Expression of the Carcinoembryonic Antigen Gene Is Inhibited by SOX9 in Human Colon Carcinoma Cells

Research Article

Philippe Jay, Philippe Berta, and Philippe Blache

Institut de Ge´ne´tique Humaine, Centre National de la Recherche Scientifique UPR1142, Montpellier, France

Abstract serum and glutamine. HT29Cl.16E cells were transfected using Tfx-50 reagents (Promega, Madison, WI). N-terminally flagged wild-type SOX9 The human carcinoembryonic antigen (CEA) is overexpressed and C-terminally truncated DC206SOX9 expression constructs (11, 12) in many types of human cancers and is commonly used as a were used to generate stable transfectant HT29Cl.16E cells inducibly clinical marker. In colon cancer, this overexpression protects expressing SOX9 or a dominant-negative form of SOX9 (DC206SOX9; T-Rex cells against apoptosis and contributes to carcinogenesis. System, Invitrogen, San Diego, CA). The ‘‘SOX-luciferase’’ reporter Therefore, CEA-expressing cells as well as CEA expression construct consists of seven copies of the AACAAAG sox-binding sequence, itself constitute potential therapeutic targets. In this report, inserted upstream of a minimal herpes simplex thymidine kinase we show that the transcription factor SOX9 down-regulates promoter, and the control ‘‘SAC-luciferase’’ construct consists of seven CEA gene expression and, as a probable consequence, induces copies of the CCGCGGT sequence (generous gift from Prof. H. Clevers). apoptosis in the human colon carcinoma cell line HT29Cl.16E. The pCMV-Luc and CEA424Luc vectors (13) were provided by Dr. W. Zimmermann. (Cancer Res 2005; 65(6): 2193-8) Reverse Transcription-PCR. Total RNA was prepared with RNeasy kit (Qiagen, Courtaboeuf, France). One microgram of total RNA was used Introduction to prepare cDNA with M-MuLV reverse transcriptase (New England The carcinoembryonic antigen (CEA), a member of the Biolabs, Beverly, MA). Primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are forward 5V-GAC CAC AGT CCA TGC CAT CAC immunoglobulin superfamily, was originally identified in human T-3V and reverse 5V-TCC ACC ACC CTG TTG CTG TAG-3V; and for CEA, fetal colon and colorectal cancers, and is widely used as a clinical forward 5V-GGG CCA CTG TCG GCA TCA TGA TTG G-3V and reverse 5V- tumor marker. This cell-surface glycoprotein is normally expressed TGT AGC TGT TGC AAA TGC TTT AAG GAA GAA GC-3V. GAPDH primer in a variety of epithelial tissues such as the urogenital, respiratory, set was amplified at 95jC for 30 seconds, 60jC for 30 seconds, and 72jC for and gastrointestinal tracts. In the healthy human colon, the CEA 1 minute for 15 cycles followed by a 10-minute extension at 72jC. CEA protein is restricted to the apical side of the well-differentiated primer set was amplified at 95jC for 30 seconds, 60jC for 30 seconds, and epithelial cells forming the luminal surface (1–3). Little is known 72jC for 1 minute for 30 cycles followed by a 10-minute extension at 72jC. about CEA function, but it acts at least as a homotypic adhesion Amplified PCR products were electrophoresed on a 2% agarose gel molecule and is implicated in cell aggregation (3). CEA is containing ethidium bromide (0.5 Ag/mL). overexpressed in numerous human cancers, where, in contrast Quantitative Reverse Transcription-PCR. RNA extraction and cDNA synthesis were done as above. Amplification was conducted in a LightCycler with healthy tissues, it is present on the entire