Epigenetic Silencing of the Sulfate Transporter Gene DTDST Induces Sialyl Lewis Expression and Accelerates Proliferation of Colo

Published OnlineFirst May 11, 2010; DOI: 10.1158/0008-5472.CAN-09-2383 Published OnlineFirst on May 11, 2010 as 10.1158/0008-5472.CAN-09-2383

Tumor and Stem Cell Biology Cancer Research Epigenetic Silencing of the Sulfate Transporter Gene DTDST Induces Sialyl Lewisx Expression and Accelerates Proliferation of Colon Cancer Cells

Akiko Yusa1,2, Keiko Miyazaki1,2, Naoko Kimura1, Mineko Izawa2, and Reiji Kannagi1,2

Abstract Colon cancer cells express the carbohydrate determinant sialyl Lewisx, while they exhibit markedly decreased the expression of its sulfated derivative, sialyl 6-sulfo Lewisx. In contrast, normal colonic epithelial cells strongly express sialyl 6-sulfo Lewisx, but they virtually do not express sialyl Lewisx. Impaired sulfation was therefore suggested to occur during the course of malignant transformation of colonic epithelial cells and was assumed to be responsible for the increased sialyl Lewisx expression in cancers. To elucidate the molecular biological background of the impaired sulfation in cancers, we studied the expression levels of mRNA for 6-O-sulfotransferase isoenzymes, PAPS synthases and transporters, and a cell membrane sulfate transporter, DTDST, in cancer tissues. The most striking decrease in cancer cells compared with nonmalignant epithelial cells was noted in the transcription of the DTDST gene (P = 0.0000014; n = 20). Most cultured colon cancer cells had a diminished DTDST transcription, which was restored when cultured with histone deacetylase inhibitors. Suppression of DTDST transcription under the control of a tet-off inducible promoter resulted in increased sialyl Lewisx expression and reduced sialyl 6-sulfo Lewisx expression. Unexpectedly, the growth rate of the cancer cells was markedly enhanced when transcription of DTDST was suppressed. These results show that the decrease in the transcription of the sulfate transporter gene is the major cause of decreased expression of sialyl 6-sulfo Lewisx and increased expression of sialyl Lewisx in colon cancers. The results also suggest that the diminished DTDST expression is closely related to enhanced proliferation of cancer cells. Cancer Res; 70(10); 4064–73. ©2010 AACR.

Introduction bohydrate determinant sialyl 6-sulfo Lewisx, but they hardly express any of its nonsulfated form, sialyl Lewisx (10). In con- Malignant transformation is frequently associated with a trast, colon cancer cells strongly express sialyl Lewisx, while drastic alteration in the surface expression of carbohydrate they exhibit markedly decreased sialyl 6-sulfo Lewisx expres- determinants (1–4). Some carbohydrate determinants that sion compared with normal colonic epithelial cells. Impaired are preferentially expressed in cancers are clinically applied 6-sulfation was therefore suggested to occur during the course for diagnosis of cancers (5, 6). However, the underlying mech- of malignant transformation of colonic epithelial cells and to anism that leads to expression of altered carbohydrate deter- be responsible for the preferential expression of sialyl Lewisx minants, as well as their pathophysiologic significance, is not in cancers (8–10). The sialyl Lewisx thus appearing in cancer always clear. cells plays an important role in E-selectin–mediated cancer The sialyl Lewisx determinant is a typical cancer-associated cell adhesion to vascular endothelial cells during the course glycan (1–3, 7–9), but the molecular mechanism responsible of tumor angiogenesis (11) and distant metastasis (1, 7, 8). In for preferential expression of the determinant in cancer has this study, we tried to elucidate the molecular biological back- not been fully elucidated. Previously, we showed that differen- ground of the impaired 6-sulfation in cancers. It has long been tiated normal colonic epithelial cells express the sulfated car- known that carbohydrate sulfation is generally suppressed in colon cancers compared with nonmalignant colonic epithelial Authors' Affiliations: 1Department of Molecular Pathology, Aichi Cancer cells (12–14) with a few exceptions (15), and this was expected Center, Nagoya, Japan and 2Research Complex for the Medicine to be related to the decreased 6-sulfation in cancers. Frontiers, Aichi Medical University, Aichi, Japan Note: Supplementary data for this article are available at Cancer Materials and Methods Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Reiji Kannagi, Research Complex for the Medi- cine Frontiers, Aichi Medical University, 21 Karimata, Yazako, Nagakute- Clinical samples, real-time reverse transcription-PCR cho Aichi-gun, Aichi 480-1195, Japan. Phone: 81-561-623311 ext 1436; analyses, and immunohistochemical staining. Tumor spe- Fax: 81-561-611896; E-mail: [email protected]. cimens were obtained from 20 patients with primary colorec- doi: 10.1158/0008-5472.CAN-09-2383 tal cancer at surgical operation and processed as described ©2010 American Association for Cancer Research. previously (16, 17). The median age of patients was 59.8 years.

