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[Cancer Research 64, 6973-6977, October 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Down-Regulation of the Homeodomain Factor Cdx2 in Colorectal Cancer by Collagen Type I

An Active Role for the Tumor Environment in Malignant Tumor Progression

Thomas Brabletz1, Simone Spaderna1, Jochen Kolb1, Falk Hlubek1, Gerhard Faller1, Christiane J. Bruns2, Andreas Jung1, Jens Nentwich1, Isabelle Duluc3, Claire Domon-Dell3, Thomas Kirchner1 and Jean-Noel Freund3

1 Department of Pathology, University of Erlangen-Nürnberg, Erlangen, Germany; 2 Department of Surgery, University of Munich, Munich, Germany; and 3 INSERM, Unit 381, Strasbourg, France


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The homeobox transcription factor Cdx2 specifies intestinal development and homeostasis and is considered a tumor suppressor in colorectal carcinogenesis. However, Cdx2 mutations are rarely found. Invasion of colorectal cancer is characterized by a transient loss of differentiation and nuclear accumulation of the oncoprotein ß-catenin in budding tumor cells. Strikingly, this is reversed in growing metastases, indicating that tumor progression is a dynamic process that is not only driven by genetic alterations but also regulated by the tumor environment. Here we describe a transient loss of Cdx2 in budding tumor cells at the tumor host interface, and reexpression of Cdx2 in metastases. Cell culture experiments show that collagen type I, through ß1 integrin signaling, triggers a transient transcriptional down-regulation of Cdx2 and its intestine-specific target gene sucrase isomaltase, associated with a loss of differentiation. These data indicate an active role for the tumor environment in malignant tumor progression.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The hallmarks of malignant tumor transformation are invasion and metastasis. In colorectal adenocarcinomas, invasion is associated with budding of tumor cells at the tumor host interface. In contrast to tumor cells from central tumor areas, these cells show a loss of epithelial differentiation, resembling an epithelial-mesenchymal transition (1 , 2) . The grade of dedifferentiation (or budding) inversely correlates with clinical prognosis, demonstrating its significance for cancer progression (3) . However, epithelial re-differentiation is found again in growing metastases. This fact indicates that tumor progression is not a linear process that could be explained solely by accumulation of irreversible genetic alterations (4) . Rather, it is a dynamic process characterized by ongoing changes in tumor cell differentiation, pointing out an additional regulatory role for the tumor microenvironment. Recently, we were able to link distinct expression patterns of the oncoprotein ß-catenin to these phenotype switches: Membranous co-localization of ß-catenin and E-cadherin is detectable in differentiated central areas of primary tumors and again in metastasis. However, a number of transient molecular changes occur in the dedifferentiated tumor cells at the invasive front. These include nuclear accumulation of ß-catenin and cytoplasmic accumulation of its target gene, laminin-{gamma}2 chain (5) .

The caudal-related cdx2 homeobox gene encodes an intestine-specific transcription factor, which is crucial for development and homeostasis of the intestinal epithelium (6) . The homoeotic function of Cdx2 in defining intestinal identity is indicated by phenotypes resulting from Cdx2 haploinsuffiency and gain of function in mouse models. Cdx2+/– mice develop noncancerous hamartomous colon lesions characterized by gastric heteroplasia (7) , whereas ectopic expression of Cdx2 in the stomach triggers intestinal heterodifferentiation (8) . The function of Cdx2 in defining the intestinal phenotype is further supported by the observation that ectopic expression of Cdx2 is found in intestinal metaplasia of the gastric mucosa and the intestinal type of gastric cancers (9 , 10) . In adult tissue, Cdx2 inhibits cell growth and stimulates differentiation by activating intestine-specific genes, e.g., the enzyme sucrase isomaltase (SI; ref. 11 ). Additionally, an intestine-specific tumor suppressor role of Cdx2 has recently been demonstrated in mice. Indeed, we have shown that the reduced expression level of Cdx2 in Cdx2+/– facilitates tumor progression in a mouse model for sporadic colorectal cancer (12) , which was later confirmed by Aoki et al. (13) in a model for familial adenomatous polyposis. This role is also supported by the observation that Cdx2 expression decreases with tumor grade of human colorectal cancers and in chemically induced rat tumors (14) . However, genetic alterations affecting the cdx2 gene locus are rarely found in human colorectal neoplasia (15) . Thus, a regulatory process instead of genetic alterations might be responsible for changes in the Cdx2 expression level in most colorectal cancers.