surface of cancer (Roche, Idaho Falls, ID) using the QuantiTect SYBER Green PCR kit cells. Up-regulation of CEA occurs at the microadenoma stage in (Qiagen). Primers specific for GAPDH are forward 5V-GAG AAG GCT GGG the colon of patients with APC mutations (4), and activation of GCT CAT-3V and reverse 5V-TGC TGA TGA TCT TGA GGC TG; for CEA, oncogenic c-Ki-ras proteins during colon cancer progression results forward 5V-GGG CCA CTG TCG GCA TCA TGA TTG G-3Vand reverse 5V-TGT in up-regulation of CEA expression and disruption of basolateral AGC TGT TGC AAA TGC TTT AAG GAA GAA GC-3V; for human mucin 2, polarity (5). Recently, Wirth et al. (6) have shown that CEA has forward 5V-TGG GTG TCC TCG TCT CCT ACA-3V and reverse 5V-TGT TGC antiapoptotic and prometastatic roles in colon cancer cells, and CAA ACC GGT GGT A; for human intestinal alkaline phosphatase, forward Ordonez et al. (7) reported that overexpression of CEA can protect 5V-CTC CAA CAT GGA CAT TGA CG and reverse 5V-CAG TGC GGT TCC tumor cells from undergoing anoikis (apoptosis induced by loss of ACA CAT AC-3V; for human upstream stimulatory factor-1, forward 5V-ACC V V cell contact with the extracellular matrix). Although decreasing CAA CCA GTG TGG CTA TTG-3 and reverse 5-GGG TCA TGG ATT GAG TGG CA-3V; and for human specificity protein 1, forward 5V-CCT GGT CAT CEA expression in cancer cells might lead to new approaches for ACT GTG GGA AAC G and reverse 5V-CAC TCC TCA TGA AGC GCT TAG G- the management of cancers of the colon and other organs (8, 9), 3V. The cycling variables were 15 minutes at 95jC, followed by 40 cycles of little is known about the regulation of CEA expression. We show 15 seconds at 94jC, 25 seconds at 62jC, and 15 seconds at 72jC. GAPDH,a here that forced expression of SOX9, a novel intestinal crypt common housekeeping gene, was used as an internal control for an equal transcriptionfactor(10),inhibitsCEA gene expression and induces amount of starting material. apoptosis in a human colon carcinoma cell line. Western Blot Analysis. After Bradford quantification, equal amounts of total cellular protein extracts were electrophoresed on an acrylamide Materials and Methods denaturing gel and transferred by electroblotting onto a nitrocellulose membrane. Primary antibodies used were rabbit anti-SOX9 (11), rabbit Cell Culture and Transfection. HT29Cl.16E cells (courtesy of C. anti-CEA (Neomarkers, Fremont, CA) and M2 monoclonal antibody anti- Laboisse) were cultured in DMEM supplemented with 10% fetal bovine Flag (Sigma, St. Louis, MO). Secondary antibodies were from Amersham Bioscience Europe (Saclay, France). Blots were developed using the enhanced chemiluminescence procedure (Amersham Bioscience Europe). Immunohistochemistry. Sections of paraffin-embedded normal human Requests for reprints: Philippe Blache, Institut de Ge´ne´tique Humaine, Centre colon were generously provided by Dr. C. Marty-Double (CHU, Nıˆmes, France). National de la Recherche Scientifique UPR1142, 141 rue de la Cardonille, 34396 For immunohistochemistry, Envision+ (DAKO, Trappes, France) was used as Montpellier cedex 5, France. Phone: 33-499-61-99-43; Fax: 011-33-499-61-99-42; E-mail: [email protected]. a secondary reagent, staining was developed with 3,3V-diaminobenzidine I2005 American Association for Cancer Research. (brown precipitate), and a haematoxylin counterstain was used. www.aacrjournals.org 2193 Cancer Res 2005; 65: (6). March 15, 2005

