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CD44 glycoproteins in colorectal cancer; expression, function and prognostic
value
Wielenga, V.J.M.
Publication date 1999
Document Version Final published version
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Citation for published version (APA):
Wielenga, V. J. M. (1999). CD44 glycoproteins in colorectal cancer; expression, function and prognostic value.
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CD44 glycoproteins in
colorectal cancer
expression, function
and prognostic value
Y
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n iHIT
Il il II II INI II
Il II II II II II II INI II II II II II II
CD44 GLYCOPROTEINS IN COLORECTAL CANCER; EXPRESSION, FUNCTION AND PROGNOSTIC VALUE
This thesis was prepared at the Department of Pathology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands, and supported by grants from The Dutch Cancer Society and Het
Praeventiefonds.
CD44 GLYCOPROTEINS IN COLORECTAL CANCER; EXPRESSION, FUNCTION AND PROGNOSTIC VALUE
Academisch proefschrift
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus
prof.dr J.J.M. Franse
ten overstaande van een door het college voor promoties ingestelde commissie,
in het openbaar te verdedigen in de Aula der Universiteit op 29 Juni 1999, te 11.00 uur
door Vera Jacqueline Marita Wielenga geboren te Wettingen
Faculteit der Geneeskunde Promotores
Prof. dr S.T. Pals
Prof. dr G.J.A. Offerhaus Promotiecommissie
Prof, dr A. Berns Prof, dr H.C. Clevers Prof, dr R. Fodde Prof dr F.J.W. ten Kate Prof. dr C.J.F, van Noorden Prof. dr J.H.P. Wilson
Contents Abbreviations
Chapter 1 General introduction 7 Chapter 2 Expression of CD44 variant proteins in human colorectal
cancer is related to tumor progression.
Cancer Res 1993, 53:4754-4756 49 Chapter 3 Expression of mutant P53 protein and CD44 variant proteins
in colorectal tumorigenesis
Gut 1995, 36:76-80 53 Chapter 4 CD44 splice variants as prognostic markers in colorectal
cancer.
Scan J Gastroenterol 1998, 33:82-87 59 Chapter 5 Expression of CD44 in Ape and Tcf mutant mice implies
regulation by the Wnt- pathway.
Am J Pathol 1999, 154:515-524 67 Chapter 6 Heparan sulfate-modified CD44 promotes hepatocyte growth
factor/scatter factor-induced signal transduction through the receptor tyrosine kinase c-Met.
JBiolChem 1999,274:6499-6506 91 Chapter 7 Co-expression of c-Met and heparan sulfate proteoglycan
forms of CD44 in colorectal cancer 101 Chapter 8 CD44 glycoproteins in colorectal cancer; expression,
function and prognostic value.
Adv Cancer Res, In press 129
Summary/Samenvatting 159 Acknowledgements/Dankwoord 169
Abbreviations
APC Adenomatous polyposis coli
ACF Abberant crypt focus
CD44s CD44standard
CD44v CD44variants
ECM Extracellular matrix
ERM Ezrin, radixin, moesin
FAP Familial adenomatous polyposis
FGF-2 Fibroblast growth factor-2
GAG Glycosaminoglycans
HA Hyaluronic acid
HS Heparan sulfate
HGF/SF Hepatocyte growth factor/scatter factor
PG Proteoglycan
Chapter 1
INTRODUCTION General introduction.
Colorectal cancer is the second leading cause of cancer related death in the western world (American Cancer Society, 1994). Mortality due to colorectal cancer is almost always the result of tumor metastasis. Roughly half of the patients with apparent localized disease at the time of diagnosis, who are operated with the intention to cure, will nevertheless have an eventual recurrence of the disease, due to undetected micrometastasespresent at the time of surgery (Coleman et al, 1993; Greenson et al, 1994). In order to improve prognosis it is imperative that the molecular determinants of metastasis are identified. This would enable differentiation between patients with actual localized disease and patients in which the disease has spread already and who might benefit from aggressive adjuvant therapy. The aim of this thesis therefore, is to study the expression and regulation of CD44 and its isoforms, molecules which have been implicated in metastasis formation in animal models, and to explore their usefulness in predicting the natural history of colorectal cancer.