The aim of this study was to identify microenvironmental regulators of tumor cell de- and re-differentiation in malignant tumor progression and define molecular characteristics of budding, dedifferentiated tumor cells at the invasive front. Therefore, differential expression of Cdx2 and a potential regulatory role of extracellular matrix factors were analyzed in colorectal cancers.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Immunohistochemistry and Immunocytochemistry.
Forty-five formalin-fixed colorectal carcinomas and corresponding metastases were retrieved from the archives of the Institute of Pathology, University of Erlangen-Nürnberg. Immunohistochemistry was performed as described previously (1) . Mouse monoclonal antibodies (mAbs) against laminin-{gamma}2 (1:100; Chemicon International, Temecula, CA) and Cdx2 (1:100; BioGenex Laboratories, San Ramon, CA) were used. For immunofluorescence histochemistry or cytochemistry, slides were pretreated with pronase for 10 minutes at 37°C for laminin/CK18 double staining or hylase for 60 minutes at 37°C for collagen type I/CK18 double staining. After two washes in PBS, slides were blocked for 30 minutes in 2% goat serum and incubated overnight with primary antibodies [rabbit anti-collagen type I (1:100; Quartett, Berlin, Germany), rabbit anti-laminin (1:50; Sigma, St. Louis, MO), and mouse mAb anti-CK18 (1:800; clone CY-90; Sigma)]. For immunocytochemistry, cells were directly fixed in the microwells [4% paraformaldehyde in PBS (pH 7.2)] for 30 minutes. After washing in PBS, cells were treated with 0.5% Triton X-100 for 5 minutes, blocked for 30 minutes with 2% goat serum, and incubated overnight at room temperature with primary antibodies [rabbit anti ß-catenin (1:1000; Sigma), mouse anti–E-cadherin (1:100; clone 36; Becton Dickinson, San Jose, CA)] diluted in 2% goat serum. After washing in PBS, cells or slides were incubated for 4 hours at room temperature in the dark with secondary antibodies [Alexa488-coupled goat antirabbit immunoglobulin (1:500; Molecular Probes, Eugene, OR) or Cy3-coupled goat antimouse immunoglobulin (1:500; Dianova, Hamburg, Germany)]. After two washes, nuclei were stained with Hoechst 33342 (Molecular Probes), washed twice, and coverplated.

Cell Culture, DNA Constructs, and Reporter Assays.
DLD1, Caco-2, and LS174T colorectal carcinoma cell lines and HEK293 epithelial cells (American Type Culture Collection, Manassas, VA) were grown and transfected with the indicated vectors as described previously (16) in 12-cluster well plates. DNA clones used were as follows: Cdx2 promoter (–907/+117) luciferase construct (named pCdx2–1luc) described previously (17) ; pcDNA3.1FAK and pcDNA3.1FRNK [encoding the dominant-negative COOH-terminal domain of focal adhesion kinase (FAK) called dnegFAK; provided by D. Schlaepfer]; and pcDNA3.1ILK and pcDNA3.1kinase dead integrin-linked kinase [ILK (ILKmut; provided by S. Dedhar)]. One hour after transfection, cells were harvested using 0.5 mmol/L EDTA in PBS, washed, and incubated on a shaker for 45 minutes at room temperature with a ß1 integrin blocking mAb (clone LIA 1/2; Immunotech, Krefeld, Germany) or control antibody (1:4 dilution in 1 mL of Dulbecco’s modified Eagle’s medium/10% fetal calf serum). Then the cell suspension (104 cells in 0.1 mL/well) was seeded directly onto the indicated extracellular matrix-coated microwells [coated with either purified human collagen type I, human laminin-1, or bovine serum albumin (BSA; Cytomatrix adhesion strips; Chemicon International)]. After 20 hours of exponential growth, cells were used for the various assays. Reporter assays were done as described previously (16) . Luciferase activity was normalized with Renilla activity of a cotransfected pCMV Renilla construct (Promega) for control of transfection efficiency. Experiments were done in triplicates.

Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction, RNA Interference, and Immunoblot.
Total cellular RNA was prepared using the RNAeasy kit (Qiagen, Chatsworth, CA) and reverse transcribed with oligo(dT)15 primers, random primers, and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). cDNA was used for quantitative real-time polymerase chain reaction analysis with SYBR Green polymerase chain reaction master mix (PE Biosystems, Darmstadt, Germany). Values for CDX2 (sense, 5'-CTGGAGCTGGAGAAGGAGTTTC; antisense, 5'-ATTTTAACCTGCCTCTCAGAGAGC) and SI (sense, 5'-CATCCTACCATGTCAAGAGCCAG; antisense, 5'-GCTTGTTAAGGTGGTCTGGTTTAAATT) were measured in triplicates using ABI PRISM 7700 and a Sequence Detector V1.7a program (PE Biosystems) and normalized to ß-actin expression as a housekeeping control (sense, 5'-TTGCGGATGTCCACGTCA; antisense, 5'-GCCCTGAGGCACTCTTCCA). To determine the absolute copy number of the target transcript, cloned plasmid DNAs were used to produce a standard curve. For specific knockdown, double-stranded small interfering RNA (siRNA) oligonucleotides for FAK (smart pool mix; Dharmacon, Lafayette, CO) or green fluorescent protein as control (sense, 5'-AAGCUACCUGUUCCAUGGCCAdTdT) were transfected in DLD1 cells using TransMessenger Transfection Reagent (Qiagen). Specific knockdown of FAK was shown by standard immunoblots using a mAb against FAK (clone H-1; 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and an anti ß-actin mAb as control (clone C-15; 1:1000; Sigma).

Orthotopic Animal Model.
Male athymic nude mice (Balb/C nu/nu) were purchased from Charles River Laboratories (Sulzfeld, Germany). The mice were housed and maintained in laminar flow cabinets under specific pathogen-free conditions and used between 8 and 12 weeks of age. For the orthotopic tumor cell injection, cells were harvested from subconfluent cultures by treatment with 0.25% trypsin and 0.02% EDTA. Trypsinization was stopped with medium containing 10% FBS, and the cells were washed once in serum-free medium and resuspended in Hanks’ balanced salt solution. Only single cell suspensions with >90% viability were used for injections (as determined by trypan blue exclusion). Mice were anesthetized by intraperitoneal injection of both Ketanest and Rompune under sterile conditions; a midline abdominal incision was performed, and the cecum was exteriorized. Using a 30-gauge needle and a 1-ml disposable syringe, 50 µL of the tumor cell suspension (1 x 106 cells) were injected into the cecal wall. To prevent leakage, a cotton swab was held over the injection site for 1 minute. The abdominal wound was closed in one layer with single sutures. The animals tolerated the surgical procedure well, and no anesthesia-related deaths occurred. Mice were not treated with antibiotics, and there were no incidences of infection. After 6 weeks, mice were sacrificed by cervical dislocation after intraperitoneal anesthesia with Ketanest and Rompune. The cecal wall tumors were removed and fixed in 5% paraformaldehyde for immunohistochemical analyses.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dynamic Pattern of Cdx2 in the Progression of Human Colorectal Adenocarcinomas.
We analyzed 45 well-differentiated to moderately differentiated colorectal adenocarcinomas. All of them expressed Cdx2; however, as described previously for ß-catenin (4) , the intratumor expression pattern was heterogeneous: We found a selective down-regulation of Cdx2 expression in dedifferentiated tumor cells predominantly at the invasive front (Fig. 1E)Citation when compared with the central differentiated tumor areas (Fig. 1D)Citation . This was coupled with loss of E-cadherin, nuclear translocation of ß-catenin (data not shown), and the expected overexpression of the ß-catenin target gene laminin-{gamma}2 (ref. 5 ; Fig. 1BCitation ). However in central areas of lymphatic or distant metastases, a reexpression of Cdx2 was found (Fig. 1F)Citation and associated with membranous redistribution and loss of nuclear ß-catenin (data not shown) as well as laminin-{gamma}2 (Fig. 1C)Citation .