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The carcinomas were staged according to Dukes classifica- Expression vectors for DTDST. Human DTDST cDNA tion (18). Malignant and nonmalignant tissues of each spec- was obtained from Quick-Clone human placenta cDNA imen were used for RNA extraction. Nonmalignant mucosa (from Clontech). Transient transfections were done with was scraped off using slide glasses, and tissue specimens of the pIRES/DTDST vector and Lipofectamine 2000 accord- cancer were carefully excised to eliminate noncancerous tis- ing to the manufacturers' protocols. Lentivirus-mediated sue components. Specimens were powdered in liquid N2, and shRNA knockdown of the DTDST gene was done in LS174T total cellular RNA was extracted with guanidine isothiocya- colon cancer cells using pLKO.1-based lentiviral DTDST nate and purified by cesium chloride gradient centrifugation. shRNA vectors purchased from Sigma-Aldrich. Tetracycline- Total cellular RNA was reverse transcribed into cDNA using repressible expression (37) was carried out using RevTet- SuperScript II with Oligo(dT) primer per manufacturer's in- Off Gene Expression Systems (Clontech) according to the structions (Invitrogen). Real-time reverse transcription-PCR manufacturers' protocols. DTDST cDNA was excised with (RT-PCR) analyses were done using ABI PRISM 7000 (Applied EcoRI from pIREShygro, blunted, and cloned into the HpaI Biosystems). The TaqMan probe IDs (Applied Biosystems) for site of the pRevTRE vector containing a hygromycin resis- five 6-sulfotransferases (GlcNAc6ST-1, HEC-GlcNAc6ST, tance gene to generate the plasmid pRevTREhygro/DTDST. I-GlcNAc6ST, GlcNAc6ST-4, and C-GlcNAc6ST; refs. 19–23), The pRev/Tet-Off-neo vector and pRevTREhygro/DTDST two PAPS synthases (PAPSS1 and PAPSS2; refs. 24, 25), two vector were transfected separately into RetroPack PT67 PAPS transporters (PAPST1 and PAPST2; refs. 26, 27), packaging cells, and the resulting virus-containing super- a sulfate transporter (DTDST; ref. 28), and two hexosamine natants were used to serially infect HT-29 cells. G418 6-sulfatases (29–31) are listed in Supplementary Table S1. (500 μg/mL) was used for screening of HT-29 cells in the The results were calculated using the comparative Ct method. initial pRev/Tet-Off virus infection. Infection of HT-29 cells −Δ Relative transcripts were determined by the formula 2 Ct.The with pRevTRE virus and screening with G418 and hygro- ΔCt value was determined by subtracting the average GAPDH mycin were done in the presence of 0.5 μg/mL doxycycline Ct value from the average target Ct value. The results of real- to keep DTDST expression suppressed. Clones were tested time RT-PCR were ascertained also with conventional RT-PCR, for DTDST mRNA expression on removal of doxycycline. and the conditions and primers for conventional RT-PCR are Most clones showed a dramatic increase in DTDST mRNA summarized in Supplementary Table S1. levels. Frozen sections of 10-μm thickness for immunohisto- Chromatin immunoprecipitation assays. Chromatin im- chemical examination were prepared from surgical speci- munoprecipitation assays were done using ChIP-IT Express mens for immunohistochemistry. Anti-DTDST