COLORECTAL CARCINOGENESIS
Cancer of the large bowel mucosa develops through a series of morphologically well described precursor lesions, known as the adenoma-carcinoma sequence. The mucosal changes are the result of progressive genetic alterations, that occur at specific time points during carcinogenesis, as summarized in "the Vogelgram"(Fig.l) (Muto et al, 1975; Vogelstein et al, 1988; Fearon et al, 1990). Four types of genes can be mutated, with different mutational routes. Stimulatory genes -proto-oncogenes- become hyperactive due to mutations with a dominant effect: only one of the gene copies need to undergo the change, and the altered gene is called an oncogene. Otherwise, inhibitory genes can be made inactive, and this type of mutation has a recessive effect: both gene copies must be inactivated or deleted to free the cell from inhibition, and the lost gene is called a tumor suppressor gene. A third
APC (5q) B-catenin (3p) K-ras (12p) SMAD2 (18q) SMAD4 (18q) p53 (17p) normal early polyp Tatï polyp
m
\i i/
mismatch repairm
Figure 1. The adenoma-carcinoma sequence.
mechanism is the obstruction of the DNA mismatch proof reading system genes due to mutations in both gene copies of the mismatch-repair genes. Mismatch-repair genes repair DNA replication errors that occur during the S-phase. Disruption of the repair mechanism results in a hypermutable phenotype, leading to accumulation of other somatic genetic mutations at an accelerated rate. The last type of genes that can be mutated are the mitotic breakpoint genes, which are implicated in correcting failures during mitotic segregation of the chromosomes.
Although the majority of colorectal cancers occurs sporadically, well described inherited predispositions to colorectal cancer as, for example, familial adenomatous polyposis (FAP) and hereditary non polyposis colorectal cancer (HNPCC), have greatly increased the insight in the molecular genetics underlying sporadic colorectal cancer. Specific pathways are disrupted by molecular genetic changes of which the sequential order in the adenoma-carcinoma sequence results in progressive disturbance of cell growth and motility.
The ras oncogenes.
Three cellular transforming ras oncogenes genes have been described,
K-ras, U-ras and N-ras (Weinberg et al, 1994). The ras genes encode
GTP-binding proteins (Bourne et al, 1991). A point mutation at an appropriate site in a ras gene creates a Ras protein that fails to hydrolyze its bound GTP and thereby persists in its active state, resulting in a growth advantage during tumorigenesis. Point mutations of ras genes are acquired during adenoma progression, and are rarely present in adenomas smaller than 1 cm but are found in 50% of adenomas larger than 1 cm and in 50% of carcinomas (Vogelstein et al, 1988; Poylak et al, 1996). Ras mutations occur frequently in adenomas, but are not neoplastic per se, since they are also found in the majority of the self-limiting non-dysplastic aberrant crypt foci (ACF).
The GTP-ase activity of wild-type Ras is stimulated by GTP-ase activating protein (GAP), while mutant Ras does not respond. Recently, the gene responsible for neurofibromatosis type I (NF1) was found to have GAP activity. This is of interest since a colon tumor without a ras mutation was reported to have a mutation of NF1 (Xuetal, 1990; Li etal, 1992). Hence, in part of the ras mutation negative colon tumors, deregulation of the same pathway by mutation of a different component might play a role
The p53 tumor-suppressor gene.
The p53 gene is located on chromosome 17p and encodes a 53kD nuclear phosphoprotein identified as a tumor suppressor gene. The wild-type
p53 gene product was shown to function as a transcription factor causing arrest
of progression through the cell cycle in Gl and repair of genotoxic damage or otherwise pushing the cell into apoptosis (Kern et al, 1994). Targets regulated by p53 include the apoptosis-inducing BAX gene (Miyashita et al, 1994) and the growth-arrest genep21/WAFl/CIPI(El-Deiry WS et al, 1993; Harper et
al, 1993).
Loss of 17p was reported in 75% of the colorectal cancers, and in 30% of the late adenomas but rarely in early adenomas, suggesting a role during the transformation of in situ to invasive carcinoma during the adenoma-carcinoma sequence (Monpezat et al, 1988; Vogelstein et al, 1988; Baker et al, 1989,
1990). In concordance with the properties of tumor-suppressor genes, the normal function of p53 is disrupted in colorectal carcinomas mostly as a result of loss of one 17p allele, coupled with a mutation of the remaining copy of p53 (Baker et al, 1989, 1990). However, in some cases p53 mutant protein has a dominant-negative effect, due to formation of tetramers by mutant and wild-type molecules (Vogelstemen/., 1992). Disruption of the p53 pathway leads to increased cell proliferation with fixation of the genotoxic damage into the DNA and to clonal expansion of the tumor cells.
The tumor-suppressor genes on chromosome 18q.
Loss of 18q occurs in 73% of colorectal cancer and in 47% of late adenomas (Thiagalingam et al, 1996). Several (candidate) tumor-suppressor genes have been identified in this region and recently the importance of the
Smad-genes on 18q was discovered. Mutations in Smad2 (also known as
DPC4) and Smad4 (also known as JV18-1, MADR2, and hMAD-2) were reported in approximately 7% and 20% of the colon carcinomas, respectively (Riggins et al, 1996; Eppert et al, 1996). Inactivating mutations in the Smad genes are postulated to disrupt the growth inhibitory signal transduction pathway of the TGF-ßll receptors (Liu et al, 1996; Yingling et al, 1996). The TGF-ßll receptor gene has a poly-A-repeat and therefore, disruption of this pathway is frequently found in colorectal tumors with microsatellite instability. This observation supports the idea that the TGF-ßll receptor is a critical target gene of inactivation in mismatch-repair deficient tumors (Parsons etal, 1995).
