequence of gain-of-function mutations leading to excessive signaling. For example mutations within the Patched gene causes the hereditary disease Gorlin’s syndrome, which is characterized by a high incidence of basal cell carcinoma and medulloblastomas [124,125]. Activating mutations within the Hedgehog pathway are also frequently found in sporadic cancers of the skin, the cerebellum and skeletal muscles [126]. Recent analysis of tumors of the upper digestive tract (including oesophagus and stomach) showed increased Hedgehog pathway activity due to increased Shh and Ihh expression. In addition, cyclopamine suppressed tumor cell growth in vitro and caused durable regression of xenograft tumors in vivo [127].

INTESTINAL CANCER Colorectal cancer is the second most frequent cause for cancer death in the Western society after lung cancer [128,129]. The incidence rates in countries such as Asia, Africa and Latin America are relatively low (65%) suggesting that environmental exposure is a critical factor. This is further supported by population studies showing that immigrants moving from low- to high-risk countries rapidly acquire an increased cancer risk [130,131]. Epidemiological studies have suggested that specific components of the western diet such as red meat and fat are part of the increased risk factors for developing colorectal cancer, while diets based on vegetables, fruits and fibers are protective (World Cancer Research Panel 1997).

Radtke et al.

A substantial amount of our current knowledge concerning the molecular mechanisms causing colorectal cancer is derived from familial cancer syndromes, although they only make up approximately 20% of all cancer patients. Hereditary colorectal cancer syndromes are generally classified into two categories based on the presence or absence of polyposis, exemplified by the familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNCC) also known as Lynch’s syndrome.

FAMILIAL (FAP)

ADENOMATOUS

POLYPOSIS

FAP is an autosomal dominant inherited disorder characterized by the emergence of hundreds to thousands of colorectal adenomatous polyps starting at the mean age of 16. The incidence of FAP has been estimated to be approximately 1/10000, with men and women being equally affected [132]. Although adenomas are generally considered as benign, the large number of polyps prone to acquiring additional mutations confers FAP patients a 100% chance of developing colorectal cancer by the age of 40 [133]. The FAP syndrome is caused by mutations within the adenomatous polyposis coli (APC) tumor suppressor gene. APC mutations are also found in the vast majority of patients with sporadic colorectal cancer [134,135]. The APC gene maps to the long arm of human chromosome 5 in band q21. A mutational hot spot has been identified within the 5’ region of the last coding exon of the APC gene. Most mutations lead to premature stop codons causing truncated APC proteins [48,136]. Other mutations have been localized to either the 5’ region of the APC gene [137] or to the 3’ part of exon 15 [138]. Both types of mutation correlate with an attenuated form of FAP (AAPC), characterized by fewer polyps and later onset of colorectal cancer compared to classic FAP patients [139]. As mentioned previously, APC is part of the destruction complex that negatively regulates βcatenin, the central signaling molecule of the classical Wnt pathway. Mutations within the APC protein result in stabilization of β-catenin, and consequently lead to enhanced proliferation of the intestinal epithelium. In addition, the C-terminus of APC has been involved in maintaining chromosomal stability during mitosis [140,141] by facilitating the binding of spindle microtubules to kinetochores. APC mutant cells have an abundance of spindle microtubules that fail to connect to kinetochores resulting in chromosomal instability (CIN) [142].

HEREDITARY NON-POLYPOSIS RECTAL CANCER (HNPCC)

COLO-

HNPCC also known as Lynch’s syndrome is the most common form of hereditary colorectal cancer [143]. This autosomal dominant cancer syndrome is



285

Fig. (4). A model for the sequence of events associated with colorectal cancer development. The transition from normal intestinal epithelium to colorectal cancer can be correlated with successive histological and genetic alterations. In this series of events epithelial cells of the gut acquire mutations within components of the Wnt pathway (such as APC, βcatenin or Axin) leading to the development of dysplastic aberrant crypt foci followed by early, intermediate and late adenomas. These events are characterized by additional genetic alterations, such as chromosomal and microsatellite instability and mutations within the RAS and TGF-β-pathway. The transition from a late adenoma to the carcinoma stage is frequently associated with mutations in the p53 gene. Additional genetic alterations might then be responsible for the development of metastases. CIN: Chromosomal instability. MIN: Microsatellite instability.

characterized by early onset of colorectal cancer, typically around the age of 45, which is approximately 20 years earlier than that for sporadic colorectal cancer. These tumors show no or only very low numbers of polyps compared to FAP, hence the name of the disorder. In addition this syndrome is associated with other adenocarcinomas such as ovarian, endometrial, gastric and urinary tract cancer [144]. The vast majority of HNPCCs are characterized by Microsatellite instability (MIN) [145].