antibody was (Active Motif) according to the manufacturer's instructions. obtained from Abnova. The avidin-biotin complex technique The antibodies for acetylated-H3K9 (H3K9Ac), trimethylated- for the immunohistochemical study was done as described in H3K9 (H3K9me3), dimethylated-H3K9 (H3K9me2), trimethy- the kit instructions (Vectastain, Vector; ref. 10). lated-H4K20 (H4K20me3), trimethylated-H3K4 (H3K4me3), Cell culture and flow cytometric analyses. Cultured hu- and trimethylated-H3K27 (H3K27me3) were purchased from man colon cancer cell lines, C-1, Caco-2, Colo201, Colo205, Abcam. Anti–dimethylated H3K27 (H3K27me2) was from Colo320, CoR-1, HCT15, HCT116, HT29, LoVo, SW480, Cell Signaling Technology. Anti–RNA polymerase II (Active SW1083, T84, and WiDr, were from American Type Culture Motif) and control anti-IgG were from Active Motif. The im- Collection (ATCC) and Japanese Collection of Research Bio- munoprecipitation mixtures containing the sheared chroma- resources. The cells were cultured in the presence of butyrate tin, protein-G magnetic beads, and each antibody were (4–8 mmol/L), trichostatin A (100 ng/mL), FK228 (2.5– incubated for 4 hours at 4°C. The DNA-histone complexes – μ 10.0 ng/mL), 5-aza-2-deoxycytidine (5 30 mol/L), or NaClO3 with or without immunoprecipitation were incubated for (12.5–25 mmol/L) for 3 days and subjected to RT-PCR anal- 15 minutes at 94°C for reverse cross-linking and then treated ysis. The G72 and G152 antibodies (murine IgM), directed to with proteinase K. The DNA fragments were purified with sialyl 6-sulfo Lewisx determinant, were prepared as described QIAquick PCR Purification kit (Qiagen). PCR for the DTDST previously (32–34). A classic anti–sialyl Lewisx antibody, promoter region −258 to −363 was done with the primers 5′- CSLEX-1, which does not cross-react to sialyl 6-sulfo Lewisx, GGGGCCTCAATTTCCCGTTCTACAAAG-3′ and 5′- was obtained from ATCC (33, 35). The antibodies KN412 and GAGTTTTGAAGTGCTCAGCGTTTGCG-3′, using GeneAmp KN171 (both murine IgM) recognizing an anonymous pan-6- Fast master mix at 38 cycles of amplification. sulfated antigen were produced by immunizing BALB/c i.p. with2×107 SW480/HEC-GlcNAc6ST cells twice at 2-week Results intervals. The splenic cells were fused 3 days after final immunization with mouse myeloma P3/X63-Ag8U1. These Real-time RT-PCR analysis of 6-sulfotransferase gene antibodies were not reactive to parental or mock-transfected expression in human colon cancer tissues and nonmalig- SW480 cells but were reactive to SW480 cells transfected nant colonic epithelial cells. Figure 1A shows a typical with GlcNAc6ST-1, HEC-GlcNAc6ST, or I-GlcNAc6ST (36); example of distribution patterns of sialyl Lewisx,acancer- hence, they were designated pan-6-sulfo antibodies. Flow associated glycan, and its sulfated derivative, sialyl 6-sulfo cytometric analyses were done with FACScan (Becton Lewisx, in cancer tissue prepared from a patient with colon Dickinson) with a 1:200 dilution of FITC-conjugated goat an- cancer. Nonmalignant colonic epithelial cells expressed the ti-mouse Ig (Silenus Laboratories) at 4°C for 30 minutes. carbohydrate determinant sialyl 6-sulfo Lewisx, but virtually