Mismatch-repair genes
Errors in DNA replication during the S-phase result in single base pair mismatches or mispaired loops that occur at repetitive sequences such as microsatellites. These errors are repaired by mismatch-repairgenes. Disruption of the repair mechanism, due to mutations in these genes, result in accumulation of other somatic genetic mutations with an acceleration of the carcinogenic process. Patients with an inherited mutation of these mismatch-repair genes will develop HNPCC with formation of colorectal tumors distinct from the ones as initially described in the Vogelgram.
The APC tumor-suppressor gene.
The previously described genetic alterations accompany the adenoma-carcinoma sequence but are not a prerequisite for this process. Mutations involving components of the Wnt-Wingless signaling cascade i.e. APC and
ß-catenin (Fig. 1, Fig.2), however, appear to be a precondition for the initial
neoplastic transformation of colon epithelium. The APC gene, located on chromosome 5q, is mutated in 80% of sporadic intestinal tumors, whereas mutations of ß-catenin are found in most of the remaining tumors. The
APCIß-catenin mutations are already present in early adenomas. Familial adenomatous
polyposis (FAP) patients, individuals with a germline mutation in the APC gene, develop multiple colorectal adenomas (Powell et ai, 1992; Ishii et ai, 1992; Levy et ai, 1994; Vogelstein et ai, 1988), with virtually 100% chance of malignant transformation after a few decades. Like FAP patients, APC
163 8N and Min mice that are heterozygous for an A PC mutation at codon 163 8 and 850, respectively, will develop multiple adenomas of the intestine, following deletion of the remaining wild type allele of APC, thus confirming the role of APC as the gatekeeper in colorectal cancer (Smits et ai, 1997; Moser et ai, 1995; Luongo et ai, 1994; Levy et ai, 1994; Ishii et ai, 1992).
APC is part of the Wnt- pathway (Fig. 2) (Peifer et ai, 1996), the human homologue of the Wingless (WG)-pathway in Drosophila. One of the molecules that interact with APC in this pathway is ß-catenin (Rubinfeld et ai,
1993; Behrens et ai, 1996). ß-Cateninwas originally identified on the basis of its association with Cadherin adhesion molecules (Birchmeier et ai, 1994), but is now also recognized as an essential component of the Wnt-Wingless cascade. Under physiological circumstances APC and ß-catenin form a complex and the activity of ß-catenin as a signal transducer, is tightly regulated by this binding. During intestinal carcinogenesis,however, as the result of APC (or ß-catenin) mutations, this complex is no longer formed and large amounts of free ß-catenin are available. This ß-catenin will bind the transcription factor TCF-4 and the ß-catenin/TCF complex will enter the nucleus, resulting in uncontrolled transcription of TCF-4 target genes (Korinek et ai, 1997; Gumbiner et ai, 1990; Behrens et ai, 1996; Molenaar et ai, 1996). The mechanisms by which deregulation of the Wnt-pathway induces cancer, even
The Cell
Figure 2. The Wnt-pathway. In this cascade, ß-catenin functions as a transciptional
co-activator when complexée! with members of the Tcf family of DNA binding proteins. The amount of ß-catenin, however, is regulated by binding of APC and subsequent degradation. On the left, the Wnt-pathway is shown under physiological conditions. In the non-activatedpathway, ß-catenin is bound to APC and phosphorylation of APC by GSK3 ß results in degradation of ß-catenin. On the right side, the Wnt-pathway is constitiutively activated as in colorectal cancer. Due to mutations in either APC or in
ß-catenin, the APC/ß-catenin complex is not formed resulting in uncontrolled amounts
of nuclear complexes between co-activator ß-catenin and Tcf-4 with transcription of Tcf-4 target genes in intestinal epithelium.
as the target genes of the ß-catenin/Tcf-4 complex, are still largely unknown. However, transfection of mutated APC was shown to inhibit apoptosis of the transfected cells (Morin et al., 1996) and recent studies have identified c-MYC as potential target gene (He et al., 1998).
Dukes A
mucosa
submucosa
Figure 3. The Dukes' stages. Dukes A: the carcinoma is confined to the bowel wall. Dukes B: malignant cells penetrate through the muscularis propria. Dukes C: malignant cells metastasize to regional lymph nodes. Dukes D: malignant cells metastasize to
The morphological changes in colorectal cancer.