Detection of Apoptotic Cells by Terminal Deoxynucleotidyl Trans- absorptive cells are induced (16, 17). During both confluence- ferase–Mediated Nick End Labeling Assay. Apoptotic nuclei of cultured induced or NaB-induced differentiation of HT29Cl.19E cells, the cells were visualized with DeadEnd Colorimetric Apoptosis Detection level of SOX9 protein progressively decreased as cells reached (Promega). confluence and differentiated (Fig. 1C). Simultaneously, we Statistical Analysis. Differences between groups of data were analyzed observed a strong increase of CEA protein expression (Fig. 1C), using the Student’s t test. which is consistent with previous reports (15). Down-regulation of SOX9 expression and up-regulation of CEA expression, Results and Discussion observed during the differentiation of HT29Cl.16E cells, mimic the SOX9 and CEA regulation observed in the tissue when SOX9 Expression in the Human Colon. The epithelium of the epithelial cells differentiate and migrate from the base to the top adult human colon is a flat surface with many invaginations of the crypts. Indeed, SOX9 is strongly and specifically expressed known as crypts. This epithelium is continuously renewed from in crypt cells (Fig. 1A and B) whereas CEA is expressed only in undifferentiated multipotent stem cells located at the base of the the differentiated cells constituting the colon surface (2). crypts. Stem cells divide to produce three types of daughter cells SOX9 Down-regulates CEA Expression. With the aim of (absorptive, goblet, and endocrine). Goblet cells are the most determining whether the increase of CEA protein expression abundant cell type in the colon. during differentiation was linked to the SOX9 down-regulation, SOX transcription factors belong to the superfamily of high we generated inducible stable transfectant cell lines expressing mobility group proteins, and are involved in tissue development either a Flag-tagged wild-type SOX9 (HT29Cl.16E-SOX9) or a and cell fate choice (14). Recently, we have shown that the SOX9 Flag-tagged dominant-negative form of SOX9 (HT29Cl.16E- protein is expressed in the mouse intestine epithelium where it DC206SOX9). The SOX9 dominant-negative protein still binds plays a crucial role in the preservation of its integrity, and that the to DNA but does not activate transcription of target genes h Wnt/ -catenin/TCF4 pathway is needed for SOX9 expression in the because the C-terminal transcription activation domain is intestine epithelium (10). As a consequence, SOX9 is also strongly lacking (11, 12). After doxycycline treatment of the expressed in all the colon carcinoma cell lines tested, which present HT29Cl.16E-SOX9 cells, the exogenous SOX9 protein was readily h a constitutive activity of the -catenin/TCF4 complex, due to detected using an antibody against the FLAG tag (Fig. 2A, top). activating mutations in components of the Wnt pathway. We used Induction of exogenous SOX9 expression resulted in a clear immunohistochemistry to study SOX9 expression in sections of increase of the total SOX9 protein (Fig. 2A, bottom). The C- healthy human colon. As shown in Fig. 1A, a strong nuclear staining terminally truncated SOX9 protein (DC206SOX9) could not was observed only in epithelial cells, with an expression gradient be detected with the anti-SOX9 antibody, directed against the from the bottom of the crypts to the mid-crypts. No nuclear SOX9 deleted C-terminal region, but the expression of the doxycycline- staining was detected in the mature epithelial cells constituting the induced DC206SOX9 protein was efficiently detected with the colon surface and we have previously shown that the slight anti-FLAG antibody (Fig. 2A). To analyze the transcriptional cytoplasmic staining observed at the top of the crypts is not activity of the doxycycline-induced SOX9 and DC206SOX9 specific for SOX9 (10). Staining for SOX9 was observed in all the proteins, inducible HT29Cl.16E-SOX9 and HT29Cl.16E- epithelial cells around the crypts from colon cross-sections (Fig. 1B). DC206SOX9 cells were transfected with a ‘‘sox-luciferase’’ reporter SOX9 and CEA Expression during Differentiation of construct consisting of consensus Sox binding sites and a HT29Cl.16E Cells. This human colon carcinoma cell line, thymidine kinase minimal promoter, controlling the expression originally cloned from the HT29 cell line, spontaneously differ- of a luciferase coding sequence. The basal luciferase activity was entiates as goblet-like cell lineage when maintained in confluent strongly increased after induction of SOX9 expression by culture (15). Alternatively, when treated with sodium butyrate doxycycline whereas it decreased by 50% on induction of (NaB) aspects of this lineage are repressed and markers of DC206SOX9 expression (Fig. 2B). This indicates that the

