Human Cancer Biology

The PutativeTumor Suppressor Cdx2 Is Overexpressed by Human Colorectal Adenocarcinomas Matthew E. Witek,1, 2 Karl Nielsen,1, 2 Rhonda Walters,3 Te r r y H y s l o p , 1, 2 Juan Palazzo, 3 Stephanie Schulz,1, 2 and Scott A. Waldman1, 2

Abstract Purpose:The current paradigm suggests that the homeodomain Cdx2, which directs the development and maintenance of the intestinal , is a tumor suppressor in the colon and rectum. Although a cardinal property of tumor suppressors is their inactivation during carcinogenesis, the expression of Cdx2 in colorectal tumors has not been compared with that in normal mucosa. Here, Cdx2 expression and function was quantified in tumors and matched normal mucosa from patients with colorectal cancer. Experimental Design: Cdx2 expression was quantified by reverse transcription-PCR, immuno- blot analysis, and immunohistochemistry. Transcriptional activity was explored by quantifying expression of an endogenous downstream target of Cdx2, guanylyl cyclase C (GCC), in tissues by quantitative reverse transcription-PCR and expression of exogenous Cdx2-specific luciferase promoter constructs in epithelial cells isolated from tumors and normal mucosa. Results: Most (>80%) colorectal tumors overexpressed Cdx2 mRNA and comparedwith normal mucosa, with median fold increases of 3.6 and1.4, respectively (P < 0.002). Concomitant- ly, immunohistochemistry revealed elevated levels of Cdx2 in nuclei of tumor cells compared with normal epithelial cells. Further, tumors exhibited increased expression of GCC compared with normal mucosa. Moreover, cells isolated from tumors overexpressed a Cdx2-specific luciferase promoter construct compared with normal mucosal cells. Conclusion:These observations show, for the first time, the structural and functional overexpres- sion of Cdx2 by human colorectal tumors compared with matched normal mucosa.They suggest that loss of Cdx2 expression or transcriptional activity is an infrequent event during tumorigenesis, which does not contribute to molecular mechanisms underlying initiation and progression of most colorectal tumors.

The caudal-related Cdx2 encodes a homeo- including adhesion (6), proliferation (7), cell cycle regulation domain transcription factor required for development and (8), and apoptosis (9). The homeotic function of Cdx2 is maintenance of the intestinal epithelium (1–3). Cdx2 expres- underscored by loss of the cognate enterocyte phenotype in sion in adults is restricted to intestinal epithelial cells, where it intestinal neoplasms arising in the proximal colon of Cdx2+/À maintains the differentiated phenotype of mature enterocytes mice upon biallelic inactivation of Cdx2 (10, 11). Conversely, by regulating expression of intestine-specific , including ectopic expression of Cdx2 in squamous epithelial cells of the guanylyl cyclase C (GCC; ref. 4), (5), sucrase-isomaltase forestomach induces intestinal metaplasia in transgenic mice (1), and LI-cadherin (6). Also, Cdx2 regulates the expression of (12, 13). ubiquitous genes important in controlling cell dynamics, Beyond its homeotic role, Cdx2 may be an important tumor suppressor in colorectal carcinogenesis (14, 15). Thus, expres- sion of Cdx2 mRNA and protein is reduced (16, 17) or lost Authors’ Affiliations: 1Division of Clinical Pharmacology, Departments of (9, 16, 18–20) in some human colorectal tumors. However, in 2 3 Pharmacology and Experimental Therapeutics; Medicine; and Pathology, some analyses, immunohistochemistry revealed Cdx2 protein Anatomy, and Cell Biology,Thomas Jefferson University, Philadelphia, Pennsylvania Received 7/27/05; revised 9/17/05; accepted 9/28/05. expression in most human colonic adenocarcinomas examined Grant support: NIH grants CA79663 and CA95026 andTargeted Diagnostics and (21, 22). In animal models, biallelic inactivation of Cdx2 Therapeutics, Inc. results in the formation of hamartomatous tumors character- The costs of publication of this article were defrayed in part by the payment of page ized by gastric heteroplasia in the proximal colon (11, 23). charges. This article must therefore be hereby marked advertisement in accordance Similarly, whereas adenomatous polyposis coli heterozygous with 18 U.S.C. Section 1734 solely to indicate this fact. +/À Note: S.A. Waldman is the Samuel M.V. Hamilton Professor of Medicine ofThomas (APC ) mice develop adenomatous polyposis of the small +/À +/À Jefferson University. intestine, APC Cdx2 mice develop polyposis of the colon Requests for reprints: Scott A. Waldman, Division of Clinical Pharmacology, (24). Moreover, the procarcinogen azoxymethane induced Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson invasive adenocarcinoma of the distal colon in Cdx2+/À mice University, 132 South 10th Street, 1170 Main, Philadelphia, PA 19107. Phone: 215- 955-6086; Fax: 215-955-7006; E-mail: [email protected]. but not wild-type littermates (10). Further, overexpression of F 2005 American Association for Cancer Research. Cdx2 in human colon cancer cells induces a less malignant doi:10.1158/1078-0432.CCR-05-1624 phenotype, inhibiting proliferation, invasion, and migration