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  		Fig. 1. Loss of Cdx2 expression in dedifferentiated tumor cells at the invasive front. Shown are serial sections from central, invasive regions of a typical colorectal adenocarcinoma and a corresponding liver metastasis (immunohistochemical staining is red for the ß-catenin target laminin-{gamma}2 and brown for Cdx2; nuclei are counterstained in blue; magnification, x200). Central, differentiated tumor areas (A and D) are characterized by formation of tubular structures (arrow) and lack laminin-{gamma}2 but strongly express Cdx2. At the invasive front, budding tumor cells dedifferentiate and form small clusters or isolated tumor cells (B and E). These tumor cells overexpress laminin-{gamma}2 but lose expression of Cdx2 (arrows), in contrast to organized tumor cells toward the tumor center (arrowheads). Central areas of metastases show the same expression patterns as central areas of the primary tumor (lack of laminin-{gamma}2, but reexpression of Cdx2). Shown is a liver metastasis; the same patterns were seen in lymph node metastases.

 
Collagen Type I Induces Dynamic Phenotypic Changes in Colorectal Tumor Cells.
The above results imply that the behavior of tumor cells may depend on their microenvironment. In an attempt to look for environmental component(s) involved in this regulation, we focused on extracellular matrix factors, in particular, on collagen type I, which has been reported to induce a transient down-regulation of E-cadherin in pancreatic tumor cells (18) . We analyzed 31 of the 45 colorectal adenocarcinomas by fluorescence immunohistology. Collagen type I is expressed in the stroma of all of the analyzed cases with an increase at the invasive front (Fig. 2Citation , right column). However, laminin-1 staining shows that tumor cells in central differentiated areas are still surrounded by a basement membrane, preventing contact with collagen type I, which is not a component of basement membranes (Fig. 2Citation , row I). Strikingly, budding or completely dissociated tumor cells in invasive regions (both showing nuclear ß-catenin and loss of Cdx2) were associated with interruptions (Fig. 2Citation , row II) or complete lack of basement membranes, allowing direct interaction with collagen type I (Fig. 2Citation , row III). This staining pattern was seen in 26 of the 31 (84%) analyzed cases.



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  		Fig. 2. Expression of collagen type I and loss of basement membranes at the invasive front of colorectal carcinomas. Shown is a typical example of the 31 analyzed cases [serial sections (x200) of three different regions of the same colorectal adenocarcinoma]: row I, differentiated central area; row II, invasive front with beginning dissociation of tumor cells; and row III, invasive region with strong dedifferentiation. Shown are stainings for laminin-1 (green) to emphasize basement membranes (left two columns) and collagen type I (green; right column). Co-staining for CK18 (red; middle and right columns) points out the tumor cells. Central tumor areas (row I) are built up by differentiated, polarized tumor cells forming tubules and surrounded by a basement membrane (arrows) that separates the tumor cells from collagen type I (stars indicate laminin staining of blood vessel walls). Tumor cells budding from tubules are seen in areas where the basement membrane is interrupted (arrowheads, row II). In invasive areas with completely dissociated and dedifferentiated tumor cells (row III) a basement membrane is completely gone, which might allow direct contact of the tumor cells with highly expressed collagen type I in the surrounding tissue.

 
The potential restriction of direct contact between tumor cells and collagen type I to invasive tumor areas without basement membrane prompted us to investigate the regulatory effect of collagen type I in cell culture models to mimic effects of the changing tumor environment. We selected three colorectal cancer cell lines that show an epithelial growth pattern, characterized by clearly detectable cell–cell attachments and expression of membranous E-cadherin/ß-catenin in adherens junctions. Two of them (DLD1 and Caco-2) have truncating mutations in the APC-gene, and one (LS174T) has an activating ß-catenin mutation. LS174T was used to exclude a dependence of extracellular matrix effects on the basic genetic alteration affecting degradation of ß-catenin (APC or ß-catenin mutation). In comparison with control BSA or laminin-1 (lam1), growth on collagen type I induced a strong change in the phenotype of all three colorectal tumor cell lines (Fig. 3)Citation . On BSA or laminin-1, colon cancer cells grow in round, epithelial clusters without cellular protrusions. In contrast, collagen type I led to a flattening of the clusters, followed by spreading of single tumor cells or small cell groups with protruding lamellipodia. Transfection of cells with luciferase clones for reporter assays had no influence on the phenotype changes induced by collagen type I (data not shown). Furthermore, changes in the expression patterns of E-cadherin and ß-catenin, very similar to those of the dedifferentiated tumor cells at the invasive front of colorectal carcinomas (4) , were detectable on collagen type I, in particular in DLD1 and Caco-2 cells. Treating the cell cultures with an anti-ß1 integrin blocking antibody completely reversed the phenotype changes induced by collagen type I, indicating that ß1 integrin signaling mediates the effects. Thus, independent of the basic genetic alteration, the cell culture models demonstrate a phenotypic variability induced by environmental factors, similar the tumor cells in human colorectal carcinomas.



