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Clinical Cancer Research 14, 995, February 15, 2008. doi: 10.1158/1078-0432.CCR-07-1588
© 2008 American Association for Cancer Research
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Human Cancer Biology

Differential Wnt Pathway Gene Expression and E-Cadherin Truncation in Sporadic Colorectal Cancers with and without Microsatellite Instability

Paloma Ortega1, Alberto Morán1, Carmen de Juan1, Cristina Frías1, Susana Hernández2, Jose-Antonio López-Asenjo2, Andrés Sánchez-Pernaute3, Antonio Torres3, Pilar Iniesta1 and Manuel Benito1

Authors' Affiliations: 1 Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense, and 2 Servicios de Anatomía Patológica and 3 Cirugía, Hospital Clínico San Carlos, Madrid, Spain

Requests for reprints: Manuel Benito, Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense, Madrid, Spain. Phone: 34-91-394-17-77; Fax: 34-91-394-17-79; E-mail: benito{at}farm.ucm.es.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Purpose: Alterations in the Wnt pathway play a major role in colorectal cancer with high (MSI-H) or low microsatellite instability (MSS/MSI-L). However, the differential impact of the Wnt pathway components on these tumors is poorly understood. MMP-3 (stromelysin-1) promoter is a target of the mutator phenotype in sporadic colorectal cancer. Among MMP-3 targets, we investigated E-cadherin integrity status in both groups of tumors. Because beta-catenin is the main effector of the Wnt pathway, we have also investigated the differential cellular status of beta-catenin.

Experimental Design: Expression profiles of 114 genes related to the Wnt pathway were analyzed by oligo microarrays in 48 tumors classified by their MSI status. In addition, we analyzed 48 sporadic colorectal cancers for E-cadherin integrity status. We performed investigation of beta-catenin and cyclin D1 by immunohistochemistry using tissue arrays containing 96 tumors.

Results: Our data show that a group of genes that negatively regulate Wnt signaling are downregulated in MSS/MSI-L as compared with MSI-H colorectal tumors. E-cadherin truncation was significantly higher in MSS/MSI-L as compared with MSI-H tumors. Moreover, MSI-H tumors showed low or null beta-catenin nuclear presence, whereas the group of tumors classified as MSS or MSI-L displayed a high content of the nuclear beta-catenin location.

Conclusions: Our results suggest that the differential expression of genes that negatively regulate the Wnt pathway, as well as the status of E-cadherin and beta-catenin in MSI-H or MSS/MSI-L colorectal tumors, shed some light on the different clinical behavior showed by the two groups.

Colorectal cancer (CRC) represents a major public health problem in Western countries. The colorectal tumorigenesis process is characterized by multiple mutations in common oncogenes and tumor suppressor genes, as well as genomic instability attributable to mismatch repair gene defects (1).

High instability in microsatellite sequences (MSI-H) is a characteristic molecular finding in most cases of hereditary nonpolyposis colorectal cancer (HNPCC) and in nearly 13% of all cases of sporadic CRC (2–4). Microsatellite instability (MSI) evolves through mutations or epigenetic alterations of the DNA mismatch repair genes and constitutes a very early event in both HNPCC and sporadic colorectal tumorigenesis (5). Carcinomas with MSI develop an increased intrinsic mutation rate, and it has been suggested that MSI-positive and MSI-negative colorectal tumors might evolve through different molecular mechanisms and pathways (6, 7). CRCs showing MSI-H display distinctive pathologic features and behave less aggressively than microsatellite stable (MSS)- or low-microsatellite-instability (MSI-L) colorectal tumors. Thus, similar to HNPCC tumors, sporadic MSI-H colorectal tumors seem to have a better prognosis, and most of them are localized to the right colon (8, 9). Moreover, right-sided tumors and MSI-H CRCs have had a better response to adjuvant chemotherapy (10). At present, the molecular basis for the different clinical behavior of MSI-H and MSS or MSI-L CRC is not completely understood.