Figure 1. Immunohistochemical staining of SOX9 in healthy human colon tissue. A, longitudinal section of colon crypts reveals a nuclear SOX9 staining in cells of the lower part of the crypt. B, cross section of colon crypts reveals a nuclear SOX9 staining of the cells around the bottom of the crypt. Original magnification, Â500. C, Western blot analysis of SOX9 and CEA expression in HT29Cl.16E cells during spontaneous and NaB-induced differentiation.

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Figure 2. Down-regulation of CEA expression by SOX9 in HT29Cl.16E cells. A, Western blot analysis of SOX9 expression on doxycycline induction in HT29Cl. 16E-SOX9 cells with anti-SOX9 and anti-FLAG antibodies. B, effect of forced SOX9 and DC206SOX9 expression on the ‘‘SOX-luciferase’’ reporter activity. Columns, mean (n = 3); bars, SE. C, RT-PCR analysis of CEA expression on doxycycline induction in HT29Cl.16E-SOX9 cells after 2 days of culture in nonconfluent conditions. D, quantification by real-time RT-PCR analysis of CEA mRNA levels on doxycycline induction in HT29Cl.16E-SOX9 cells after 2 days of culture in nonconfluent conditions. After normalization to GAPDH, the results (closed column) are expressed as the fold increase of expression compared with the control (open column). Columns, mean of three determinations; bars, SE. *, P < 0.001. E, Western blot analysis of CEA and Flag-SOX9 expression on doxycycline induction in HT29Cl.16E-SOX9 during spontaneous and NaB-induced differentiation.

exogenous SOX9 and DC206SOX9 proteins affect the transcrip- expression is forced. Expression of the CEA protein was also strongly tion of the SOX reporter gene, as expected. increased when cells with a secretory phenotype were induced to Next, to analyze the effect of inducing the expression of SOX9 on switch to an absorptive phenotype by NaB treatment and this CEA expression level, HT29Cl.16E-SOX9 cells were grown for 2 expression was also reduced on induction of SOX9 expression. Thus, days, in nonconfluent conditions, in the presence and absence of independently of the cell lineage, the down-regulation of SOX9 doxycycline. Reverse transcription-PCR (RT-PCR) analysis of CEA expression is required to allow the up-regulation of CEA expression expression revealed that the basal level of the CEA mRNA associated with differentiation of colon cancer cells. decreased after induction of SOX9 expression (Fig. 2C). A typical Endogenous SOX9 Limits the Basal CEA Expression Level in experiment of real-time RT-PCR is represented in Fig. 2D. HT29Cl.16E Cells. Inducible HT29Cl.16E-DC206SOX9 cells were Quantification indicated a 3.45-fold reduction (three determina- grown for 2 days in nonconfluent conditions in the presence or tions; P < 0.001) of the CEA mRNA levels on induction of SOX9 absence of doxycycline. RT-PCR analysis revealed that the CEA expression. Thus, SOX9 inhibits the expression of CEA. mRNA level was increased in cells expressing the DC206SOX9 HT29Cl.16E cells differentiate along the secretory lineage when protein (Fig. 3A) and real-time RT-PCR quantified this increase maintained in confluent culture, and when treated with NaB, aspects at 1.46-fold (three determinations; P < 0.001; Fig. 3B). An of this lineage are repressed and markers of an absorptive cells may increase of CEA protein level was also observed when be induced (17). In both cases, as this differentiation results in a DC206SOX9 expression was induced for 5 days (Fig. 3B). This strong increase of CEA expression and a decrease of SOX9 indicates that the endogenous SOX9 protein present in expression, we asked whether forced SOX9 expression during the exponentially growing colon cancer cells limits the expression differentiation process, along either of the two lineages, would affect of CEA. Interfering with the function of this endogenous SOX9 the expression level of the endogenous CEA protein. To test this, the with a dominant-negative protein abrogates this inhibition and CEA protein expression was analyzed before and after confluence- results in increased CEA expression. Thus, in exponentially induced or NaB-induced differentiation and with or without growing HT29Cl.16E cells, strong SOX9 expression limits CEA doxycycline induction of SOX9 expression (Fig. 2E). After 5 days of transcription. During differentiation, SOX9 expression is down- culture, when the cells were not yet confluent, the level of CEA regulated, probably as a result of the decrease of h-catenin/TCF4 protein was significantly lower in cells with forced expression of signaling which was already described in Caco-2 cells (18). SOX9. After 23 days of culture, when the cells are differentiated into This down-regulation of SOX9, in turn, derepresses the goblet-like cells and endogenous SOX9 has been down-regulated, expression of CEA. Consistent with this, in the human colon, the CEA protein was strongly expressed in non-doxycycline-treated SOX9 is expressed specifically in actively proliferating cells cells whereas it was almost undetectable in cells where SOX9 located at the bottom of the crypts, where CEA is absent. This www.aacrjournals.org 2195 Cancer Res 2005; 65: (6). March 15, 2005