The result of these genetic alterations, is the evolution of the colorectal epithelium through a series of morphologically well described neoplastic lesions. The first morphologically detectable anomaly of the mucosa is the dysplastic aberrant crypt focus (ACF) (Nucci et al., 1997). Impaired maturation of the dysplastic epithelium leads to a disturbed architecture with atypical cells present throughout the crypt, up to the mucosal surface. When more crypts become involved, the mucosal surface will develop a polyploid aspect resulting in the formation of an adenomatous polyp. When the tumor cells penetrate the basement membrane, by definition^ carcinoma has developed. The subsequent extension of the carcinoma can be separated in distinctive stages as originally described by CE. Dukes in 1932 (Fig.3). Initially the carcinoma will be
confined to the bowel wall (stage Dukes A). When the carcinoma cells penetrate through the muscularis propria (stage Dukes B) they eventually metastasize, first to regional lymph nodes (stage Dukes C) and finally to distant organs (stage Dukes D).
THE
CD44
GENE AND ITS PROTEIN PRODUCTS.
The oncogenes and tumor suppressor genes, previously mentioned, are all involved in the regulation of tumor growth and/or apoptosis. The main cause of cancer related death, however, is not primary tumor growth, but formation of distant metastases. Although relatively little is known about molecular mechanisms underlying this complicated process, a large body of studies indicate an important role of CD44 in metastasis (Lesley et al, 1993; Herrlich et al, 1993; Naor étal, 1997).
CD44 was originally described as a homing receptor on lymphocytes, mediating lymphocyte interactions with high endothelial venules (HEV) (Jalkanen et al, 1986, 1987). Metastasizing tumor cells and recirculating (activated) lymphocytes share several properties including invasive behavior, migration involving reversible adhesive contacts, accumulation and expansion in draining lymph nodes, release into the circulation, and adhesion to vascular endothelium and extravasation. This analogy between lymphocyte recirculation and tumor dissemination prompted the hypothesis that malignant cells might "misuse" molecules like CD44 for metastasis formation (Herrlich et al., 1993). Support for this hypothesis has come from experimental studies in laboratory animals showing a causal role of specific CD44 isoforms in metastasis formation (Güntherte/ al, 1991), as well as from clinical studies documenting deregulated CD44 expression in human cancer, including colorectal cancer (Lesley et al, 1993; Herrlich et al, 1993; Koopman et al, 1993; Naor et al,
1997; Matsumura et al, 1992; Heider et al, 1993; Tanabe et al, 1993 Wielengae/a/., 1993; Finn et al, 1994; Kim et al, 1994; Mulder et al, 1994 Kaufmann et al, 1995; Orzechowski et al, 1995; Rodriguez et al, 1995 Imazeki et al, 1996; Yamaguchi et al, 1996).
The structure of CD44.
All CD44 family members are encoded by a single gene on chromosome 11 p 13 that consists of 19 exons (Fig.4) (Stamenkovic et al., 1989; Dougherty et al, 1991 ; Günthert et al, 1991 ; Screaton et al., 1992; Lesley et
al., 1993). They share the N-terminal cartilage link protein homology domain,
that binds hyaluronic acid (HA) (Aruffo et al., 1990; Stamenkovic et al., 1989; Peach et al, 1993), as well as the C-terminal (transmembrane, cytoplasmic) domains encoded by exon 1-5 and exon 15-19, respectively. However, they differ in the extracellular membrane proximal part as a result of extensive alternative splicing of exons 6-14 (also referred to as exons v2-v 10) (Dougherty
etal, 1991; Günthert et al, 1991; Screaton étal, 1992; Tölg étal, 1993). In
this way, more than a thousand CD44 variants (CD44v) can be potentially generated. Further diversity of CD44 results from post-translational modifications with N-and O-linked sugars and with glycosaminoglycan(GAG) side chains like chondroitin- and heparan-sulfate. The CD44 cytoplasmic domain associates with the actin cytoskeleton via ankyrin, as well as via the ERM-family proteins ezrin, radixin, and moesin (Bourguignon et al, 1993; Tsukita et al., 1994; Isacke et al, 1994). In T-lymphocytes, CD44 is physically
and functionally associated with the Src-family tyrosine kinase p56lck(Taher et
al, 1996).
The expression of CD44.
CD44 is a family of type I transmembrane glycoproteins that is widely expressed on a variety of cells including cells of epithelial, mesenchymal, and hematopoietic origin. The expression of CD44 isoforms is tissue specific, for example, the shortest isoform of CD44 (CD44s), which lacks v2-vl0, is themost common form on hematopoietic cells, while larger CD44 splice variants dominate on several normal and on neoplastic epithelia. In normal colorectal epithelium, for example, CD44 expression is low and confined to the base of the crypt, while during malignant transformation, the expression is greatly enhanced (Heider et al, 1993; Lesley et al, 1993; Wielenga et al,
1993; Fox et al, 1994). CD44 splice variants are also found on activated lymphocytes and malignant lymphomas (Arch et al, 1992; Koopman et al,
Cartilage link protein homology domain
Growth factors: HGF/SF FGF
P3 a a) 'JK% Plasma mantrane •
Figure 4. Schematical representation of the CD44 gene and its encoded proteins. The
extracellular domain and cytoplasmic tail of CD44 isoforms vary in size as the result of alternative splicing. The alternative spliced exons are indicated by open boxes. The human vl exon contains a stop codon. In the model of the potein, all putative glycosylation sites are indicated: 0-glycosylation(open circles);yV-glycosylation(closed circles); chondroitin sulfate (open squares); heparan sulfate chain (rod). As indicated, the heparan sulfate binding site in exon v3 has the ability to bind growth factors. In addition, the HA-binding sites (double lines); the disulfide bonds (S-S); the ankyrin binding site (...); the ezrin binding sites (—); the phosphorylation sites (P); and the putative interaction sites for SRC-family kinases, are indicated.