It has been proposed that alterations in the Wnt signaling pathway may be involved in carcinogenesis of CRC with MSI (11). However, to date, no expression analyses on the Wnt pathway components in these tumors have been reported. In addition, previous works have shown that the promoter of the MMP-3 gene (stromelysin-1) is a target of the mutator phenotype (12, 13). Thus, in MSI-H tumors, MMP-3 expression is impaired due to a characteristic mutation of the mutator phenotype. This reduced expression allows that molecules that are usually cleaved by MMP-3 maintain their integrity status, as we have shown with MMP-9, one of the proteolytic targets of MMP-3. Another important proteolytic target of MMP-3 is the adhesion protein E-cadherin (120 kDa). E-cadherin is a single transmembrane domain protein with an extracellular N-terminus and an intracellular C-terminus (14, 15). E-cadherin is bound indirectly to the cytoskeleton, via a linkage of its intracellular C-terminal portion to β-catenin, which in turn, is linked to alpha-catenin, which binds the actin cytoskeleton. MMP-3 cleaves the extracellular domain of E-cadherin, originating two fragments, one of them soluble, of about 80 kDa, and the other one bound to the cytoplasmic membrane, of about 40 kDa (16, 17). E-cadherin was initially considered a structural protein. However, it also acts as a tumor suppressor with expression that is silenced in many types of cancers. Its loss permits or enhances the invasion of adjacent normal tissues and its re-expression can induce morphologic reversion (14, 18). More recently, it has also been recognized to regulate signaling. Thus, it is implicated in the Wnt signaling pathway through its relation with beta-catenin, a key component of this pathway (19, 20). Dissociation of beta-catenin and E-cadherin can be mediated by MMPs, and this dissociation can lead to accumulate beta-catenin in the cytoplasm. This accumulation increases the formation of beta-catenin/LEF-1 DNA complexes within the nucleus (21).

Our main interest is focused evaluating molecular differences between CRCs with and without MSI that could be useful to better understand the differential clinical behavior of MSI-L/MSS and MSI-H CRCs. Thus, we hypothesized that the better prognosis associated with MSI-H CRCs may be related to the Wnt pathway function. Thus, in this work, we investigated the Wnt pathway gene expression in a sporadic CRC population previously classified in the function of its MSI status. Following, considering connexion between E-cadherin and Wnt pathway, as well as data from previous works from our group, we determined the differential E-cadherin integrity status in colorectal tumors from the mutator pathway (MSI-H) as compared with stable or low MSI (MSS/MSI-L) colorectal tumors. Due to the important link between E-cadherin and beta-catenin and because beta-catenin is the main effector of the Wnt pathway, we have also investigated the expression and cellular localization of beta-catenin and its transcriptional target cyclin-D1 in tumor cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Patients and tissue samples
Every tumor sample analyzed in this work corresponded to primary sporadic colorectal adenocarcinomas that were obtained from patients who underwent surgery between 2000 and 2005, at San Carlos Hospital in Madrid, Spain. In all cases, the corresponding normal tissues were also obtained in order to be used as control samples. Previously to surgery, none of these patients had received adjuvant treatment. Informed consent was obtained from patients prior to investigation, and this study was approved by the hospital's ethical committee. After surgical resection, all tissue samples were snap-frozen in liquid nitrogen and stored at –80°C until processed. Cryostat sectioned, H & E stained samples from each tumor block were examined microscopically by two independent pathologists to confirm the presence of more than 80% tumor cells. Paired normal tissues from the same patient, used as controls, were obtained at least 10 cm away from the margin of the tumor and microscopically confirmed.

Wnt signaling pathway expression assays
Gene expression screening by Wnt arrays. This study was established for 48 CRCs in which we had previously determined the MSI status. Thus, according to established criteria (12), 37 tumors were classified as MSS/MSI-L, and 11 were MSI-H. We also evaluated the expression profile of a pool obtained from the corresponding normal samples. Thus, the expression profile of 114 genes related to the Wnt signaling pathway was analyzed by using the Wnt Signaling Pathway Oligo GEArray in HybTube Format (OHS-043; SuperArray Bioscience Corporation) according to the manufacturer's instructions. To normalize results, we referred expression data obtained from these genes to expression of housekeeping GAPDH, also included in the arrays. Thus, genes showed a more constant expression value in tumor samples.

Gene expression analysis by real-time quantitative PCR. Expression data from arrays were confirmed investigating gene expression by real-time quantitative PCR, using Taqman probes (MGB "Assay on Demand" probes; Applied Biosystems, NJ) following the manufacturer's instructions. The study was performed for those genes showing expression differences of ± threefold in tumors as comparing with the control pool in the array analysis. Some interesting genes with expression differences between ± two- and ± threefold were also included. The comparative threshold cycle (Ct) method was used to calculate the relative expression. For quantification of gene expression, the target genes values were normalized to the expression of the endogenous reference (GAPDH). Thus, the amount of target, normalized to GAPDH and relative to a calibrator (normal pool expression), is given by 2–{Delta}{Delta}Ct [{Delta}Ct = Ct (target gene) – Ct (GAPDH); {Delta}{Delta}Ct = {Delta}Ct for any sample – {Delta}Ct for the calibrator].