activity (19). Footprinting identified five putative cis-acting elements in the 1,098 bp sequence situated upstream of the initiation codon (ref. 19; Fig. 5A). Among the five cis-acting elements found in the CEA promoter, four are positive regulatory elements (FP1-FP4) and one is a negative regulatory element (FP5; ref. 19). When transiently transfected into HT29Cl.16E cells, the full- length SOX9 efficiently activated transcription of a SOX-luciferase reporter construct (three different experiments; P < 0.001). The SOX9 protein contains a high mobility group domain, which mediates sequence-specific DNA binding, and a C-terminally located transcription activation domain (12). The DC206SOX9 construct, lacking the C-terminal transcription activation domain (Fig. 5B), did not activate the SOX-luciferase reporter. On the contrary, the DC206SOX9 construct acted as a dominant-negative protein (three different experiments; P = 0.005) interfering with the basal luciferase activity driven by endogenous SOX9 (Fig. 5B). Next, these two constructs were transiently transfected into HT29Cl.16E cells together with the CEA minimal promoter (CEA424Luc). Cotransfection of the CEA424Luc vector with the full-length SOX9- Figure 3. A, RT-PCR analysis of CEA expression on doxycycline induction in expressing vector produced a 3-fold significant decrease (three HT29Cl.16E-DC206SOX9 cells after 2 days of culture in nonconfluent conditions. different experiments; P = 0.002) of the luciferase activity (Fig. 5C) B, quantification by real-time RT-PCR analysis of CEA mRNA levels on doxycycline induction in HT29Cl.16E-DC206SOX9 cells after 2 days of culture in whereas cotransfection of the DC206SOX9 truncated protein had nonconfluent conditions. After normalization to GAPDH, the results (closed no significant effect (three different experiments; P = 0.464). These column) are expressed as fold increase of expression with relation to the control experiments show that SOX9 inhibits the CEA minimal promoter (open column). Columns, mean of three determinations; bars, SE. *, P < 0.001. C, C, Western blot analysis of CEA and Flag-DC206SOX9 expression on activity in HT29Cl.16E cells and that the transcription activation doxycycline induction in HT29Cl.16E-DC206SOX9 cells in nonconfluent culture. domain of SOX9 is required for this inhibition. Moreover, this inhibition is not due to the FP5 silencer element, which is absent suggests that CEA expression might also be inhibited by SOX9 from the 424 bp minimal CEA promoter. in vivo, but this remains to be shown. When we analyzed the 424 bp sequence of the CEA minimal SOX9 Inhibits Differentiation of Both Secretory and promoter, we found that it lacks the SOX9 optimal binding Absorptive Cell Lineage. We have previously shown that SOX9 sequence AGAACAATGG (20). Furthermore, the SOX consensus inhibits expression of the mucin 2 (MUC2) gene, a goblet cell- binding sequence AACAAAG, present in the ‘‘SOX-luciferase’’ specific differentiation marker (10), in human colon carcinoma reporter construct, was not found in the 424 bp sequence. This goblet-like cells. We then asked whether SOX9 is also able to analysis was then extended to the 10.8 kb of the CEA gene, situated inhibit the differentiation of HT29Cl.16E-SOX9 cells into the upstream of the initiation codon (Genbank accession no. Z21818), absorptive phenotype. For that purpose, we treated the and again no putative SOX9 or SOX consensus binding site could HT29Cl.16E-SOX9 cells with NaB, which is known to promote be detected. differentiation of the HT29Cl.16E cells along the absorptive phenotype (17). The expression of the intestinal alkaline phosphatase gene (ALP), which is an enterocyte-specific marker, was analyzed by real-time quantitative RT-PCR upon induction of SOX9 expression with doxycycline. As previously reported (17), expression of alkaline phosphatase was increased by NaB treatment, and this increase was inhibited by forced SOX9 expression (Fig. 4). CEA expression, which is normally found in both the secretory and the absorptive cell lineages, was increased by NaB, and this increase was strongly inhibited by SOX9 (Fig. 4). As basal expression of mucin 2 was weak and not significantly modified by NaB (Fig. 4) or forced SOX9 expression, we concluded that the observed decrease in alkaline phosphatase expression upon SOX9 overexpression was not a consequence of a SOX9-mediated reorientation of the enterocyte fate into a goblet fate. Instead, SOX9 inhibits differentiation of HT29Cl.16E- SOX9 cells into both the secretory and absorptive cell lineages. SOX9 Repression of the CEA Gene Promoter. The minimal CEA promoter was first described as a 424 bp sequence upstream of the translation initiation site (À424 to +1) conferring cell-type- Figure 4. SOX9 inhibits the differentiation of HT29Cl.16E cells into an absorptive specific expression of a reporter gene (13). Later, it was shown that phenotype. After 3 days of culture in nonconfluent conditions, HT29Cl. the sequence between À403 and À124 bp directed high levels of 16E-SOX9 were treated with NaB in the absence or presence of doxycycline for 3 additional days. Quantifications of CEA, intestinal alkaline phosphatase, and expression in colon carcinoma cells and that inclusion of an mucin 2 transcripts were done by real-time RT-PCR. After normalization to additional upstream sequence (À1,098 to À403 bp) repressed all the GAPDH, the results are expressed as fold increase of expression.