www.aacrjournals.orgClin Cancer Res 2005;11(24) December8549 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Human Cancer Biology while promoting the expression of genes characteristic of mature enterocytes (2, 9, 17, 25, 26). Table 1. Amplicon sizes,Y intercepts, and slopes from Thus, the prevalent paradigm suggests that Cdx2 is a tumor standard curves for quantitative RT-PCR amplification suppressor whose reduced expression and/or function contrib- of marker genes utes to initiation and progression of sporadic adenocarcinomas of the colon and rectum (14, 15, 24). This hypothesis presumes Marker Amplicon Standard curve size (bp) that promotion of colorectal carcinogenesis reflects loss of Slope Intercept LOD Cdx2 expression and/or function in tumor cells relative to (copies) normal intestinal epithelium. However, with the exception of one study (19), expression of Cdx2 in human colorectal tumors Cdx2 50-150* À3.072 F 0.139 42.82 F 0.67 100 has not been compared with matched normal mucosa (15). In h-Actin 99 À3.515 F 0.205 42.95 F 1.07 200 the present study, Cdx2 expression and transcriptional activity GCC 84 À3.344 F 0.108 43.33 F 0.49 200 in colorectal tumors were compared with matched normal mucosa from patients. Unexpectedly, Cdx2 mRNA, protein, Abbreviation: LOD, limit of detection. and activity were elevated in most colorectal tumors compared *Proprietary information of Applied Biosystems. with matched normal mucosa. These observations challenge the prevailing paradigm and suggest that loss of Cdx2 amplify and detect Cdx2 RNA (Applied Biosystems). The primer and expression or function is not a dominant mechanism under- probe sequences used to amplify and detect h-actin and GCC transcripts lying initiation or promotion of colorectal carcinogenesis. were as follows (5V-3V): h-actin sense, CCACACTGTGCCCATCTACG; h-actin antisense, AGGATCTTCATGAG-GTAGTCA-GTCAG; probe Materials and Methods (FAM)ATGCCC-X (TAMRA)-CCCCCATGCCATCCTGCGTp (X indicates a linker-arm nucleotide and p indicates phosphorylation) and GCC Clinical specimens. Specimens were obtained from the Department sense, ATTCTAGTCCATCTTTTCAATGACC; GCC antisense, CGTCA- of Pathology, Anatomy and Cell Biology of Thomas Jefferson University GAACAAG-GACATTTTTCATA; and probe (6FAM)TACTTGGAGG- Hospital (Philadelphia, PA) under an Institutional Review Board– ACAATGTCACAGCCCTTC(TAMRA). Reactions were incubated at approved protocol (control no. 98.0614). Slides, stained with H&E, 50jC for 2 minutes, 60jC for 30 minutes, 95jC for 5 minutes, and were reviewed by a pathologist (J. Palazzo) to confirm that all tumors then subjected to 45 cycles at the following conditions: 94jC for were adenocarcinoma and all normal mucosal samples were free of 20 seconds and 62jC for 1 minute. disease. Total RNA was extracted from specimens with Tri-Reagent Immunohistochemistry. Sections (5 Am) of paraffin-embedded (Molecular Research Center, Inc., Cincinnati, OH). Cytoplasmic and tissues were mounted on Superfrost Plus-charged slides. Routine nuclear protein fractions were isolated using NE-PER extraction reagents deparaffinization from xylene to 95% alcohol was done on a Leica (Pierce, Rockford, IL). Autostainer (Leica, Inc., Deerfield, IL) and included a 30-minute Primary cell isolation. Following resection, tumor and normal methanolic peroxide step to block endogenous peroxidase activity. mucosal specimens were incubated at 37jC in DMEM/F12 supple- Slides were rehydrated and antigen recovery was done at 70% power in mented with 350 units/mL collagenase XI (Sigma, St. Louis, MO). a 1,050 W microwave oven (model EM-F3400, Sanyo, Chatsworth, CA) Disaggregated cell suspensions were centrifuged, resuspended in Eagle’s in EDTA buffer (pH 8.0) for two intervals of 5 minutes each. After a MEM containing 10% fetal bovine serum, insulin (0.1 Ag/mL), 30-minute cooling period, slides were washed in deionized water and epithelial growth factor (50 ng/mL), L-glutamine (2 mmol/L), placed in PBS for 5 minutes. Immunostaining was conducted using a penicillin/streptomycin (100 units/mL), gentamicin (40 Ag/mL), and DAKO autostainer (model LV-1, DAKO Corporation, Carpinteria, CA). fungizone (125 Ag/mL), passed through 40 Am nylon cell strainers (BD Slides were incubated for 15 minutes each with avidin and blocking Biosciences Clontech, Palo Alto, CA), and seeded in tissue culture solutions (Vector Laboratories, Burlingame, CA), followed by Cdx2 plates. primary antibody (1:1,600; BioGenex, San Ramon, CA) for 60 minutes. Quantitative reverse transcription-PCR cRNA standards. RNA har- After a PBS wash, slides were incubated with DAKO LSAB secondary vested from T84 human colon carcinoma cells (American Type Culture antibody for 30 minutes followed by DAKO LSAB streptavidin– Collection, Manassas, VA) was used in a reverse transcription reaction horseradish peroxidase reagents (DAKO). After another PBS wash, to produce Cdx2, GCC, or h-actin cDNA. The synthesized cDNA was slides were incubated for 5 minutes in 3,3V-diaminobenzidine solution amplified by PCR and subsequently cloned into the Bluescript vector (DAKO), washed in deionized water, counterstained with Harris (Stratagene, La Jolla, CA). Purified plasmid DNA was linearized with hematoxylin (SurgiPath, Richmond, IL), and dehydrated with xylene appropriate restriction enzymes. The conserved 5VT7 promoter site was before mounting. used to transcribe target genes using the RiboMAX Large Scale RNA Reporter gene analysis. The transcriptional activity of Cdx2 was Production System-T7 (Promega, Madison, WI). Transcription reactions examined using luciferase reporter constructs, including a pGL-3-Basic were treated with DNase to remove template DNA fragments and the luciferase vector (negative control; Promega), a pGL3 construct cRNA purified on an RNeasy Mini-Column (Qiagen, Foster City, CA). containing fragment À835 to +117 of the GUCY2C promoter (GCC- The purity and concentration of cRNA stock solutions was determined Luc), and the identical construct with a TTT to CCC mutation in the by UV spectrophotometry. Stock cRNA solutions underwent serial 10- Cdx2 consensus site (DGCC-Luc; ref. 4). Following seeding, primary fold dilutions to generate working standards to establish calibration cells at 30% to 50% confluency were cotransfected with 0.5 Agof curves for absolute analyte quantification by quantitative reverse luciferase reporter constructs, modified from pGL3-Basic, and 0.005 Ag transcription-PCR (RT-PCR). Slopes and intercepts of calibration curves of the Renilla luciferase control reporter, pRL-TK, driven by a viral for Cdx2, GCC, and h-actin showed that amplification reactions for thymidine kinase promoter (Promega) with FuGENE 6 Transfection these analytes were of comparable high efficiency (Table 1). Reagent at 3 AL/Ag DNA (Roche Diagnostics, Indianapolis, IN). One Reverse transcription-PCR. RNA (1 Ag) isolated from specimens was day after transfection, cells were harvested and assayed using the used in a one-step reverse transcription PCR. The reaction was done protocol and materials in the Dual-Luciferase Reporter Assay system using Taqman EZ RT-PCR Core Reagents (Applied Biosystems, Inc., (Promega). Luminescence was measured with a TD-20/20 luminometer Foster City, CA) and the 7000 Sequence Detection System (Applied (Turner Designs, Sunnyvale, CA). Luciferase expression was normalized Biosystems). A commercially available primer and probe set was used to to pRL-TK expression for standardization of transfection efficiency