Analysis of E-cadherin expression by Western blotting
Forty-eight sporadic colorectal tumors were subjected to Western-blotting analysis to analyze E-cadherin expression. Thirty-seven cases were MSS/MSI-L tumors, and 11 corresponded to the group showing MSI-H tumors. Briefly, after SDS-PAGE, proteins from tumors and their corresponding normal controls were transferred to immobilon membranes and blocked using 5% nonfat dried milk in 10 mM Tris/HCl, 150 mM NaCl, pH 7.5. Incubation with E-cadherin antibody (mouse anti-human, clone NCH-38; ref M3612, DAKO, Glostrup, Denmark) was performed in 0.05% Tween 20, 10 mM Tris/HCl, 150 mM NaCl, pH 7.5. Immunoreactive bands were visualized using the ECL Western blotting protocol (Amersham Biosciences). E-cadherin antibody recognizes the mature 120-kDa form and the fragment of 40 kDa of E-cadherin. Normalization with beta-actin was performed.

Tissue array study of beta-catenin, E-cadherin, and cyclin D1 expression by immunohistochemistry
These analyses were performed in 96 sporadic colorectal tumors, and all were previously classified in our laboratory as MSS/MSI-L or MSI-H tumors (12). This tumor population included the 48 cases in which Wnt pathway and E-cadherin had previously been evaluated.

In order to perform the immunohistochemistry study, we elaborated two tissue-array blocks, using a MTA1 arrayer (Beecher Instruments Inc.) Each block contained 110 cylinders of tissue; 48 corresponded to samples and seven to controls, all of them duplicated. These tissue cylinders (tissue "donors") were included into paraffin blocks ("receptors"). We used 1-mm-diameter needles for picking up the cylinders. Cylinders of tissue embedded in paraffin were separated by 1.5 mm between them in both the X- and Y-axis. Blocks were heated for 30 minutes at 37°C and then cooled at 4°C. We made 4-µm-thick cuts with a Leica RM2145 microtome. Sections were mounted onto sialinized slides for optimum tissue fixing. Results for beta-catenin were expressed as percentage of stained nuclei. Results for E-cadherin and cyclin D1 cellular status were expressed as positive or negative, considering that more than 10% of staining was considered as positive case. The E-cadherin antibody used for tissue arrays was mouse antihuman, clone NHC-38, dilution 1:50 (DAKO). Antibodies for cyclin D1 and beta-catenin were purchased from Novocastra Laboratories Ltd. (Newcastle upon Tyne, United Kingdom): cyclin D1, clone P2D111F11 ready to use; beta-catenin clone 17c2, dilution 1:25.

Statistical analysis
We performed statistical analyses using windows SPSS version 11.5 software. Associations of categorical variables were assessed using the {chi}2 test. The differences among multiple groups were analyzed by ANOVA, Student t, and Mann-Whitney tests. Correlations were evaluated by Pearson's test. P < 0.05 was judged significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Differential Wnt pathway gene expression in MSS/MSI-L and MSI-H colorectal tumors. Expression profile analyses of genes related to the Wnt signaling pathway were performed in 48 tumor samples and in a pool of the corresponding normal tissues (Fig. 1 ; Table 1 ). Expression differences between profiles obtained from MSI-H and MSS/MSI-L tumor populations were established. The results indicated that 10 genes showed an overexpression higher than threefold in the MSI-H tumor group compared with MSS/MSI-L cancers (Table 2 ). These genes were CTNNB1 (catenin beta 1), DVL2 (dishevelled, dsh homolog 2), FRAT2 (frequently rearranged in advanced T-cell lymphomas 2), GSK3B (glycogen synthase kinase 3 beta), KREMEN2 (kringle-containing transmembrane protein 2), PPP2R1B (protein phosphatase 2 regulatory subunit A, beta isoform), FBXW4 (F-box and WD repeat domain containing), TLE2 (transducin-like enhancer of split 2), WIF1 (WNT inhibitory factor 1), and CSNK1D (casein kinase 1 delta). All of these genes showed significant expression differences between the two groups of cancer considered in this work. Moreover, we detected significant differences in expression values in the case of TLE-3 (transducin-like enhancer of split 3), CTNNBIP1 (catenin-beta interacting protein 1), and CXXC4 (CXXC finger 4; Table 2).


Figure 1
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  		Fig. 1. A, B, representative array results of the two CRCs subgroups established in function of MSI status. A, tumors showing MSI-H. B, tumors showing null or MSS/MSI-L.