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  		Fig. 3. Collagen type I induces changes in the phenotype of colorectal cancer cells. Indicated colorectal carcinoma cell lines were grown on different substances and stained for ß-catenin (green) and E-cadherin (red); yellow indicates co-localization. Cells grow in compact epithelial clusters on BSA or laminin-1 (lam1) and express membranous ß-catenin/E-cadherin. Also, weak expression of nuclear ß-catenin can be seen in DLD1 cells. In contrast, collagen type I induces a completely different growth pattern with spreading cells, showing protruding lamellipodia. Nuclear ß-catenin is more prominent (in DLD1 and Caco-2 cells), and in some cells, E-cadherin is found in dot-like complexes surrounding the nuclei (arrows). Treatment with anti-ß1 integrin blocking antibody (col I + anti ß1 Int) completely reversed the phenotype on collagen type I.

 
ß1 Integrin Signaling Suppresses the Expression of Cdx2 and Its Target Gene, SI.
We next wanted to determine whether the phenotype induced by collagen type I in cell cultures was associated with changes in the level of Cdx2 expression. Real-time reverse transcription-polymerase chain reaction analyses indicated that collagen type I led to a reduction in Cdx2 mRNA level to 55% and 58% in Caco-2 and DLD1, respectively, in comparison with growth on BSA. This was paralleled by a reduction of mRNA for SI, an intestine-specific target gene of Cdx2, to 46% in Caco-2 cells (Fig. 4A)Citation . In addition, reporter assays revealed a significant reduction of the Cdx2 promoter activity by collagen type I in Caco-2 (33%), DLD1 (56%; Fig. 4BCitation ), and LS174T cells (data not shown). These results correlated with the phenotype changes induced by collagen type I (Fig. 3)Citation . Again, treatment with the ß1 integrin blocking antibody reversed the collagen type I effect on both mRNA level (Cdx2 mRNA to 100% and SI mRNA to 103% in Caco-2; Cdx2 mRNA to 104% in DLD1) and promoter activity (Cdx2 promoter to 109% and 107% in Caco-2 and DLD1, respectively). In contrast, laminin-1 had only a weak inhibitory effect on the Cdx2 promoter activity (72% and 84% in Caco-2 and DLD1, respectively), almost resistant to blocking of ß1 integrin signaling (79% and 89% of BSA activity in Caco-2 and DLD1, respectively).



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  		Fig. 4. Down-regulation of Cdx2 mRNA and promoter activity by collagen type I and ß1 integrin signaling. A. Quantitative real-time PCR showing reduced mRNA levels for Cdx2 ({blacksquare}) and SI ({cjs2108}) in Caco-2 and DLD1 cells grown on collagen type I compared with growth on BSA. No reduction on collagen type I is seen after blocking ß1 integrin signaling. B, luciferase reporter assays using Caco-2 ({blacksquare}) or DLD1 cells ({cjs2108}) after growth under the indicated conditions. Significant reduction is seen on collagen type I, which is again not seen after blocking ß1 integrin signaling. Growth on laminin-1 had only a weak inhibitory effect, which is hardly reversed by the ß1 blocking antibody. C. HEK293 epithelial cells were cotransfected with the Cdx2 promoter luciferase construct and the indicated expression vector. Note the strong reduction by FAK and an increase in activity by dominant-negative (dneg) FAK. ILK had no effect. D. Immunoblot showing specific reduction of FAK in DLD1 cells by FAK siRNA compared with control siRNA for green fluorescent protein. Cdx2 mRNA is up-regulated in cells with reduced FAK expression. Bars indicate SDs of experiments performed in triplicate. The relative mRNA amounts and luciferase activities on BSA or with control transfections are set to 100%.