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no sialyl Lewisx. In contrast, colon cancer cells strongly nificant levels of other 6ST isoenzymes in cancer cells, such express sialyl Lewisx, while they exhibit markedly decreased as GlcNAc6ST-1 and HEC-GlcNAc6ST, capable of synthesiz- sialyl 6-sulfo Lewisx expression compared with normal co- ing sialyl 6-sulfo Lewisx, would detract from the contribution lonic epithelial cells. From the comparison of the structures of I-GlcNAc6ST in explaining the decreased sialyl 6-sulfo of the two glycans, impaired sulfation at the C6 position of Lewisx expression in cancer cells. In addition, it was previ- the N-acetylglucosamine moiety was assumed to have ously reported that I-GlcNAc6ST was not able to generate occurred during the course of malignant transformation of the sialyl 6-sulfo Lewisx epitope in transfected cells (38). colonic epithelial cells, and this was thought to be responsi- We also obtained experimental results suggesting the very ble for the increased expression of sialyl Lewisx in cancers. low efficiency, if any, of this enzyme in glycan 6-sulfation, To elucidate the molecular biological background of the whereas GlcNAc6ST-1 and HEC-GlcNAc6ST could efficiently impaired 6-sulfation in cancers, we first studied the expres- synthesize sialyl 6-sulfo Lewisx in the same cells (see Supple- sion levels in cancer tissues of mRNA for 6-O-sulfotransferase mentary Fig. S1). These findings suggested that the decreased (6ST) isoenzymes. Results of RT-PCR analyses of five 6ST sialyl 6-sulfo Lewisx expression in cancer cells may not be as- transcripts in colorectal cancer tissues and nonmalignant co- cribed solely to the suppressed transcription of the gene for lonic epithelial cells are shown in Fig. 1B. Preliminary results I-GlcNAc6ST. Accordingly, we studied the expression of on nonmalignant epithelial cells indicated that the major 6ST genes other than 6STs that are involved in carbohydrate sul- species in the human colon was I-GlcNAc6ST, followed by fation in human colon cancer tissues. GlcNAc6ST-1. The amount of mRNA for the other three iso- RT-PCR analysis of genes involved in carbohydrate 6- enzymes was almost negligible in nonmalignant epithelium. sulfation in human colon cancer tissues and nonmalig- A marked decrease of I-GlcNAc6ST mRNA was detected in nant colonic epithelial cells. We turned our attention to cancer cells compared with nonmalignant epithelial cells two PAPS synthases (PAPSS1 and PAPSS2) producing the prepared from the same patient (P = 0.000007; n = 20), where- donor substrate for sulfotransferases, two PAPS transporters as GlcNAc6ST-1 mRNA showed no statistically significant (PAPST1 and PAPST2) in the Golgi membrane, a sulfate difference (Fig. 1B). transporter (DTDST) localized at the cell membrane, and These results suggested a possibility that the suppressed two 6-sulfohexosamine sulfatases (Fig. 1C). Transcripts for transcription of the I-GlcNAc6ST gene, the major 6ST in PAPSS1 showed a moderate increase in cancer (P ≤ 0.05; the intestine, may account for the decreased expression of n = 20), whereas sulfated glycan expression decreased. Tran- sialyl 6-sulfo Lewisx in colon cancer cells. However, the sig- scripts for PAPST1 showed no significant change. PAPSS2 and