 

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  		Table 1. Genes included in expression analyses by arrays

 

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  		Table 2. Gene expression screening by Wnt arrays: significant differences between CRCs with or without MSI

 
In order to validate these results, qRT-PCR was performed for the genes mentioned above. With this technique, we confirmed significant differential expression values for DVL2, KREMEN2, PPP2R1B, FBXW4, CSNK1D, and TLE3. Transcriptional expression for all of these genes was higher in MSI-H tumors, as compared with the MSS/MSI-L group (Table 3 ). If we considered expression values for these genes after GAPDH normalization, we found that MSI-H CRCs showed expression profiles nearly to the values detected in the pool of control samples. However, in MSS/MSI-L tumors, expression levels significantly diminished in relation to normal samples. Therefore, sporadic CRCs from the mutator phenotype pathway and normal colorectal mucosa displayed similar transcriptional profiles for genes described above. In contrast, CRCs from the suppressor pathway showed downregulated transcriptional profiles. Of interest, KREMEN2 displayed undetectable expression levels in the control tissues. Therefore, transcriptional profiles for this gene are shown in Table 3 as {Delta}Ct values.


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  		Table 3. Significant Wnt expression differences between MSS/MSI-L and MSI-H colorectal tumors, after validation by qRT-PCR

 
Differential E-cadherin truncation in MSS/MSI-L and MSI-H colorectal tumors. We next investigated E-cadherin truncation in 48 CRCs. Thirty-seven of these tumors corresponded to the suppressor pathway (MSS or MSI-L tumors), and 11 corresponded to the mutator phenotype pathway (MSI-H tumors). To perform the analysis, we considered that E-cadherin is an important proteolytic target of MMP-3 and, as our group showed previously, MMP-3 expression is impaired due to a characteristic mutation of the mutator phenotype in MSI-H tumors (12, 13). Moreover, E-cadherin has been implicated in the Wnt signaling pathway through its relation with beta-catenin, a key component of this pathway. Thus, by Western-blot analysis (Fig. 2 ), we were able to detect the entire E-cadherin form (120 kDa) and the truncated form (about 40 kDa) that remains bound to the membrane after the cleavage. We used the concept of E-cadherin inactivation, defined as the relation between the densitometry values of the 40-kDa and the 120-kDa bands (22). Then, we calculated the relation between the E-cadherin inactivation of the normal tissue control and its corresponding tumor samples. If this ratio is higher than one, it means that there is low E-cadherin inactivation in tumor cells. If this ratio is lower than one, there is high E-cadherin inactivation in tumor cells. The values obtained for this ratio were considerably higher than one for the group of MSI-H tumors (1.85 ± 0.73). In contrast, the ratio was about one for MSS/MSI-L tumors (1.08 ± 0.74; Table 4 ). Differences between the two groups of cancers were statistically significant (P = 0.008). The inactivation ratio was higher than one in every MSI-H tumor. However, the MSS/MSI-L population presented a higher heterogeneity although most tumors showed a ratio lower than one. These results correlated to MMP-3 expression levels that we analyzed previously (12). Thus, MSI-L/MSS tumors showed significantly higher MMP-3 expression than those tumors from the mutator phenotype pathway (MSI-H) (P = 0.03). Of interest, tumors in which the active E-cadherin form was detected conferred a clinical outcome significantly better than those showing E-cadherin truncation (data not shown; P = 0.03, by log-rank test).


Figure 2
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  		Fig. 2. Western blot of E-cadherin, showing the 120-kDa E-cadherin form and the truncated form (about 40 kDa). Normalization with beta-actin was performed.

 

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  		Table 4. Relation between E-cadherin truncation values of the control tissues and the corresponding tumor samples

 
Differential beta-catenin and cyclin-D1 expression within the nucleus. Next, we developed two tissue arrays, each one containing 48 tumor samples and seven controls, all of which were duplicated. On these arrays, we performed an immunohistochemistry study using specific antibodies for E-cadherin and beta-catenin, to assess their cellular status. We detected a significant correlation between the presence of E-cadherin active form and membrane location for beta-catenin (P = 0.01), as expected. Also, positive staining for E-cadherin in membrane correlates with lack of beta-catening nuclear staining (P = 0.03). We found no differences in E-cadherin staining between MSI-H, MSS/MSI-L tumors, and the control tissue. Our data also indicated significant differences between the two groups of CRCs considered (MSS/MSI-L and MSI-H) regarding beta-catenin nuclear status (Fig. 3A ). Thus, tumors from the mutator phenotype pathway showed low or null beta-catenin nuclear presence (the mean of stained nuclei was 6.25 ± 3.31% of stained nuclei, range 0-30%). By contrast, the group of tumors classified as MSS or MSI-L displayed a high content of beta-catenin immunopositive nuclei (mean of stained nuclei 25.65 ± 3.62%, range 0-80%). The difference between both groups reached statistical significance (P = 0.027).