 
To further specify the molecules relevant in the down-regulation of Cdx2 promoter activity, we analyzed downstream components of the ß1 integrin signaling pathway by overexpressing ILK or FAK. For higher transfection efficiency, we used epithelial HEK293 cells in these experiments. FAK overexpression led to a strong reduction in Cdx2 promoter activity to 25% compared with vector control. Conversely, the FAK COOH-terminal mutant known to act as a selective dominant-negative form of FAK (19) induced an increase in activity to 133% (Fig. 4C)Citation . In contrast, ILK had no significant effect on the Cdx2 promoter. A role of FAK was further demonstrated by knocking down FAK expression using siRNAs. This led to an up-regulation of Cdx2 mRNA to 160% compared with transfection with an unrelated control siRNA in DLD1 growing on collagen type I (Fig. 4D)Citation .

Regulated Expression of Cdx2 and ß-Catenin in an Orthotopic Nude Mouse Model.
Next we questioned whether the observed changes at the invasive front of colorectal carcinomas can be induced by environmental host factors. For this purpose, we injected LS174T tumor cells orthotopically in the cecum wall of nude mice. After 4–6 weeks, invasive colon tumors, which were morphologically similar to human colon carcinomas, had grown (Fig. 5A)Citation . As in human tumors, we found a clustered epithelial-like growth pattern with membranous/cytoplasmic ß-catenin and expression of Cdx2 in central tumor areas (Fig. 5B and C)Citation . As in human tumors, tumor cells at the surrounding invasive region detached from the tumor mass, accumulated nuclear ß-catenin (Fig. 5D)Citation , and lost Cdx2 expression (Fig. 5E)Citation .



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  		Fig. 5. Loss of Cdx2 expression at the invasive front of orthotopic tumors in nude mice. A. LS174T cells injected into the colon of nude mice grow to an invasive tumor very similar to human colorectal carcinomas [x12.5; cytokeratin 18 staining; arrow marks normal mucosa; right box marks central area; left box marks invasive area magnified (x200) in B–E]. The central tumor area (cent) shows no nuclear ß-catenin but shows membranous ß-catenin (B) and nuclear expression of Cdx2 (C). At the invasive front (inv), tumor cells detach from the tumor mass (arrowheads), accumulate nuclear ß-catenin (D), and lose expression of Cdx2 (E). Specific immunohistochemical staining of serial sections is brown; nuclear counterstaining is blue.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Dynamic changes of tumor cell phenotype and the expression pattern of the oncoprotein ß-catenin characterize the malignant progression of colorectal adenocarcinomas during tumor cell invasion and metastasis (4) . In addition, here we describe a loss of Cdx2 expression in dedifferentiated tumor cells at the invasive front and a reexpression of Cdx2 in metastases. In cell culture models, we showed that collagen type I induced phenotype changes through the ß1 integrin signaling pathway that are accompanied by reduced Cdx2 mRNA expression and promoter activity. Based on the fact that Cdx2 is one of the most specific factors of intestinal differentiation, our data suggest that dissociated, mesenchyme-like tumors cells have actually undergone dedifferentiation. The potential significance of collagen type I as a driving force for human colorectal carcinogenesis is indicated by the observations that dedifferentiated tumor cells in invasive regions lose their basement membranes (see Fig. 2Citation ). This might subsequently allow direct cell contact to abundantly expressed collagen type I in the tumor environment. Recently, the two genes encoding collagen type I {alpha}1- and {alpha}2-chain were found among eight genes overexpressed in the primary tumor of metastatic adenocarcinomas of various origins (20) . Our results could help to explain a postulated role of collagen type I for tumor progression. Moreover, human colon tumor cell lines orthotopically grafted in nude mice recapitulate the phenotype changes observed in human colorectal cancers. This suggests that changes in the expression patterns of Cdx2, ß-catenin, and target genes are regulated by the tumor environment rather than due to clonal accumulation of novel genetic alterations.

Changes in the expression of the molecules analyzed in the present study may have decisive effects for the tumor cells. ß1-containing integrins and their downstream pathways, among which is FAK (19) , are involved in malignant progression. In particular, this was demonstrated previously for breast cancer (21) . The Cdx2 homeobox gene acts as a tumor suppressor and is involved in colorectal cancer. A regulated loss of Cdx2 at the invasive front may lead directly to transient tumor cell dedifferentiation, which, together with nuclear accumulation of ß-catenin, may trigger dissemination of tumor cells through blood and lymphatic vessels. We found a reexpression of Cdx2 in re-differentiated tumor areas of metastases, suggesting that Cdx2 reexpression may be a driving force re-differentiation. Our finding of a correlation between Cdx2 expression and intestinal differentiation in the different areas of colorectal carcinomas is supported by the findings of Almeida et al. (9) and Mizoshita et al. (10) . They found a strong association of the grade of intestinal differentiation in gastric cancer with ectopic expression of Cdx2 and its intestine-specific target gene MUC2.