Clin Cancer Res 2005;11(24) December 15, 2005 8550 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Cdx2 Overexpression in Colorectal Tumors

(ratio). Promoter activity was normalized by subtracting the pGL3 Cdx2 mRNA is overexpressed by colorectal tumors compared control vector ratio from the respective promoter fragment–containing with matched normal mucosa. External calibration standards reporter vector ratio. Cdx2-transactivating activity was quantified as the (Fig. 1A and B) were used in quantitative RT-PCR to quantify difference between normalized values for GCC-Luc activity and DGCC- Cdx2 and h-actin mRNA expression in tissues (Table 2). For Luc activity. clinical specimens, mRNA levels were not routinely normalized Immunoblot analysis. Tissues were homogenized in Laemmli h buffer and 30 Ag of total lysate were separated on 12% to -actin, reflecting the heterogeneity of contribution of SDS-polyacrylamide gels under reducing conditions and transferred to epithelial and stromal cells to different specimens; the nitrocellulose (Osmonics, Inc., Minnetonka, MN). After transfer, sheets variability of expression of housekeeping genes in different were subjected to Ponceau S (Sigma) staining, which was consistently cells, tissues, and patients; and the general acceptance of identical in all lanes. Following incubation in 5% nonfat dry milk in normalization to amount of total RNA analyzed (31). However, PBS with 0.5% Tween 20 (Sigma) to block nonspecific protein binding, specimens yielding <103 copies of h-actin mRNA per micro- proteins of interest were identified by incubating with affinity-purified gram of total RNA were deemed insufficient and omitted from rabbit anti-Cdx2 antibody (1:500; Biosource International, Hopkinton, further analysis. MA) and rabbit polyclonal anti-a-tubulin antibody (1:10,000; Santa Expression of Cdx2 mRNA was compared in 30 paired Cruz Biotechnology, Santa Cruz, CA) in TBS with 0.1% Tween and 5% colorectal tumors and histologically normal adjacent mucosa nonfat milk (blotting buffer) overnight at 4jC. After several washes, membranes were incubated in horseradish peroxidase–conjugated goat (Fig. 1C and D; Table 2). Quantitative RT-PCR revealed anti-rabbit antibody (1:10,000; Jackson ImmunoResearch Laboratories, substantial interindividual variation in expression of Cdx2 6 6 West Grove, PA) in blotting buffer at room temperature for 45 minutes. mRNA in normal mucosa (1.2 Â 10 F 1.9 Â 10 copies/Ag 6 Membranes were washed and protein antibody complexes were total RNA; mean F SD) and adenocarcinomas (7.7 Â 10 F visualized by chemiluminescent detection using SuperSignal West Pico 1.9 Â 107 copies/Ag total RNA; mean F SD), with a range of f2 Chemilumenescent Substrate (Pierce). Staining intensities on immuno- orders of magnitude. However, Cdx2 mRNA was significantly blots were quantified with Kodak ID (v.3.5.4) software (Eastman overexpressed in 25 of 30 adenocarcinomas (f83%) compared Kodak, New Haven, CT). with matched normal adjacent mucosa (P < 0.002; Fig. 1C Fluorescence immunohistochemistry. Cells in 24-well plates were and D). Indeed, the median difference in expression of Cdx2 fixed and permeabilized with À20jC methanol for 20 minutes, washed j mRNA between tumors and matched normal adjacent mucosa, with TBS, blocked with preimmune goat or rabbit serum at 25 C for 5 6 7 1 hour, and then incubated overnight at 4jC with rabbit anti-Cdx2 8.3 Â 10 , with a range of À5.0 Â 10 to 7.9 Â 10 copies, or goat anti-LI-cadherin antibody (1:50; Santa Cruz Biotechnology) reflected a 3.6-fold median increase in Cdx2 mRNA expression in TBS supplemented with 3% bovine serum albumin. Subsequently, in tumors compared with matched normal mucosa. Although cells were washed with TBS and incubated at room temperature most tumors were moderately differentiated and were from for 45 minutes in rhodamine (TRITC)–conjugated goat anti-rabbit or patients with stage B or C disease, Cdx2 mRNA copy number Cyan2+-conjugated rabbit anti-goat antibody (1:100; Jackson Immu- did not obviously correlate with tumor grade or stage (Table 2). noResearch Laboratories) in TBS with 3% bovine serum albumin. Of significance, four of five tumor specimens that expressed less Antibody complexes were visualized using fluorescence microscopy Cdx2 mRNA compared with matched normal adjacent mucosa (Kodak DC290 ZOOM Digital Camera; Adobe Photoshop 7.0). were of an early stage (stage A and B; Table 2) in contra- Statistical analysis. Determinations were done in duplicate or triplicate and results are representative of at least three experiments distinction to suggestions that Cdx2 expression decreases unless otherwise indicated. Cdx2 and GCC mRNA and protein during tumor progression (16, 17). expression in tumors and matched normal mucosal specimens were Cdx2 protein is overexpressed by colorectal tumors compared compared using the Wilcoxon signed-rank test (27). Correlation of with matched normal mucosa. Immunoblot analysis revealed Cdx2 with GCC expression was quantified by the Spearman correlation elevated Cdx2 protein in tumors that overexpress Cdx2 mRNA coefficient, a nonparametric estimate (28). Cdx2 transcriptional activity compared with matched normal adjacent mucosa (Fig. 2A). In in tumors and matched normal mucosal specimens were compared every tumor examined in which Cdx2 mRNA was over- using the Student’s t test. In all analyses, P < 0.05 was considered expressed (n = 10), Cdx2 protein also was elevated compared statistically significant. with matched normal mucosa. The mean increase in Cdx2 protein in tumors was f150% compared with matched Results normal mucosa (P < 0.002; Fig. 2B). Further, overexpression of Cdx2 mRNA and protein detected by immunoblot analysis Patient characteristics. Patients ranged in age from 48 to 94 was associated with an increase in Cdx2 detected by years (72 F 12.9 years) and there were no significant immunohistochemistry in tumors compared with matched differences in the ages of females (53%; range = 48-94 years; normal mucosa (n = 5; Fig. 2C). Cdx2 expressed by tumors 75 F 11.8 years) and males (47%; range = 51-93 years; 68 F and normal mucosa was appropriately localized in the nucleus 13.4 years). All patients had adenocarcinoma of the colon or (Fig. 2C). rectum and their anatomic distribution included the right colon Cdx2 transcriptional activity is elevated in colorectal cancer (43%), transverse colon (7%), left colon (7%), sigmoid colon compared with matched normal mucosa. Cdx2 transcriptional (23%), rectosigmoid colon (10%), and rectum (10%). The activity, which regulates expression of proteins that define the distribution of disease stage (29) included stage A (30%), stage differentiated enterocyte phenotype (1–4, 10, 32), can be B (37%), stage C (20%), and stage D (10%), comparable with examined in intestinal cells by quantifying the expression of distributions observed previously (30). Moreover, the distribu- genes that are downstream targets (17, 25). GCC is an epithelial tion of tumor grade included well-differentiated (10%), cell in the intestine that regulates fluid and electrolyte moderately well differentiated (3%), moderately differentiated homeostasis (33) and proliferation along the crypt-villus axis (70%), moderately poorly differentiated (13%), and poorly (34–36) whose tissue-specific expression is defined by Cdx2 differentiated (3%; Table 2). (4). Thus, Cdx2 transcriptional activity in colorectal tumors and