Figure 3
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  		Fig. 3. A, representative tissue array results for beta-catenin detection by immunohistochemistry. Nuclear localization for beta-catenin was detected for MSS/MSI-L tumors but not for MSI-H tumors or control tissues. B, expression of cyclin-D1 by immunohistochemistry using tissue arrays. As it is shown, cyclin-D1 expression levels were null or low in MSI-H tumors, whereas higher expression signals were obtained in MSS/MSI-L tumors.

 
We then tried to determine if a transcriptional product of the Wnt signaling pathway, cyclin D1, was differentially expressed in MSI-H tumors versus MSS/MSI-L tumors. We performed an immunohistochemistry study using specific antibodies on the tissue arrays previously developed (Fig. 3B). These experiments showed a borderline correlation between beta-catenin and cyclin D1 expression nuclear status in colorectal tumors. Thus, 70% of tumors without significant beta-catenin presence in the nucleus showed a low expression of cyclin D1, whereas 43% of positive beta-catenin nuclear staining cases presented a positive cyclin D1 expression (P = 0.05). In addition, a trend to correlation was detected between E-cadherin inactivation, as indicated Western-blot experiments and high levels of cyclin D1 expression (P = 0.1).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
The Wnt signaling pathway is essential in different biological processes. Misregulation of this pathway is a hallmark of many human cancers, including colorectal carcinomas (23, 24). Briefly, in the absence of a Wnt signal, cells degrade beta-catenin by a multiprotein complex consisting of the adenomatous polyposis coli (APC) tumor suppressor protein, axin and the glycogen synthase kinase (GSK3β). This kinase phosphorylates beta-catenin marking it for subsequent ubiquitination and degradation. When the Wnt ligand docks to its Frizzled (Fz) receptor, the degradation complex is destabilized, and beta-catenin accumulates in the cytoplasm and translocates to the nucleus, where it functions as a cofactor for TCF/LEF (T-cell factor/lymphoid-enhancing factor). There are positive and negative regulators of the pathway in most of its steps. The vast majority of colorectal tumors contain sporadic or inherited truncations of the APC tumor suppressor (25) that remove the binding sites for axin and destabilize the destruction complex resulting in the permanent induction of Wnt target genes, such as c-Myc (26, 27). Stabilizing mutations in beta-catenin are also frequent in cancer and have the same final effect on Wnt target genes. However, the impact of other components of the Wnt pathway on colorectal tumors is poorly understood. Recent data support the hypothesis that Wnt/beta-catenin gene expression may contribute to the malignant progression of CRC (28).

Data reported here clearly show that a group of genes directly or indirectly related to the Wnt pathway are downregulated in MSS/MSI-L as compared with MSI-H colorectal tumors. Thus, the expression of PPP2R1B (protein phosphatase 2 regulatory subunit A, beta isoform) is downregulated in MSS/MSI-L as compared with MSI-H colorectal tumors. PP2A is a serine-threonine phosphatase that has been reported to bind to axin, a component of the beta-catenin degradation complex axin-APC-GSK3beta-beta-catenin (29, 30). PP2A acts as a tumor-suppressor gene (31, 32), through a mechanism not completely elucidated. CSNK1D (casein kinase 1 delta) is a member of the casein kinase I family (CKI). CKI phosphorylates APC, axin, and beta-catenin. CKI phosphorylates beta-catenin serine 45 residue, which allows its progressive phosphorylation by GSK3beta and ubiquitinization (33). Thus, the downregulation of CSNK1D in MSS/MSI-L versus MSI-H colorectal tumors may contribute to the differential activation of the Wnt signaling in both groups of tumors. TLE-3 (transducin-like enhancer of split 3) is a transcriptional repressor that binds to TCF/LEF1 within the nucleus in the absence of Wnt signaling (34). Thus, stable colorectal tumors showing lower expression levels of TLE3 as compared with unstable tumors, would have a more active TCF/beta-catenin nuclear transcriptional complex. DVL2, in its phosphorylated form, docks to the Fz receptor leading to its endocytosis (35). Thus, lower expression of DVL2 would induce the Wnt signaling as comparatively observed in MSS/MSI-L versus MSI-H colorectal tumors. FBXW4 (F-box and WD repeat domain containing), as other F-box-containing proteins, is involved in ubiquitination-dependent proteolysis (36). Thus, a lower expression of FBXW4 would imply a higher availability of beta-catenin, as it seems to be the case in MSS/MSI-L colorectal tumors described above. Finally, KREMEN2 forms a ternary complex with DKK1/DKK2 (Dickkopf 1/2) and low-density lipoproteins receptors LRP5-LRP6 (co-receptors of the Fz receptor). This fact allows coreceptor internalization and causes Wnt pathway inactivation (37). Thus, the lower expression of KREMEN2 observed in MSS/MSI-L tumors would result in an enhanced Wnt signaling. Overall, colorectal tumors without MSS/MSI-L downregulate a group of genes that negatively regulate Wnt signaling, as compared with unstable tumors (MSI-H). These results are entirely consistent with the much poorer prognosis of MSS/MSI-L versus MSI-H colorectal tumors described earlier.