Factors that regulate expressionand alternative splicing of CD44 during physiological and non-physiological circumstances are still largely unknown. Activation of the AP-1 binding site in the CD44 promoter has been shown to induce CD44 expression (Lamb et al, 1997; Kogerman et al, 1996). CD44 overexpression was furthermore described in a embryonic intestinal cell line as result of ras and src transfections (Jamal et al, 1994). Finally, several growth factors were reported to induce upregulationof CD44v, as for example TGF-1 and PDGF with involvement of PI 3-kinase and PKC (Fichter et al,
1997) and HGF/SF (Hiscox et al, 1997). In epithelial cell lines, alternative splicing of CD44 is positively regulated by Trans- acting factors (König et al,
1996).
Functions of CD44.
CD44 is involved in a number of important biological processes, including embryogenesis (Wheatley et al, 1993 ; Ruiz et al. ,1995; Sherman et
al, 1998) hematopoiesis, angiogenesis (Trochon et al, 1996), lymphocyte
homing and activation (Arch et al, 1992; Koopman et al, 1990; Naujokas et
al., 1993), survival from apoptosis (Ayroldi et al, 1995), and tumor metastasis
(Lesley et al, 1993; Herrlich et al, 1993; Naor et al, 1997). Importantly, a number of studies have indicated that CD44 can function as a signal-transducing receptor. In lymphocytes, outside-in signaling through CD44 costimulates antigen-specific lymphocyte activation and proliferation. Furthermore, CD44 engagement leads to activation of integrins on the lymphocyte cell-surface (Miyakae/ al, 1990; Haynes etal, 1989; Shimizu et
al, 1989; Koopman et al, 1990).
A major obstacle towards a better insight into the functions of the heterogenous CD44 family is the thus far limited number of ligands that has been identified, the only extensively studied interaction being that between CD44 and hyaluronate (HA) (Lesley et al, 1993). CD44 acts as a major receptor for this glycosaminoclycan (GAG) that is abundant in the ECM of mesenchymal tissues and that is believed to play a regulating role in cell migration (Lesley et al, 1993; Knudson et al, 1993; Aruffo et al, 1990). Although the binding site for HA is located on the constant N-terminal part of CD44, present on all isoforms, HA-binding capacity is not a constant feature
of CD44, but is subject to complex regulation by mechanisms involving both alternative splicing, modulation of cytoskeletal interaction, and posttranslational modification of CD44 (Aruffo et al, 1990; Culty et al, 1990; Lesley et al, 1992; Bourguignon et al, 1993; Bennett et al, 1995a; Katoh et
al, 1995; Stamenkovic étal, 1991; Takahashi étal, 1996; Uffetal., 1995;
van der Voort et al, 1995; Liu et al, 1996; Sleeman et al, 1996; Pure et al, 1995) By altering cellular interactions with HA, CD44 may facilitate tumor metastasis at least at two distinct levels: It may promote cell migration through the ECM. Alternatively, it may facilitate rolling of tumor cells on HA expressed on the surface of vascular endothelium at inflammatory sites and thereby enhance extravasation (DeGrendele et al, 1996, 1997). Indeed, in studies by Bartolazzi et al (1994), HA binding of melanoma cell lines was reported to be essential for the metastasis promoting effect of CD44s. However, properties other than HA binding must also play a role, as the HA-binding capacity and metastatic potential of pancreatic carcinoma cell lines were not related (Sleeman et al, 1996). Other molecules that have been reported to interact with CD44 are collagen IV, fibronectin, serglycin, invariant chain, and osteopontin, (Jalkanen et al, 1992; Lesley et al, 1993; Toyama et
al, 1995; Weber et al, 1996). The role of CD44 in tumorigenesis.