www.aacrjournals.orgClin Cancer Res 2005;11(24) December8551 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Human Cancer Biology

Table 2. Patient demographics, tumor characteristics, and Cdx2 mRNA copy number

Sex Age (y) Site Stage Grade Cdx2 copy number (104) Dukes’ TNM Mucosa Tumor

F65Rightcolon A T2N0Mx Poor 1.0 4.1

F71Rightcolon B T3N0Mx Poor-Moderate 110 1,200

F79Rightcolon AT1N0Mx Poor-Moderate 89 79

M87Rightcolon C T3N1Mx Poor-Moderate 0.4 22

M58Rightcolon D T3N2M1 Poor-Moderate 50 180

F 73 Rectum C T3N1Mx Moderate 14 64

M74Rightcolon A T2N0Mx Moderate 340 800

M 82 Rectosigmoid colon B T3N0Mx Moderate 2 10 8,100

M 93 Rectosigmoid colon B T3N0Mx Moderate 70 670

F 91 Transverse colon B T3N0Mx Moderate 38 54

M54Rightcolon D T3N1M1 Moderate 75 610

F 88 Sigmoid colon B T3N0Mx Moderate-Well 11 54

M 59 Sigmoid colon C T3N2Mx Moderate 79 130

F60Rightcolon A T1N0Mx Well 34 550

M 54 Rectum B T3N0Mx Well 89 49

M 51 Rectum A T2N0Mx Moderate 28 120

M 78 Sigmoid colon B T3N0Mx Moderate 6.6 56

F73Rightcolon BT2N0Mx Moderate 3.4 180

F69Rightcolon B T3N0Mx Moderate 29 170

F 85 Transverse colon A T2N0Mx Moderate 18 340

M67Leftcolon C T3N1Mx Moderate 1.4 110

F84Rightcolon B T3N0Mx Moderate 1.1 0.8

F 75 Sigmoid colon C T3M1Mx Moderate 15 720

F75Rightcolon AT2N0Mx Moderate 2.5 5.2

M 57 Sigmoid colon C T3N1Mx Moderate 330 1,600

F 48 Sigmoid colon C T3N2Mx Moderate 370 1,100

M 73 Sigmoid colon D T4N1Mx Moderate 5.0 58

M71Leftcolon A T2N0Mx Moderate 500 6.8

F 76 Rectosigmoid colon A T1N0Mx Moderate 490 4.9

F94Rightcolon B T3N0Mx Well 2.7 46

matched normal mucosa (n = 26) was compared by quantify- isolated tumor cells and matched normal enterocytes by ing the expression of GCC using quantitative RT-PCR. reporter gene analysis. In these studies, Cdx2-specific trans- Calibration curves of in vitro transcribed cRNA encoding activation was quantified as the difference between luciferase GCC were used in quantitative RT-PCR to quantify GCC mRNA activities generated by a construct driven by the wild-type GCC expression levels (Fig. 3A; Table 2). GCC mRNA levels promoter (À835 to +117) and the identical construct with were significantly elevated in tumors exhibiting overexpression a TTT-to-CCC mutation in the Cdx2 consensus site (4). of Cdx2 mRNA compared with matched normal mucosa (P < Following surgical resection, colorectal tumors and matched 0.01; Fig. 3B and C). The median difference in expression of GCC normal adjacent mucosa were disaggregated and established in mRNA in tumor compared with matched normal mucosa, 6.0 Â culture. To confirm identity, isolated cells were stained with 104 with a range of À9.2 Â 104 to 1.0 Â 106 copies, reflected antibody against Cdx2 and LI-cadherin, nuclear and membrane a median f2.4-fold increase in GCC mRNA expression in proteins exclusively expressed by intestinal epithelial cells tumors compared with matched normal mucosa. The precursor- (Fig. 4A). Cdx2-specific transcriptional activity was f2-fold product relationship between Cdx2 and GCC is highlighted by greater in isolated tumor cells compared with matched normal the close correlation of the quantities of their cognate transcripts adjacent mucosal cells (P < 0.05; Fig. 4B). in individual tissues (r = 0.53; P < 0.01; Fig. 3D). The foregoing is consistent with a mechanism in which Discussion increased expression of GCC mRNA reflects overexpression of Cdx2 in tumors compared with matched normal mucosa. A cardinal property defining tumor suppressors is their However, expression of GCC mRNA reflects the integrated inactivation in cancer cells compared with normal cells, activities of multiple transcriptional regulatory proteins at the resulting in disequilibrium in molecular mechanisms regulat- promoter and differences in expression could reflect the ing proliferation and survival (38). The current paradigm activities of transcriptional factors other than Cdx2 (4, 37). suggests that Cdx2 is a tumor suppressor (14, 15, 24) whose Thus, Cdx2 transcriptional activity was directly quantified in loss of function, reflecting loss of expression (16), mutation

Clin Cancer Res 2005;11(24) December 15, 2005 8552 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Cdx2 Overexpression in Colorectal Tumors

Fig. 1. A and B, in vitro transcribed cRNA was used to generate quantitative RT-PCR standard curves to quantify Cdx2 and h-actin mRNA expression levels. Points, mean of at least five independent experiments done in duplicate or triplicate; bars, SE. C and D, total RNA (1 Ag) isolated from paired human colorectal tumor and normal adjacent mucosa samples was analyzed by quantitative RT-PCR to quantify expression of Cdx2 mRNA. Box and whisker plot in (D) exhibit the median Cdx2 mRNA copy number (horizontal line) surrounded by the second and third quartiles of those values (50% of the values; box), and the extreme values (whiskers). RT-PCR analysis of paired tissue samples (n =30;*,P < 0.002).