We previously reported the occurrence of mutations characteristic of the mutator phenotype in the promoter of the matrix metalloproteinase-3 in MSI-H tumors. These mutations, which were completely absent in MSS/MSI-L tumors, cause a decrease in protein expression of MMP-3. This decrease may affect different proteolytic processes usually carried out by MMP-3, as the MMP-9 activation (12, 13). In this work, we have studied another proteolytic target of MMP-3, E-cadherin (16). E-cadherin is an important adhesion molecule closely related to tumoral processes (14, 38). Quite a few differential studies of E-cadherin in MSI-H colorectal tumors versus MSS/MSI-L colorectal tumors are available. Thus, previous works studied the expression of E-cadherin (39) or its promoter hypermethylation (40) in both groups of tumors. However, E-cadherin integrity status in both populations of colorectal tumors remained unknown. Our data showed marked differences between colorectal tumors with or without MSI regarding E-cadherin integrity status. MSI-H tumors showed little E-cadherin truncation, whereas truncation in MSS/MSI-L tumors was significantly higher. Based on the well-known molecular linkage between E-cadherin and beta-catenin and the relationship between beta-catenin and the Wnt pathway, we have also studied the cellular status of beta-catenin in both groups of colorectal tumors. Thus, we have detected an almost complete absence of nuclear beta-catenin in MSI-H tumors. In contrast, MSS/MSI-L tumors showed a significantly higher presence of beta-catenin within the nucleus. These data may be related to the maintenance of E-cadherin integrity status in MSI-H compared with MSS/MSI-L tumors. On the other hand, E-cadherin associates with beta-catenin at the plasmatic membrane. Thus, in DLD-1 CRC cells, which show constitutive activation of Wnt signaling and exhibit E-cadherin–based cell contacts, it has been described that knockdown of E-cadherin leads to translocation of beta-catenin to the nucleus and an enhancement of beta-catenin/TCF–dependent reporter activity (41). Moreover, Orsulic et al. (42) showed that E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1–mediated transactivation. Thus, truncation of E-cadherin or downregulation of E-cadherin may cooperate through the enhancement of cytosolic beta-catenin to its translocation to the nucleus. Previous works in CRC reported that increased nuclear beta-catenin expression and loss of membranous E-cadherin were independently associated with poor patient clinical outcome (43).

The dual role of beta-catenin in cell adhesion and Wnt signaling (effector) led us to study cyclin-D1 nuclear expression, a transcriptional target of beta-catenin/TCF heterodimer. Although at the border of significance, our results showed a trend toward correlation between beta-catenin and cyclin D1 expression nuclear status in colorectal tumors. Although this incomplete correlation is similar to information in a recent article by Herzig et al. (44), our results support the concept that that E-cadherin regulates tumor growth by modulating beta-catenin transcriptional activity (45). Thus, truncation of E-cadherin or downregulation of E-cadherin and upregulation of nuclear beta-catenin transcriptional activity may be cooperative in the pathogenesis of CRCs.


   Conclusion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
In conclusion, our results suggest that the differential expression of genes that negatively regulate the Wnt pathway, as well as the status of E-cadherin, beta-catenin, and cyclin-D1 in MSI-H or MSS/MSI-L colorectal tumors shed light on the different clinical behavior showed by the two tumor groups. In addition, our data could be useful to establish therapeutic protocols based on the beta-catenin nuclear status.


   Footnotes
 
Grant support: Ministerio de Sanidad y Consumo (FIS PI050039) and Fundación de Investigación Médica Mutua Madrileña.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: P. Ortega and A. Morán equally contributed to this work.