Most cases of human colorectal adenocarcinomas have mutations in the APC gene. In contrast, mutations in the Cdx2 gene are very rare (15 , 22) , which further supports that loss of Cdx2 in dedifferentiated tumor cells is transient and due to regulation. Interestingly, a subtype of replication error-positive colorectal cancers shows mutations in repetitive elements of both alleles of Cdx2 (22) . These tumors are characterized by a homogenous growth pattern of poorly differentiated tumor cells and therefore have a completely different morphology compared with the common colorectal adenocarcinomas included in our study. The overall poor differentiation of these tumors may be explained by a complete loss of Cdx2 expression. These observations and our findings provide support for a hypothesis that the molecular pathogenesis of both tumor types is distinct. Accordingly, we postulate two principle mechanisms of tumor progression, a "regulated" type and a "genetic" type: The subtype of replication error-positive tumors with irreversible, poor differentiation represents a prototype of colon cancers with genetic alterations as main driving force. In contrast, malignant progression of well-differentiated to moderately differentiated colorectal adenocarcinomas, characterized by stages of a transient dedifferentiation and Cdx2 loss, is also driven by regulatory input of the microenvironment. Cdx2 can be used as a marker molecule to support this view and to differentiate these types at the molecular level.

An active, regulatory role of the microenvironment for tumor progression has already been demonstrated for other tumors, e.g., breast cancer (21) . Moreover, epithelial–mesenchymal interactions are important forces in many developmental processes, including intestinal differentiation (23) . We showed that collagen type I induces phenotypic changes and a reduction in Cdx2 promoter activity in colorectal cancer cells in a ß1 integrin-dependent manner. ß1 Integrin signaling through FAK and ILK has various effects promoting tumor progression. In contrast to ILK, FAK strongly down-regulated Cdx2 promoter activity, although we could not yet define the precise FAK-responsive promoter element. As an alternative to a direct repressive effect, FAK activity could block an activator of the Cdx2 promoter. Recently, we showed that the Cdx2 promoter is activated by PTEN, antagonizing phosphatidylinositol 3'-kinase–mediated inhibition of Cdx2 expression via nuclear factor {kappa}B binding elements (17) , which is significant because FAK can activate the phosphatidylinositol 3'-kinase–Akt pathway (24) .

In summary, we have shown that expression of the tumor-suppressive homeodomain factor Cdx2 undergoes dynamic changes during invasion and metastasis in human colorectal adenocarcinomas and in an orthotopic animal model. In cell culture, these changes can be induced by collagen type I through ß1 integrin signaling. These data indicate an active regulatory role of the microenvironment for invasive and metastatic growth.


   ACKNOWLEDGMENTS
 
We are grateful to S. Dedhar and D. Schlaepfer for DNA constructs. For expert technical assistance we thank U. Suchy, S. Pfeiffer, and C. Knoll.


   FOOTNOTES
 
Grant support: Deutsche Krebshilfe grant 10-2029 Br I (to T. Brabletz and C. Bruns); Deutsche Forschungsgemeinschaft grant BR 1399/4-1 (to T. Brabletz and T. Kirchner); and INSERM and Association pour la Recherche sur la Cancer grant 3286 (to J-N. Freund). F. Hlubek was funded by the BMBF (NGFN1 Project KR-S05T02), and C. Domon-Dell was funded by the Fondation de la Recherche Médicale.

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.

Requests for reprints: Thomas Brabletz, Department of Pathology, University of Erlangen-Nürnberg, Krankenhausstrasse 8-10, 91054 Erlangen, Germany. Phone: 49/9131-8522856; Fax: 49/9131-8524745; E-mail: thomas.brabletz{at}patho.imed.uni-erlangen.de

Received 4/ 5/04. Revised 7/23/04. Accepted 7/30/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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