Initial evidence for a role of CD44 in cancer comes from studies in human non-Hodgkin's lymphomas. In this disease, the expression of CD44 was found to be associated with tumor dissemination and unfavorable prognosis (Pals et al, 1989; Horst et al, 1990; Jalkanen et al, 1991; Stauder et al, 1995). In a study by Günthert et al. in 1991, specific CD44 splice variants containing exon v6 conferred metastatic potential to a non-metastatic rat pancreatic carcinoma cell line. This observation catalyzed a large number of studies of CD44 splice variant expression in a variety of epithelial malignancies, including colorectal cancer, breast cancer, cervical cancer and bladder cancer. In many epithelial cancers, increased levels of CD44 and/or different patterns of splice variants were found in tumors in comparison with their normal counterparts (Matsumura et al, 1992 ; Heider et al, 1993 ; Herrlich
Wielenga et al, 1993; Dali et al, 1994; Finn et al, 1994; Kim et al, 1994; Mulder ef a/., 1994; Coopère/al, 1995; Kaufmänner a/., 1995; Orzechowski
étal, 1995; Rodriguez et al, 1995; Stauder étal, 1995; Imazeki étal, 1996;
Yamaguchi et al., 1996). In colorectal cancer, as well as in breast cancer, enhanced CD44 expression was reported to be associated with unfavorable prognosis (Wielenga et al, 1993; Mulder et al, 1994; Kaufmann et al, 1995; Yamaguchi et al, 1996; Ropponen et al, 1998). It identifies patients with a high propensity to develop metastases, who may benefit from adjuvant therapy. The explanation for the promoting activity of CD44 on the process of tumor dissemination remains unclear but, as mentioned before, an altered tumor cell-ECM interaction could be responsible. Furthermore, recent studies have identified a novel function for CD44 that might be highly significant for tumorigenesis, i.e. binding and presentation of growth factors.
Interaction of CD44 with growth factors.
CD44 can be modified by both chondroitin- and heparan-sulfate side chains and is thus a "facultative" cell-surface proteoglycan (Jalkanen et al,
1988; Stamenkovic et al, 1989; Brown et al, 1991; Faassen et al, 1992; Screaton et al., 1992; Lesley et al., 1993). Recent studies have shown that these modifications are associated with CD44 isoforms containing the v3 alternative exon (Jackson et al, 1995), which encodes a consensus motif SGSG for glycosaminoclycan (Bourdon et al, 1987; Greenfield et al, 1999) addition, which is evolutionary conserved between rodents and man (Screaton et al,
1992; Tölg et al, 1993). Proteoglycans are believed to play an important regulatory role in cell growth and motility (Kjellén et al, 1991; Ruoslahti et
al, 1991; Schlessinger et al, 1995). They can bind growth factors via their
GAG side chains and target these factors to their high affinity signal transducing receptors. This process has been particularly well explored for fibroblast growth factor-2 (FGF-2). Binding of FGF-2 to the low affinity proteoglycan receptor on the cell-surface presumably allows more frequent encounters with the high affinity receptor and, furthermore, formation of a multivalent FGF-proteoglycancomplexmay be required for dimerizationof the high affinity signaling receptors (Ruoslahti et al, 1991; Schlessinger et al, 1995; Yayon et al, 1991). Interestingly, specific structural modifications of
heparan sulfate side chains appear to determine their affinity for a given heparin-binding growth factor. This creates a mechanism for cell or tissue selective growth factor binding (David et al, 1993; Lindahl et al, 1998; Tanaka et al, 1998). Altered composition and expression of cell-surface proteoglycans has been reported in tumors and has been implicated in tumor invasion and metastasis (Kjellén et al, 1991; David et al, 1993; van Muijen
étal, 1995).
As for CD44, Tanaka and colleagues have shown that heparan sulfate proteoglycan forms isolated from monocytes can present the chemokine macrophage inflammatory protein-lb to T-lymphocytes resulting in activation of T-cell integrins (Tanaka etal, 1993). Subsequent studies from Bennett and colleagues demonstrated that GAG-modified CD44 splice variants can bind FGF-2 (Bennett et al, 1995b), and presentation of FGF-2 by CD44v-HS expressed on the apical epidermal ridge appears to be involved in limb bud formation during embryogenesis (Sherman et al, 1998).
THE HGF/SF-C-MET-SIGNALING PATHWAY.
Among other molecules that have been implicated in tumor invasion and metastasis are c-Met and its ligand HGF/SF (Liu et al, 1992; Yamashita
et al, 1994; Boros et al, 1995; Di Renzo et al, 1995; Tuck et al, 1996).
HGF/SF induces epithelial cells to invade collagen matrices in vitro, and NIH-3T3 cells cotransfected with c-met and HGF/SF acquire an invasive and metastatic phenotype (Rong et al, 1992, 1994; Giordano et al, 1993). Furthermore, in HGF/SF-transgenicmice, tumorigenesis was reported in many different tissues including mammary glands, skeletal muscles, and melanocytes (Liang et al, 1996; Takayama et al, 1997). In human cancer, both HGF/SF and c-Met are often overexpressed, and germline mutations in the c-met gene have recently been reported in hereditary renal cancer (Schmidt et al, 1997). These mutations lead to an enhanced enzymatic activity of the c-Met tyrosine kinase (Jeffers et al, 1996a ).