(39), posttranslational modification (40), or regulation (17), colorectal tumors retained expression of Cdx2 (17, 21, 22, 41). contributes to mechanisms underlying colorectal carcinogene- Taken together, these observations show that loss of Cdx2 sis. In stark contrast, the current study shows that Cdx2 is function is not a principle mechanism underlying the initiation overexpressed in most sporadic human colorectal tumors or promotion of most sporadic human colorectal tumors. compared with matched normal mucosa. Overexpression of The present study does not eliminate a role for Cdx2 as a mRNA and protein is associated with elevated Cdx2 transcrip- tumor suppressor in colorectal carcinogenesis. Indeed, <20% of tional activity and overexpression of GCC, an endogenous sporadic colorectal tumors exhibit reduced or absent Cdx2 downstream gene target of Cdx2 (4). These observations show expression (17, 21, 22, 41). Also, Cdx2 expression seems to be that Cdx2 is structurally and functionally overexpressed in most lost in large cell minimally differentiated colon carcinomas sporadic human colorectal tumors. They are in close agreement (16). Additionally, f10% of sporadic colorectal tumors exhibit with immunohistochemical studies in which >80% of human loss of heterozygosity with respect to Cdx2 (42, 43). Moreover,

Fig. 2. A, whole cell protein fractions extracted from paired human colorectal tumor (T)andnormal(N) adjacent mucosa samples were analyzed byWestern blot for Cdx2 protein expression (three representative paired samples). B, densitometry of Cdx2 protein expression normalized to a-tubulin (n =10; *, P < 0.002). C, paraffin-embedded colorectal tumors and matched normal adjacent mucosa specimens from patients were immunostained with Cdx2 antibody to determine expression and subcellular localization of Cdx2.

www.aacrjournals.orgClin Cancer Res 2005;11(24) December8553 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Human Cancer Biology

Cdx2 transcription is silenced by a dominant repression tion. Thus, 35% of sporadic colorectal tumors exhibit poly- mechanism in f10% of sporadic colorectal tumors (17). ploidy of 13q12.13-q12.2 where the gene These observations are consistent with a role for Cdx2 as a encoding Cdx2 resides (48). Also, most colorectal tumors tumor suppressor in a subset of colorectal tumors. However, constitutively overexpress nuclear factor-nB (49), a potent it remains unclear whether loss of Cdx2 expression in those transactivator of Cdx2 expression (50). Moreover, the Cdx2 tumors is intrinsic to mechanisms underlying carcinogenesis or promoter has Cdx2 binding sites that mediate transactivation reflects the effects of genetic instability associated with and increases in expression reflecting genetic instability will be neoplastic transformation. amplified by autoregulation (51). Although Cdx2 is not a classic tumor suppressor in most Alternatively, overexpression of Cdx2 might reflect compen- colorectal neoplasms, it may play a role in tumor progression in satory mechanisms directed at reestablishing the equilibrium in a restricted spatial and temporal window. Cells that metastasize proliferation, differentiation, and survival characteristics of from the invasive front of primary colorectal tumors undergo epithelial cells that is disrupted during carcinogenesis. Biallelic transient dedifferentiation, only to reestablish the differentiated inactivation of Cdx2 in epithelial cells results in the formation phenotype at the metastatic site (44–46). Although Cdx2 is of hyperplastic colonic polyps in Cdx2+/À mice (11, 23, 24). expressed in most colorectal cancer cells, it is transiently lost in Conversely, conditional expression of Cdx2 in colon carcinoma cells at the invasive edge of the tumor (25). Transient loss of cells in vitro suppressed their proliferation (9, 17). Further, Cdx2 is initiated by cell interaction with collagen type I, conditional expression of Cdx2 in HT-29 colon carcinoma cells mediated by cell surface h-integrin and nuclear h-catenin induced the expression of the mitogen-activated protein kinase signaling, and is presumed to be the mechanism mediating the MOK, which mediates growth arrest (52). Ectopic expression of epithelial-mesenchymal transition characterizing metastasis Cdx2 in the foregut of transgenic mice induces the development (25). Establishment of tumor metastases at distant sites results of the differentiated intestinal cell phenotype (13). Similarly, in the reexpression of Cdx2 associated with recovery of the overexpression of Cdx2 in human colon cancer cells induces the differentiated phenotype (25). In the context of the present expression of genes characteristic of differentiated enterocytes study, these observations suggest that rather than a tumor and goblet cells (2, 25, 26, 52). Moreover, conditional suppressor gene, Cdx2 may be an important metastasis expression of Cdx2 increased the sensitivity of human colon suppressor gene (47) with a role restricted spatially to the carcinoma cells to apoptosis (9). invasive front in tumors accessible to collagen type I and An intriguing possibility not considered previously is that temporally to the perimetastasis period (25). overexpression of Cdx2 in tumors reflects its role in mediating Although Cdx2 is overexpressed by most colorectal tumors, neoplastic transformation. Ectopic expression of Cdx2 in the precise mechanisms underlying its overexpression remain hematopoietic progenitor cells induces acute myeloid leuke- undefined. Overexpression may reflect the genetic instability mia in transgenic mice (53). Similarly, although ectopic characterizing carcinogenesis wherein Cdx2 expressed by expression of Cdx2 in the foregut induces intestinal metapla- tumors is a bystander in the process of neoplastic transforma- sia in mice (12, 13), these lesions ultimately progress to frank