Received 6/28/07; revised 10/15/07; accepted 10/22/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

  1. Navaratnam RM, Chowaniec J, Winslet MC. The molecular biology of colorectal cancer development and the associated genetic events. Ann R Coll Surg Engl 1999;81:312–9.[Medline]
  2. Tannergard T, Liu T, Weger A, Nordenskjöld M, Lindblom A. Tumorigenesis in colorectal tumors from patients with hereditary non-polyposis colorectal cancer. Hum Genet 1997;101:51–5.[CrossRef][Medline]
  3. Salahshor S, Koelble K, Rubio C, Lindblom A. Microsatellite instability and hMLH1 and hMSH2 expression analysis in familial and sporadic colorectal cancer. Lab Invest 2001;81:535–41.[Medline]
  4. Thibodeau S, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993;260:816–19.[Abstract/Free Full Text]
  5. Pedroni M, Sala E, Scarselli A, et al. Microsatellite instability and mismatch-repair protein expression in hereditary and sporadic colorectal carcinogenesis. Cancer Res 2001;61:896–9.[Abstract/Free Full Text]
  6. Breivik J, Gaudernack G. DNA methylation and natural selection in colorectal carcinogenesis. Semin Cancer Biol 1999;9:245–54.[CrossRef][Medline]
  7. Salahshor S, Kressner U, Pahlman L, Glimelius B, Lindmark G, Lindblom A. Colorectal cancer with and without microsatellite instability involves different genes. Genes Chromosomes Cancer 1999;26:247–52.[CrossRef][Medline]
  8. Gafà R, Maestry I, Mattenzzi M, et al. Sporadic colorectal adenocarcinomas with high-frequency microsatellite instability. Cancer 2000;89:2025–37.[CrossRef][Medline]
  9. Gryfe R, Kim K, Hsieh ET, et al. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 2000;342:69–77.[Abstract/Free Full Text]
  10. Elsaleh H, Joseph D, Grieu F, Zeps N, Spry N, Iacopetta B. Association of tumor site and sex with survival benefit from adjuvant chemotherapy in colorectal cancer. Lancet 2000;355:1745–50.[CrossRef][Medline]
  11. Shimizu Y, Ikeda S, Fujimori M, et al. Frequent alterations in the Wnt signaling pathway in colorectal cancer with microsatellite instability. Genes Chromosomes Cancer 2002;33:73–81.[CrossRef][Medline]
  12. Moran A, Iniesta P, de Juan C, et al. Stromelysin-1 promoter mutations impair gelatinase B activation in high microsatellite instability sporadic colorectal tumors. Cancer Res 2002;62:3855–60.[Abstract/Free Full Text]
  13. Moran A, Iniesta P, de Juan C, García-Aranda C, Díaz-López A, Benito M. Impairment of stromelysin-1 transcriptional activity by promoter mutations in high microsatellite instability colorectal tumors. Cancer Res 2005;65:3811–4.[Abstract/Free Full Text]
  14. Hajra KM, Fearon ER. Cadherin and catenin alterations in human cancer. Genes Chromosomes Cancer 2002;34:255–68.[CrossRef][Medline]
  15. Wheelock MJ, Johnson KR. Cadherins as modulators of cellular phenotype. Annu Rev Cell Dev Biol 2003;19:207–35.[CrossRef][Medline]
  16. Noe V, Fingleton B, Jacobs K, et al. Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1. J Cell Sci 2001;114:111–8.[Abstract]
  17. Davies G, Jiang WG, Mason MD. Matrilysin mediates extracellular cleavage of E-cadherin from prostate cancer cells: a key mechanism in hepatocytes growth factor/scatter factor-induced cell-cell dissociation and in vitro invasion. Clin Cancer Res 2001;7:3289–97.[Abstract/Free Full Text]
  18. Perl AK, Wilgenbus P, Dahl U, Semb H, Christofori G. A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature 1998;392:190–3.[CrossRef][Medline]
  19. Sharpe C, Lawrence N, Martinez-Arias A. Wnt signaling: a theme with nuclear variations. BioEssays 2001;23:311–8.[CrossRef][Medline]
  20. Brembeck FH, Rosario M, Birchmeier W. Balancing cell adhesion and Wnt signaling, the key role of beta-catenin. Curr Opin Genet Dev 2006;16:51–9.[CrossRef][Medline]
  21. Mei JM, Borchert GL, Donald SP, Phang JM. Matrix metalloproteinase(s) mediate(s) NO-induced dissociation of beta-catenin from membrane bound E-cadherin and formation of nuclear beta-catenin/LEF-1 complex. Carcinogenesis 2002;23:2119–22.[Abstract/Free Full Text]
  22. Rashid MG, Sanda MG, Vallorosi CJ, Rios-Doria J, Rubin MA, Day ML. Posttranslational truncation and inactivation of human E-cadherin distinguishes prostate cancer from matched normal prostate. Cancer Res 2001;61:489–92.[Abstract/Free Full Text]