The structure of c-Met and HGF/SF.
encodes a receptor tyrosine kinase of 190 kDa (Fig. 5) (Park et al, 1986). c-Met is synthesized as a 170 kDa precursor that is cleaved and glycosylated to give rise to a heterodimer (Giordano et al., 1989). This mature form of the receptor is composed of two disulfide-linked chains, i.e., an extracellular 50 kDa a-subunit and a transmembrane 145 kDa ß-a-subunit (Giordano et al, 1989) that is endowed with tyrosine kinase activity (Park et al, 1987, Giordano et al,
1988). Binding of the ligand HGF/SF results in activation of c-Met.
HGF/SF is a heparan-binding glycoprotein that consists of a 60 kDa a-chain and a 30 kDa ß-a-chain, linked by disulfide bonds (Fig.6) (Gherardi et al,
1989). HGF/SF belongs to the family of kringle proteins, characterized by triple disulfide loop structures (kringles) that mediate protein/protein interactions(Nakamuraet al, 1986). Like c-Met, HGF/SF is synthesized as a single chain precursor that is cleaved extracellularly by serine proteases including coagulation factor XII and HGF/SF activator (Miyazawa et al, 1993, 1996). In addition to the high affinity receptor c-Met, HGF/SF also binds to heparan sulfate (HS) proteoglycans on cell surfaces and in the extracelluar matrix (ECM). This binding, and possible sequestering and presentation of HGF/SF by HS- proteoglycans to c-Met, may enhance the activation of the HGF/SF - c-Met pathway (Zioncheck et al, 1995).
The expression of c-Met and HGF/SF.
Wereas c-Met is expressed on a variety of, predominantly epithelial, cells and is overexpressed on several carcinomas, HGF/SF is secreted by mesenchymal cells, like fibroblasts, vascular smooth muscle cells, and glial cells, as well as by macrophages and activated T-lymphocytes (Naidu et al,
1994, Rosen et al, 1990, 1994; Stoker et al, 1987). c-Met is expressed on normal intestinal epithelium and c-Met protein and mRNA are upregulated in colorectal tumorigenesis(Di Renzo et al, 1991,1995; Liu et al., 1992; Prat et
al, 1991). Inflammatory cytokines like II-la and II-Iß enhance HGF/SF
production by fibroblasts (Tamura et al, 1993), whereas TGF-ß inhibits the production (Gohda et al, 1992). Activation of the oncogenes ras and ret, may induce deregulated c-Met expression in carcinomas (Ivan et al, 1997).
Functions of the c-Met-HGF/SF pathway.
The HGF/SF-c-Met pathway induces growth and motility of target epithelial cells, endothelial cells, myoblasts, lymphocytes, and tumor cells (Stoker et al, 1987; Bottaro ef a/., 1991; Bussolino etal, 1992; Tajima et al,
1992; Weidner et al, 1990,1993; Donated al, 1994;Bladte/La/., 1995;Boros
et al., 1995 ; Brinkmann et al, 1995 ; Van der Voort et al, 1997). The pathway
is a key mediator of in mesenchymal-epithelial interactions that take place during embryogenesis (Wang et al, 1994; Sonnenberg et al, 1993), wound healing (Dignass et al, 1994), liver regeneration (Nakamura et al., 1986), and tumorigenesis (Tuck et al, 1996; Liuetal, 1992; Furukawa et al, 1995;Prat
et al, 1991; Di Renzo et al, 1991, 1995; Ebert et al, 1994). During these
processes, the c-Met pathway directs cells toward motility ("scattering"), by inducing the phosphorylation of ß-catenin and by downregulating cadherin-mediated adhesion (Shibamoto et al, 1994). Furthermore, it may mediate proliferation, and/or morphogenesis. These activities are not mutually exclusive: Studies using chimeric receptors containing the extracellular domains of other receptors fused to the cytoplasmic part of c-Met, demonstrate that c-Met can convey multiple biological functions (Weidner et al, 1993). Activation of the signaling pathways that propagate these different activities is determined at the receptor level by differential binding of cytoplasmic signaling proteins to phosphotyrosines in the cytoplasmic domain of c-Met (Koch et al, 1991; Ponzetto et al., 1994), or may be regulated more downstream.