Fig. 3. A, in vitro transcribed cRNA was used to generate a quantitative RT-PCR standard curve to quantify GCC mRNA expression. B and C, GCC mRNA copy numbers from human colorectal tumors that overexpressed Cdx2 RNA were quantified by RT-PCR (n =25;*,P < 0.01). Box and whiskers plots in (C) are similar to those in Fig.1. D, correlation of Cdx2 and GCC mRNA expression in human colorectal tumors (n =25;r =0.53;P < 0.01).

Clin Cancer Res 2005;11(24) December 15, 2005 8554 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Cdx2 Overexpression in Colorectal Tumors

adenocarcinoma (54). Although the precise role of Cdx2 in neoplastic transformation in bone marrow (53) and foregut (54) remains unclear, carcinomas arising from its ectopic expression exhibit elevated proliferating cell nuclear antigen as well as genetic instability reflected by mutations in APC and (54). Conversely, hamartomas deficient in Cdx2 arising in the proximal colon of Cdx2+/À mice fail to incorporate bromodeox- yuridine, a marker of proliferation, in contrast with normal adjacent mucosa expressing Cdx2, which suggests a proprolifer- ative function for this transcription factor (55). Moreover, conditional expression of Cdx2 in IEC-6 undifferentiated intestinal epithelial cells increased their proliferation and migration (2, 7). Further, Cdx2 induces the expression of (56), a member of the family of transcription factors that regulate G1-S phase transition and DNA replication (57). Although these observations do not establish Cdx2 as an oncogene, they underscore the potential for this transcription factor to contribute to the cancer phenotype. In the context of overexpression in tumors compared with normal mucosa, they suggest a need to define the role of Cdx2 in gastrointestinal tumorigenesis. In conclusion, Cdx2 is overexpressed in most colorectal tumors compared with matched normal mucosa. Overexpres- sion of Cdx2 mRNA was associated with increased Cdx2 protein expression localized in the nuclei of tumor cells. Similarly, increased protein expression was associated with elevated Cdx2-dependent transcriptional activity. Moreover, increased transactivation was associated with overexpression of GCC, an endogenous downstream target of Cdx2. The structural and functional overexpression of Cdx2 in tumors, compared with normal mucosa, suggests that, in contrast to the prevailing paradigm, Cdx2 does not serve as a tumor suppressor in the development of most sporadic colorectal tumors. Rather, in the context of earlier observations of its role in promoting the neoplastic phenotype in some cells and tissues, the present Fig. 4. A, immunofluorescent detection of Cdx2 and LI-cadherin protein in primary observations suggest the intriguing possibility that Cdx2 could cultures of tumor and normal adjacent mucosal cells confirms the presence of intestinal epithelial cells. B, reporter analysis of primary tumor and normal adjacent serve as an oncogene in the . The ability to mucosal cells using the promoter of GCC with wild-type (GCC-Luc) and mutated regulate proneoplastic and antineoplastic pathways highlights (TTT-to-CCC) Cdx2 binding sequences (DGCC-Luc). GCC-Luc and DGCC-Luc the importance of further studies to define the complex roles reporter activities were normalized to aTK-driven Renilla reporter.Values reflect the arithmetic difference between activities obtained with GCC-Luc and DGCC-Luc for Cdx2 in molecular mechanisms underlying human colo- (n =4;*,P < 0.05). rectal carcinogenesis.