Figure 1. Sialyl 6-sulfo glycan and 6-sulfotransferase mRNA expression in human colon cancer tissues and nonmalignant mucosa. A, typical examples of distribution of sialyl 6-sulfo Lewisx (left), nonsulfated sialyl Lewisx (middle), and H&E staining of same tissue (right). Ca, cancer cells; N, nonmalignant epithelial cells. B, real-time RT-PCR analyses of 6-sulfotransferase gene expression in human colon cancer tissues and nonmalignant mucosa (n = 20). Ca, cancer tissue; N, nonmalignant mucosa prepared from same patient. See Supplementary Table S1 for probes and nomenclature of the enzymes.

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PAPST2 showed a moderate decrease (P ≤ 0.0001 and P ≤ occurring in the right or left hemicolon (Fig. 1C). The de- 0.0005; respectively; n = 20). These changes, although not very creased expression of DTDST was ascertained also at the remarkable, are in line with the decreased expression of protein level. Immunohistochemical staining of frozen tissue sulfated glycans in cancers. Decreased expression of PAPSS2 sections prepared from colon cancer patients with anti- had reportedly also occurred in cultured human prostate DTDST antibody indicated that cancer cells express only cancer cells (25, 39). The two sulfatases showed only a low levels of DTDST protein, compared with adjacent non- modest increase in cancers (statistically not significant). malignant colonic epithelial cells. This was consistently ob- The most prominent decrease, which is compatible with served with all eight tested cases, and representative photos the loss of sulfated glycans in cancers, was observed with are shown in Fig. 1D. the transcript of DTDST (P = 0.0000014; n = 20; Fig. 1C). Its Effect of perturbation of sulfate incorporation on the ex- decreased transcription was apparent even in the relatively pression of sialyl 6-sulfo Lewisx and sialyl Lewisx glycans. early-stage cancer tissues taken from patients with Dukes As the transcription of sulfate transporter DTDST showed stages A and B and was ubiquitously observed in the cancers the most prominent decrease in cancers compared with

Figure 1. Continued. C, results of real-time RT-PCR analyses of mRNA levels of genes involved in cellular sulfate metabolism, including two PAPS synthases (PAPSS1 and PAPSS2), a PAPS transporter [PAPST1 (rel4)], DTDST (SLC26A2), and two hexosamine-6-sulfatases (G6ase and GALNS; n = 20). Bottom, effects of Dukes stages and primary tumor sites on DTDST mRNA expression. Ca, cancer tissue; N, nonmalignant mucosa prepared from same patient. A paired t test was done to ascertain statistical significance between the level in cancer tissue and that in nonmalignant mucosa in B and C. N.S., not significant. See Supplementary Table S1 for probes. D, examples of expression of DTDST protein in cancer tissue and adjacent nonmalignant mucosa. Ca, cancer tissue; N, nonmalignant mucosa. Bar, 200 μm.

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nonmalignant epithelial cells, we next tested if perturbation of cellular sulfate incorporation indeed affects the cell surface expression of sialyl 6-sulfo Lewisx and sialyl Lewisx glycans. In our preliminary experiments, cultured colon cancer cells Colo201 were found to contain a considerable amount of HEC-GlcNAc6ST mRNA, but no detectable mRNA for I-GlcNAc6ST or GlcNAc6ST-1 (see Supplementary Fig. S2), and to moderately express sialyl 6-sulfo Lewisx as well as sialyl Lewisx under the usual culture conditions. When the Colo201 cells were cultured in the presence of NaClO3, an inhibitor of eukaryotic cell sulfate incorporation (40–42), a significant attenuation of sialyl 6-sulfo Lewisx expression was observed, which was accompanied by an increase of the nonsulfated form of the glycan, sialyl Lewisx (Fig. 2A). More clear results were obtained when ECV304 cells cotransfected with the genes for GlcNAc6ST-1 and fucosyltransferase VII (34) were used in similar experiments (Fig. 2B). These cells strongly expressed sialyl 6-sulfo Lewisx and virtually no sialyl Lewisx under usual culture conditions. Addition of NaClO3 to the culture medium almost completely abro- gated the sialyl 6-sulfo Lewisx expression and induced prominent expression of sialyl Lewisx (Fig. 2B). There was a clear reciprocal relationship between expression of sialyl 6-sulfo Lewisx and sialyl Lewisx, depending on the concentrations of NaClO3 (Fig.2C).Thiswasnotobservedwiththecells transfected with the gene for fucosyltransferase only, which expressed sialyl Lewisx under normal conditions, and addition of NaClO3 produced no change in its expression (Fig. 2B). Effect of the histone deacetylase inhibitor butyrate on DTDST transcript. Most cultured human colon cancer cells were found to have decreased levels of DTDST transcript (Fig. 3A). The only exception was Caco-2 cells, which showed a moderate level of DTDST mRNA, but still less than the level observed in nonmalignant epithelial cells. Significant in- creases in DTDST mRNA were observed when these cancer cells were cultured in the presence of histone deacetylase (HDAC) inhibitors, and representative results are shown in Fig. 3B. The DNA methylation inhibitor 5-aza-2-deoxycytidine was not a potent inducer of DTDST mRNA. These results suggested that transcription of the DTDST gene was suppressed by some epigenetic silencing mechanism involving histone modification, but that DNA methylation was not closely involved.