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SOX9 expression in exponentially growing or differentiated HT29Cl.16E cells, under conditions in which CEA expression was efficiently down-regulated by SOX9. No significant variation of upstream stimulatory factor-1 or specificity protein 1 could be detected, indicating that the SOX9-mediated repression of CEA does not involve regulation of the upstream stimulatory factor-1 and specificity protein 1 transcription factors. Several other still unknown transcription factors bind to the minimal CEA promoter (FP1-FP4; Fig. 5A; ref. 19), and the possible deregulation of these nuclear factors by SOX9 might explain the inhibition of expression of CEA by SOX9. Effect of SOX9 Expression on Apoptosis. One of the well- described consequences of CEA deregulated overexpression in cancer cells is the inhibition of apoptosis (6). As SOX9 expression results in a reduction of CEA protein level, then it might be expected to induce apoptosis. To test this hypothesis, we stained the inducible HT29Cl.16E-SOX9 cells by the terminal deoxynucleotidyl transferase–mediated nick end labeling assay on induction of SOX9 expression. A typical microscope field of stained cells is shown on Fig. 6A. The analysis of a significant number of fields (n = 22) revealed that cells with forced expression of SOX9 were more inclined to undergo apoptosis (7% F 1.2 for induced cells versus 0.8% F 0.3 for the control without doxycycline; Fig. 6C). This result indicates that forced SOX9 expression increases the apoptotic rate of HT29Cl.16E cells, potentially due to the down- regulation of the CEA protein. Figure 5. SOX9 negatively regulates the CEA gene promoter. A, schematic In summary, clinical studies have suggested that CEA expression representation of the CEA promoter. Arrow, transcriptional start site. B, HT29Cl.16E cells were transfected in triplicate with the indicated constructs is correlated with metastatic growth of colon cancer, and a (50 ng) together with 500 ng of the ‘‘SOX-luciferase’’ reporter vector or of the preoperative elevated serum level of CEA is therefore associated control ‘‘SAC-luciferase’’ vector. Luciferase activities of the ‘‘SOX-luciferase’’ with a poor prognostic (23). Moreover, CEA plays a direct role in vector were normalized with the luciferase activities of the ‘‘SAC-luciferase’’ vector. Ratios are relative to the empty vector, arbitrarily set to 100%. Columns, metastatic behavior because, first, inhibition of its endogenous mean of three different experiments; bars, SE. *, P < 0.001; ** P = 0.005. level inhibits the metastatic tumor growth of colon cancer cells (6), C, HT29Cl.16E cells were transfected in triplicate with the indicated construct and second, CEA is antiapoptotic (24) and inhibits anoikis (7). All (50 ng) together with 500 ng of the CEA424Luc vector or of the CMVLuc vector. Luciferase activities of the CEA424Luc vector were normalized with the these functions of CEA in colon cancer cells are potentially luciferase activities of the CMVLuc vector. Ratios are relative to the empty vector, arbitrarily set to 100%. Columns, mean of three different experiments; bars, SE. *, P = 0.002; ** P = 0.464.