References 1. Suh E, Chen L,Taylor J,Traber PG. A homeodomain regulated by Cdx2 in the intestinal epithelium. Am J expression induces gastric intestinal metaplasia protein related to caudal regulates intestine-specific Physiol Gastrointest Liver Physiol 2002;283:G840 ^ 7. in transgenic mice. Gastroenterology 2002;122: gene transcription. Mol Cell Biol 1994;14:7340 ^ 51. 8. BaiYQ, Miyake S, IwaiT,YuasaY.CDX2, a homeobox 689^96. 2. Suh E, Traber PG. An intestine-specific homeobox transcription factor, upregulates transcription of the 14. Abate-Shen C. Deregulated homeobox gene gene regulates proliferation and differentiation. Mol p21/WAF1/CIP1gene. Oncogene 2003;22:7942 ^ 9. expression in cancer: cause or consequence? Nat Cell Biol 1996;16:619^ 25. 9. Mallo GV, Soubeyran P,LissitzkyJC, et al. Expression Rev Cancer 2002;2:777 ^ 85. 3. Traber PG, Silberg DG. Intestine-specific gene of the Cdx1 and Cdx2 homeotic genes leads to 15. GuoRJ,SuhER,LynchJP.Theroleofcdxproteinsin transcription. Annu Rev Physiol 1996;58:275 ^ 97. reduced malignancy in colon cancer-derived cells. intestinal development and cancer. Cancer Biol Ther 4. Park J, SchulzS,Waldman SA. Intestine-specific ac- J Biol Chem 1998;273:14030 ^ 6. 2004;3:593^601. tivity of the human guanylyl cyclase C promoter is reg- 10. Bonhomme C, Duluc I, Martin E, et al. The Cdx2 16. Hinoi T,Tani M, Lucas PC, et al. Loss of CDX2 ex- ulated by Cdx2. Gastroenterology 2000;119:89 ^96. homeobox gene has a tumour suppressor function in pression and microsatellite instability are prominent 5. Fang R, Santiago NA, Olds LC, Sibley E. The homeo- the distal colon in addition to a homeotic role during features of large cell minimally differentiated carcino- domain protein Cdx2 regulates lactase gene promoter gut development. Gut 2003;52:1465 ^ 71. mas of the colon. AmJPathol 2001;159:2239 ^ 48. activity during enterocyte differentiation. Gastroenter- 11. Chawengsaksophak K, James R, Hammond VE, 17. Hinoi T, Loda M, Fearon ER. Silencing of CDX2 ology 2000;118:115^ 27. Kontgen F, Beck F. Homeosis and intestinal tumours expression in colon cancer via a dominant repression 6. HinoiT, Lucas PC, Kuick R, Hanash S, Cho KR, Fearon in Cdx2 mutant mice. Nature1997;386:84 ^ 7. pathway. J Biol Chem 2003;278:44608 ^ 16. ER. CDX2 regulates liver intestine-cadherin expression 12 . Mutoh H, Hakamata Y, Sato K, et al. Conversion of 18. Okon K, Zazula M, Rudzki Z, Papla B, Osuch C, in normal and malignant colon epithelium and intesti- gastricmucosatointestinalmetaplasiainCdx2- Stachura J. -2 expression is reduced in colorectal nal metaplasia. Gastroenterology 2002;123:1565 ^ 77. expressing transgenic mice. Biochem Biophys Res carcinomas with solid growth pattern and proximal 7. Uesaka T, Lu H, Katoh O, Watanabe H. Heparin- Commun 2002;294:470 ^ 9. location, but is largely independent of MSI status. binding EGF-like growth factor gene transcription 13 . Silberg DG, Sullivan J, Kang E, et al. Cdx2 ectopic Pol J Pathol 2004;55:9 ^ 14.

www.aacrjournals.orgClin Cancer Res 2005;11(24) December8555 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Human Cancer Biology