HGF/SF induces intrinsic, tissue-specific morphogenic activities in a wide variety of epithelial cells that are grown in three-dimentional matrices. It induces colon cell lines to form crypt-like structures in (Brinkmann et al,
1995), and mammary epithelial cell lines (Brinkmann et al., 1995) and cultured mouse mammary glands (Yang et al, 1995) to form long branching ducts. Morphogenic activities of epithelial cells were reported to require ezrin, a cytoskeletal component that links the cell membrane to F-actin (Crepaldi et al,
1997). During mouse embryogenesis, c-Met mRNA is expressed in epithelial cells of various organs, while HGF/SF is present in surrounding mesenchymal tissue (Sonnenberg et al., 1993 ; Iyer et al., 1990; Kolatsi et al., 1997). HGF/SF as well as c-Met mutant mice show severe defects in liver development,
oc-chain
ß-chain
TK
DS
Figure 5. The structure of the receptor tyrosine kinase c-Met. The receptor is composed
of two disulfide- linked chains: an extracellular50 kDa a-subunit and a transmembrane 145 kDa ß-subunit. C-Met is synthesized as a 170 kDa precursor that is cleaved and posttranslationally glycosylated to give rise to the mature heterodimer with total
leading to death at E15, and in limb bud formation (Bladt et al., 1995; Schmidt
et al, 1995). The latter defect is caused by defective migration of myogenic
precursor cells.
The involvement of HGF/SF- c-Met in tumorigenesis was shown in several studies. For example, c-Met was isolated originally as the product of a human oncogene, Tpr-met, which encodes a consitutively dimerized/activated chimeric c-Met protein possessing transforming activity (Cooper et al, 1984; Rodrigues et ai, 1993). The generation of an autocrine loop as a result of
OC-chain
ß-chain
Figure 6. The structure of the HGF/SF heparin-binding growth factor. HGF/SF is a
glycoprotein that consists of a 60 kDa a-chain and a 30 kDa ß-chain, linked by disulfide bonds with triple disulfide loop structures (kringles) that mediate protein/protein and
coexpression of wild-type c-Met and HGF/SF molecules in the same cell is also oncogenic (Jeffers et al, 1996b). The tumorigenecity of both Tpr-Met and autocrine HGF/SF-Met signaling has been verified in transgenic mouse models, which develop tumors in many different tissues including mammary glands, skeletal muscles and melanocytes (Liang et al, 1996; Takayama et al. 1997). c-Met activationhas also been shown to promote the metastatic spread of cancer, a finding that most likely is due to its stimulatory effects on a variety of processes such as angiogenesis, cell motility, and protease secretion (Jeffers
et al, 1996a). Recently, missense mutations in c-Met were found to be
mutations deregulate the enzymatic activity of the receptor, thereby unleashing its oncogenic potential (Jeffers et al., 1997). Furthermore, the c-Met expression is upregulated in several carcinomas including those of the breast (Di Renzo
et ai, 1991; Tuck et al., 1996; Jin et al, 1997), kidney (Prat et al, 1991),
colorectum(DiRenzo^a/., 1991, 1995; Un et al, 1992; Prate/ al, 1991) and pancreas (Ebert et al, 1994; Furukawa et al, 1995). Congenital mutations in the tyrosine kinase domain of the c-Met gene, results in papillary renal carcinomas (Schmidt et al, 1997).
SCOPE OF THE THESIS
The aim of the work decribed in this thesis was to elucidate the role of CD44 in colorectal tumorigenesis and to assess the prognostic value of CD44 expression in patients with colorectal cancer.
CD44 splice variants play a causal role in the metastatic spread of cancer in animal models. In Chapter 2, we explored the expression of CD44 splice variants at the subsequent stages of colorectal tumor progression, i.e., in normal intestine, adenomas, and carcinomas. In Chapter 3, the relation between expression of the tumor suppressor gene p53 and CD44 splice variants containing v5 and v6 was studied.
We have previously provided evidence for an association of CD44v6 containing variants with tumor-related death in patients with colorectal cancer. In Chapter 4, we further investigated the prognostic significance of CD44 in relation to conventional prognosticators.
Overexpression of CD44 is an early event in the adenoma-carcinoma sequence. This suggests a link with disruption of APC tumor suppressor protein-mediated regulation of /?-catenin/Tcf-4 signaling, which is crucial in initiating tumorigenesis. In Chapter 5, we explore this hypothesis by analyzing CD44 expression in the intestinal mucosa of mice and humans with genetic defects in either APC or Tcf-4, leading to constitutive activation or blockade of the /î-catenin/Tcf-4 pathway, respectively.
The mechanism(s) by which CD44 promotes tumor growth and/or metastasis, is as yet poorly understood. Recent studies have shown that CD44 isoforms containing the alternatively spliced exon v3 carry heparan sulfate side
chains (HS) and are able to bind heparin-binding growth factors. In Chapter 6, we have explored the possibility of a physical and functional interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor kinase c-Met, since this pathway plays a key role in invasion and metastasis. In Chapter 7, we studied the expression of c-Met, HGF/SF and CD44-HS, along the adenoma-carcinoma sequence.
In Chapter 8 the literature on CD44 in colorectal cancer is reviewed and a model for CD44 functioning in colorectal tumorigenesis is proposed.
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