Direct binding of SOX9 to the minimal promoter of CEA, causing inhibition of its activity, is very improbable for at least three reasons. First, SOX9 function has been analyzed in a number of physiologic situations, including chondrogenesis (21), sex determi- nation (11), intestinal epithelium physiology (10), and nervous system development (22), and it has always been found to be a transcriptional activator. Second, the transcription activation domain of SOX9 is required for the observed inhibition of the CEA gene promoter. Third, sequence analysis of the CEA gene promoter (10.8 kb) did not reveal any putative SOX9 binding sequence. Taken together, these data indicate that most likely SOX9 does not regulate CEA expression by direct binding to the CEA promoter but rather regulates the expression of one or several nuclear factors implicated in CEA promoter activity. Despite its medical importance, little is known about the transcriptional regulation of the CEA gene. Only two transcription factors are known to bind the CEA promoter. Specificity protein 1 recognizes FP2 and FP3 regulatory elements, and upstream stimulatory factor-1 recognizes the FP1 regulatory element and activates the CEA gene promoter in vivo (19). To determine whether Figure 6. Detection of apoptotic cells using the terminal deoxynucleotidyl these factors are involved in the regulation of the CEA gene by SOX9, transferase–mediated nick end labeling assay. A, HT29Cl.16E-SOX9 cells cultured in the presence of doxycycline. B, control HT29Cl.16E-SOX9 we used real-time PCR to monitor upstream stimulatory factor-1 cells cultured without doxycycline. C, % apoptotic cells on SOX9 doxycycline and specificity protein 1 expressions upon doxycycline induction of induction. www.aacrjournals.org 2197 Cancer Res 2005; 65: (6). March 15, 2005

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Cancer Research reversible by SOX9, as it inhibits CEA expression. SOX9 is the first Acknowledgments tissue-specific transcription factor shown to regulate the CEA gene. Received 5/4/2004; revised 12/8/2004; accepted 1/4/2005. We also show that SOX9 induces apoptosis, and we propose that Grant support: ‘‘Centre National de la Recherche Scientifique’’, ‘‘Association pour la this is due to the abolition of the antiapoptotic effect of CEA. Recherche sur le Cancer’’, grant no. ARC3570 (P. Jay), and ‘‘Ligue Nationale Contre le Cancer, comite´ Languedoc-Roussillon.’’ Recently, it has been described that overexpression of CEA provides The costs of publication of this article were defrayed in part by the payment of page a tumorigenic contribution to colon carcinogenesis (25), and the charges. This article must therefore be hereby marked advertisement in accordance authors concluded their article by suggesting that ‘‘reversing the with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Dr. Daniel Fisher for constructive discussions and reviewing of the overexpression of CEA which occurs in so many different human manuscript, Dr. C. Laboisse (Institut National de la Sante et de la Recherche cancers represents an appealing novel approach for cancer Medicale U539, Nantes, France) for the HT26Cl16E cell line, Prof. H. Clevers (Utrecht, Netherlands) and Dr. W. Zimmermann (Department of Urology, University treatment.’’ Our study shows that the transcription factor SOX9 Clinic Grosshadern, Muenchen, Germany) for reagents, and Dr. A. Pe`legrin for is able to reverse this CEA overexpression in colon cancer cells. helpful suggestions.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Expression of the Carcinoembryonic Antigen Gene Is Inhibited by SOX9 in Human Colon Carcinoma Cells

Philippe Jay, Philippe Berta and Philippe Blache

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