19. Mallo GV, Rechreche H, Frigerio JM, et al. Molecu- reverse transcription PCR (RT-PCR): trends and prob- sequence. Analogies with embryonic . Am lar cloning, sequencing and expression of the mRNA lems. JMol Endocrinol 2002;29:23 ^ 39. J Pathol 2000;157:1113^ 21. encoding human Cdx1 and Cdx2 homeobox. Down- 32. Silberg DG, Swain GP, Suh ER,Traber PG. Cdx1and 45. BrabletzT, Jung A, Reu S, et al. Variable h-catenin regulation of Cdx1 and Cdx2 mRNA expression cdx2 expression during intestinal development. Gas- expression in colorectal cancers indicates tumor pro- during colorectal carcinogenesis. Int J Cancer 1997; troenterology 2000;119:961 ^ 71. gression driven by the tumor environment. Proc Natl 74:35 ^ 44. 33. Lucas KA, Pitari GM, Kazerounian S, et al. Guanylyl Acad Sci U S A 2001;98:10356^61. 20. Ee HC, Erler T, Bhathal PS, Young GP, James RJ. cyclases and signaling by cyclic GMP. Pharmacol Rev 46. Thiery JP. Epithelial-mesenchymal transitions in Cdx-2 homeodomain protein expression in human 2000;52:375 ^ 414. tumour progression. Nat Rev Cancer 2002;2:442 ^ 54. and rat colorectal adenoma and carcinoma. Am J 34. Pitari GM, Di Guglielmo MD, Park J, SchulzS, 47. Yoshida BA, Sokoloff MM, Welch DR, Rinker- Pathol1995;147:586^92. Waldman SA. Guanylyl cyclase C agonists regulate Schaeffer CW. Metastasis-suppressor genes: a review 21.WerlingRW,YazijiH,BacchiCE,GownAM.CDX2,a progression through the cell cycle of human colon car- and perspective on an emerging field. J Natl Cancer highly sensitive and specific marker of adenocarcino- cinoma cells. Proc Natl Acad Sci U S A 2001;98: Inst 2000;92:1717^30. mas of intestinal origin: an immunohistochemical 7846 ^ 51. 48. Douglas EJ, Fiegler H, Rowan A, et al. Array com- survey of 476 primary and metastatic carcinomas. Am 35. Pitari GM, Zingman LV,Hodgson DM, et al.Bacterial parative genomic hybridization analysis of colorectal J Surg Pathol 2003;27:303 ^ 10. enterotoxins are associated with resistance to colon cancer cell lines and primary carcinomas. Cancer Res 22. Kaimaktchiev V,Terracciano L, Tornillo L, et al. The cancer. Proc Natl Acad Sci U S A 2003;100:2695 ^ 9. 2004;64:4817^25. homeobox intestinal differentiation factor CDX2 is 36. Shailubhai K,Yu HH, Karunanandaa K, et al. Urogua- 49. Kojima M, Morisaki T, Sasaki N, et al. Increased selectively expressed in gastrointestinal adenocarci- nylin treatment suppresses polyp formation in the nuclear factor-kB activation in human colorectal carci- nomas. Mod Pathol 2004;17:1392 ^ 9. Apc(Min/+) mouse and induces apoptosis in human noma and its correlation with tumor progression. 23. Beck F, Chawengsaksophak K, Waring P, Playford colon adenocarcinoma cells via cyclic GMP. Cancer Anticancer Res 2004;24:675 ^ 81. RJ, Furness JB. Reprogramming of intestinal differen- Res 2000;60:5151 ^ 7. 50. Kim S, Domon-Dell C, Wang Q, et al. PTEN and tiation and intercalary regeneration in Cdx2 mutant 37. Swenson ES, Mann EA, Jump ML, Giannella RA. TNF-a regulation of the intestinal-specific Cdx-2 mice. Proc Natl Acad Sci U S A1999;96:7318^ 23. Hepatocyte nuclear factor-4 regulates intestinal ex- homeobox gene through a PI3K, PKB/Akt, and NF- 24. Aoki K, Tamai Y, Horiike S, Oshima M, Taketo MM. pression of the guanylin/heat-stable toxin receptor. nB-dependent pathway. Gastroenterology 2002;123: Colonic polyposis caused by mTOR-mediated chro- Am J Physiol 1999;276:G728 ^ 36. 116 3 ^ 7 8 . mosomal instability in Apc+/D716 Cdx2+/À com- 38. Sherr CJ. Principles of tumor suppression. Cell 51. Xu F, Li H, JinT. Cell type-specific autoregulation of pound mutant mice. Nat Genet 2003;35:323^ 30. 2004;116:235 ^ 46. the Caudal-related homeobox gene Cdx-2/3. J Biol 25. BrabletzT, Spaderna S, Kolb J, et al. Down- 39. Woodford-Richens KL, Halford S, Rowan A, et al. Chem 1999;274:34310^6. regulation of the homeodomain factor Cdx2 in colo- CDX2 mutations do not account for juvenile polyposis 52. Uesaka T, Kageyama N. Cdx2 homeodomain pro- rectal cancer by collagen type I: an active role for the or Peutz-Jeghers syndrome and occur infrequently in tein regulates the expression of MOK, a member of tumor environment in malignant tumor progression. sporadic colorectal cancers. Br J Cancer 2001;84: the mitogen-activated protein kinase superfamily, in Cancer Res 2004;64:6973 ^ 7. 1314 ^ 6. the intestinal epithelial cells. FEBS Lett 2004;573: 26. LorentzO, Duluc I, Arcangelis AD, Simon-Assmann 40. Rings EH, Boudreau F,Taylor JK, Moffett J, Suh ER, 147 ^ 5 4. P, Kedinger M, Freund JN. Key role of the Cdx2 ho- Traber PG. Phosphorylation of the serine 60 residue 53. Rawat VP, Cusan M, Deshpande A, et al. Ectopic meobox gene in extracellular matrix-mediated intesti- within the Cdx2 activation domain mediates its expression of the homeobox gene Cdx2 is the trans- nal cell differentiation. J Cell Biol 1997;139:1553 ^ 65. transactivation capacity. Gastroenterology 2001;121: forming event in a mouse model of t(12;13)(p13;q12) 27.Warnecke-EberzU, Hokita S, Xi H, et al. Overexpres- 14 37 ^ 5 0. . Proc Natl Acad Sci U S A sion of survivin mRNA is associated with a favorable 41. Moskaluk CA, Zhang H, Powell SM, Cerilli LA, 2004;101:817^ 22. prognosis following neoadjuvant radiochemotherapy Hampton GM, Frierson HF, Jr. Cdx2 protein expres- 54. Mutoh H, Sakurai S, Satoh K, et al. Development of in esophageal cancer. Oncol Rep 2005;13:1241 ^6. sion in normal and malignant human tissues: an immu- gastric carcinoma from intestinal metaplasia in Cdx2- 28. Chotteau-Lelievre A, Revillion F, Lhotellier V, et al. nohistochemical survey using tissue microarrays. Mod transgenic mice. Cancer Res 2004;64:7740 ^7. Prognostic value of ERM in human Pathol 2003;16:913 ^ 9. 55.TamaiY, Nakajima R, IshikawaT,Takaku K, Seldin MF, primary breast cancers. Clin Cancer Res 2004;10: 42.Wicking C, Simms LA, EvansT, et al. CDX2, a human Taketo MM. Colonic hamartoma development by 7297 ^ 303. homologue of Drosophila caudal, is mutated in both anomalous duplication in Cdx2 knockout mice. Cancer 29. Hermanek P. Colorectal carcinoma: histopathologi- alleles in a replication error positive colorectal cancer. Res 1999;59:2965 ^ 70. cal diagnosis and staging. Baillieres Clin Gastroenterol Oncogene 1998;17:657 ^ 9. 56. Uesaka T, Kageyama N, Watanabe H. Identifying 19 8 9;3 :511 ^ 29. 43.Yagi OK, AkiyamaY,YuasaY. Genomic structure and target genes regulated downstream of Cdx2 by micro- 30. Ries LAG, Eisner MP, Kosary CL, et al. SEER cancer alterations of homeobox gene CDX2 in colorectal array analysis. JMol Biol 2004;337:647 ^ 60. statistics review. 1975-2002 [cited; Available from: carcinomas. BrJ Cancer 1999;79:440 ^ 4. 57. Ren B, Cam H,Takahashi Y, et al. E2F integrates cell http://seer.cancer.gov/csr/1975_2002/. 44. Kirchner T, BrabletzT. Patterning and nuclear h- cycle progression with DNA repair, replication, and 31. Bustin SA. Quantification of mRNA using real-time catenin expression in the colonic adenoma-carcinoma G(2)/M checkpoints. Genes Dev 2002;16:245 ^ 56.

Clin Cancer Res 2005;11(24) December 15, 2005 8556 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. The Putative Tumor Suppressor Cdx2 Is Overexpressed by Human Colorectal Adenocarcinomas

Matthew E. Witek, Karl Nielsen, Rhonda Walters, et al.

Clin Cancer Res 2005;11:8549-8556.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/11/24/8549

Cited articles This article cites 56 articles, 22 of which you can access for free at: http://clincancerres.aacrjournals.org/content/11/24/8549.full#ref-list-1

Citing articles This article has been cited by 6 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/11/24/8549.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/11/24/8549. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research.