  23. Moon RT, Kohn AD, De Ferrari GV, Kaykas A. Wnt and beta-catenin signaling: diseases and therapies. Nat Rev Genet 2004;5:691–701.[CrossRef][Medline]
  24. Reya T, Clevers H. Wnt signaling in stem cells and cancer. Nature 2005;434:843–50.[CrossRef][Medline]
  25. Segditsas S, Tomlinson I. Colorectal cancer and genetic alterations in the Wnt pathway. Oncogene 2006;25:7531–7.[CrossRef][Medline]
  26. Bienz M. The subcellular destinations of APC proteins. Nat Rev Mol Cell Biol 2002;3:328–38.[CrossRef][Medline]
  27. Henderson BR, Fagotto F. The ins and outs of APC and beta-catenin nuclear transport. EMBO Rep 2002;3:834–9.[CrossRef][Medline]
  28. Hlubek F, Brabletz T, Budczies J, Pfeiffer S, Jung A, Kirchner T. Heterogeneous expression of Wnt/beta-catenin target genes within colorectal cancer. Int J Cancer 2007;121:1941–8.[CrossRef][Medline]
  29. Hsu W, Zeng L, Costantini F. Identification of a domain of Axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain. J Biol Chem 1999;274:3439–45.[Abstract/Free Full Text]
  30. Ikeda S, Kishida M, Matsuura Y, Usui H, Kikuchi A. GSK-3beta-dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by beta-catenin and protein phosphatase 2A complexed with Axin. Oncogene 2000;19:537–45.[CrossRef][Medline]
  31. Calin GA, di Iasio MG, Caprini E, et al. Low frequency of alterations of the {alpha} (PPP2R1A) and β (PPP2R1B) isoforms of the subunit A of the serine-threonine phosphatase 2A in human neoplasms. Oncogene 2000;19:1191–5.[CrossRef][Medline]
  32. Polakis P. The many ways of Wnt in cancer. Curr Opin Genet Dev 2007;17:45–51.[CrossRef][Medline]
  33. Gao ZH, Seeling JM, Hill V, Yochum A, Virshup DM. Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex. Proc Natl Acad Sci U S A 2002;99:1182–7.[Abstract/Free Full Text]
  34. Daniels DL, Weis WI. Beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation. Nat Struct Mol Biol 2005;12:364–71.[CrossRef][Medline]
  35. Chen W, ten Berge D, Brown J, et al. Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4. Science 2003;301:1391–4.[Abstract/Free Full Text]
  36. Patton EE, Willems AR, Tyers M. Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis. Trends Genet 1998;14:236–43.[CrossRef][Medline]
  37. Mao B, Wu W, Davidson G, et al. Kremen proteins are Dickkopf receptors that regulate Wnt/β-catenin signaling. Nature 2002;417:664–7.[CrossRef][Medline]
  38. Bates RC, Mercurio AM. The epithelial-mesenchymal transition (EMT) and colorectal cancer progression. Cancer Biol Ther 2005;4:365–70.[Medline]
  39. Kim JC, Koo KH, Kim HC, Kim JS, Kang GH. Geno- and pheno-typic characterization in ten patients with double-primary gastric and colorectal adenocarcinomas. Int J Colorectal Dis 2004;19:561–8.[CrossRef][Medline]
  40. Kim H, Kim YH, Kim SE, Kim NG, Noh SH, Kim H. Concerted promoter hypermethylation of hMLH1, p16INK4A, and E-cadherin in gastric carcinomas with microsatellite instability. J Pathol 2003;200:23–31.[CrossRef][Medline]
  41. Kuphal F, Behrens J. E-cadherin modulates Wnt-dependent transcription in colorectal cancer cells but does not alter Wnt-independent gene expression in fibroblasts. Exp Cell Res 2006;312:457–67.[Medline]
  42. Orsulic S, Huber O, Aberle H, Arnold S, Kemler R. E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. J Cell Sci 1999;112:1237–45.[Abstract]
  43. Lugli A, Zlobec I, Mingo P, et al. Prognostic significance of the wnt signalling pathway molecules APC, beta-catenin and E-cadherin in colorectal cancer: a tissue microarray-based analysis. Histopathology 2007;50:453–64.[CrossRef][Medline]
  44. Herzig M, Savarese F, Novatchkova M, Semb H, Christofori G. Tumor progression induced by the loss of E-cadherin independent of beta-catenin/Tcf-mediated Wnt signaling. Oncogene 2007;26:2290–8.[CrossRef][Medline]
  45. Stockinger A, Eger A, Wolf J, Beug H, Foisner R. E-cadherin regulates cell growth by modulating proliferation-dependent beta-catenin transcriptional activity. J Cell Biol 2001;154:1185–96.[Abstract/Free Full Text]



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