Microsatellites are repetitive, short DNA or single nucleotide sequences found within the genome. DNA mismatch repair proteins normally repair small sequence errors that occur during DNA replication. However, mutations within such DNA mismatch repair genes lead to the accumulation of sequence errors within these repetitive DNA sequences characterized by either shortening or elongation of such DNA repeats. The observation that the MIN in tumors of HNPCC patients resembles those seen in bacterial


and yeast cells with DNA mismatch repair gene mutations, has stimulated researchers to look for human orthologues of these genes in HNPCC. Indeed, linkage analysis of tumor samples from HNPCC patients has led to the identification of five human mismatch repair genes (MSH2, MLH1, MSH6, PMS1 and PMS2) [146-151]. Mutations within the MHL1 and MSH2 mismatch repair genes account for approximately 90% of all identified mutations. Although most of the microsatellite DNA is found in introns, some genes carry them in their coding regions. In HNPCC tumors mismatch repair defects have been identified in the human -catenin gene (CTNNB1), in the transforming growth factor receptor II (TGFBR2) gene and in the pro-apoptotic gene Bax [152].

SPORADIC COLORECTAL CANCER Basically every second person in Western society will develop adenomas by the age of 70, and in around 1 in 20 individuals these adenomas will progress to colorectal carcinoma [153]. The accessibility and co-existence of various tumor stages has allowed identification of stage specific genetic alterations within colorectal cancers. This led to a genetic model describing the transition from healthy colonic epithelia through various adenoma stages to malignant cancers [154,155] “Fig. (4)”. According to this model, colorectal tumors arise as a result of activating mutations in oncogenes, coupled with loss-of-function alterations in tumor suppressor genes. Up to 7 genetic and histological alterations have been suggested to be required for the progression from adenoma to carcinoma [156]. Although some genetic alterations appear to occur in a preferential order, it is rather the total accumulation of genetic changes that seem to be critical. Aberrant crypt foci (ACF) in colorectal mucosa have been proposed to be the earliest detectable morphological lesions during development from adenoma to carcinoma. Their histological appearance can vary from dysplastic to hyperplastic, and seem to correlate with different genetic alterations [157]. For example, most ACFs in FAP patients exhibit a high frequency of dysplasia, are located in the epithelium of upper crypts, and carry mutations in the APC gene, but have a low frequency of ras mutations. They are considered to be potential precursors of colorectal adenomas and adenocarcinomas. In contrast, sporadic ACFs resemble hyperplastic polyps, with a relatively higher frequency of mutations in the ras gene. However, they appear to rarely develop into malignant lesions [157,158]. Expansion of dysplastic lesions gives rise to larger adenomas, that often carry mutations in the ras gene itself, or B-raf, a downstream component. Subsequent acquisition of alterations within the TGFβ pathway (SMAD4 and/or TGFβR-II) adds additional malignant features to adenomas. Malignancy has also been associated with mutational loss of the TP53 gene. Approximately half of all colorectal

Radtke et al.

cancers have p53 (TP53) gene mutations, with higher frequencies observed in distal colon and rectal tumors, and lower frequencies in proximal tumors [159]. As p53 controls the expression of apoptosis and cell cycle genes, its inactivation causes additional genetic alterations, resulting in survival of cells that should undergo apoptosis. In addition, cells can no longer control the cell cycle machinery in response to DNA damage or other cellular stress which results in genetic instability [160]. Genomic instability is a hallmark of all intestinal malignancies. Although we have introduced chromosomal instability (CIN) and microsatellite instability in the paragraphs discussing hereditary forms of colorectal cancer, they clearly play important roles in sporadic colorectal cancer. CIN is characterized by allelic losses and aneuploidy [161]. As described above, mutations in the APC and TP53 genes contribute to the development of the CIN phenotype [140,141,159]. Approximately 15% of sporadic tumors carry the MIN phenotype due to inactivation of mismatch repair genes resulting from either epigenetic silencing through promoter methylation [162] or somatic mutations within the genes [163]. Genetic instability bears a great advantage for tumor cells leading to a continuous acquisition of novel mutations allowing a rapid selection for growth advantages of tumor cells.

CONCLUDING REMARKS Homeostasis of the intestine follows the classical paradigm of other self-renewing tissues such as the hematopoietic system or the skin. This includes stem cells giving rise to rapidly proliferating transient amplifying cells, which terminally differentiate into specified post mitotic cells. The pathways regulating these mechanisms seem to be the same as those responsible for embryonic pattern formation and organogenesis. Studies, not only from hereditary cancer syndromes, but also from sporadic colorectal carcinomas, combined with studies derived from rodent model systems reveal that cancer cells hijack, subvert and abuse molecules or pathways that normally ensure the physiological homeostasis self renewing tissues. Increasing evidence derived from other neoplasms, including lymphoid and myeloid leukemias, breast cancers and malignancies in the skin indicate that this is a recurrent strategy of tumor cells. The challenge for the future will be to identify strategies to specifically interfere with and modulate these pathways in tumor cells, without affecting their physiological role in normal tissues.

ACKNOWLEDGEMENTS We thank Anne Wilson for critical discussion and reading of the manuscript, and apologize to those colleagues whose work was not mentioned due to space limitations. F.R. and O.R. are in part supported by grants from Oncosuisse and the Swiss National Science Foundation.



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Received: June 06, 2005

Revised: August 15, 2005

Accepted: September 14, 2005

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