Figure 2. Effect of inhibition of cellular sulfate incorporation by sodium chlorate on expression of sialyl 6-sulfated glycans. A, cultured human colon cancer cell line Colo201 was cultured for 7 d with 12.5 mmol/L sodium chlorate and analyzed by flow cytometry for cell surface expression of sialyl 6-sulfo Lewisx defined by G72 antibody and nonsulfated sialyl Lewisx defined by CSLEX-1 antibody. B, cell surface expression of sialyl 6-sulfo Lewisx and nonsulfated sialyl Lewisx in ECV304 cells cotransfected with genes for GlcNAc6ST and fucosyltransferase VII (clone LS12; top) or in cells transfected with the gene for fucosyltransferase VII alone (clone 7ECV; bottom) was analyzed after culture for 7 d with 25 mmol/L sodium chlorate. C, inverse correlation between expression of sialyl 6-sulfo Lewisx and nonsulfated sialyl Lewisx in LS12 cells depending on sodium chlorate concentration in terms of mean fluorescence intensity ascertained by flow cytometric analyses.

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Figure 3. Epigenetic silencing of DTDST transcription in cultured human colon cancer cells. A, RT-PCR analysis of DTDST mRNA in 15 cultured human colon cancer cells. Lane N, representative result on nonmalignant colonic epithelial cells serving as a positive control. B, effect of HDAC inhibitors [butyrate, trichostatin A (TSA), and FK228] and a DNA methylation inhibitor (5-Aza-dC) on DTDST transcription in cultured human colon cancer cells. C, chromatin immunoprecipitation assays of DTDST promoter in cultured colon cancer cells. Caco-2 is an example of a moderate expresser of DTDST showing chromatin markers for active gene expression (H3K9Ac and H3K4me3). SW480, SW1083, and HT29 are representative cells exhibiting reduced DTDST transcription showing markers for inactive gene expression (H4K20me3, H3K9me2, H3K9me3, H3K27me2, and H3K27me3). See Materials and Methods for information on antibodies directed to modified H3 and H4 histones used in the assay.

Silencing of the DTDST gene in cultured colon cancer cells of sialyl 6-sulfo Lewisx (Fig. 4A), and trials for isolating stable correlated with promoter histone modification in chromatin transfectant clones were not successful; therefore, we at- immunoprecipitation assays (Fig. 3C). Only CaCo-2 cells, tempted to use a tet-off inducible construct. The transfectant which had a moderate level of DTDST transcription, showed cells thus obtained expressed only a low level of DTDST tran- significant histone H3 lysine 9 acetylation and H3 lysine 4 scripts under culture conditions with doxycycline (DOX+), methylation, which are key chromatin marks associated with but showed strongly induced expression of a large amount active gene expression, in chromatin immunoprecipitation of DTDST transcripts on doxycycline removal (DOX−; assays.Athickbandobservedwithanti–pol II antibody Fig. 4B). When transcription of DTDST was induced, a sig- was compatible with significant DTDST transcription in nificant expression of sialyl 6-sulfo Lewisx was observed, to- Caco-2 cells (Fig. 3C). On the other hand, histone H3 lysine gether with an anonymous pan-6-sulfated antigen defined by 9 deacetylation and loss of H3 lysine 4 methylation were ob- the KN412 antibody. Interruption of DTDST expression by served in cultured colon cancer cells with diminished DTDST the addition of doxycycline induced a marked increase in sia- transcription, such as SW480, SW1083, and HT29. Instead, in- lyl Lewisx expression, accompanied by the disappearance of creased methylation of H4 lysine 20, H3 lysine 27, and H3 ly- sialyl 6-sulfo Lewisx as well as the pan-6-sulfated antigen sine 9 was noted in some cultured colon cancer cells with (Fig. 4C). The presence or absence of doxycycline did not af- diminished DTDST transcription (Fig. 3C). These results sug- fect the transcription of other genes involved in the sulfated gested that aberrant epigenetic chromatin events may be the glycan synthesis, such as 6-sulfotransferases, PAPS synthases, underlying mechanism for the reduced DTDST transcription and PAPS transporters (Supplementary Fig. S3). DTDST in cancer cells. knockdown experiments with lentiviral shRNA vectors also Effects of inducible expression of DTDST on glycan indicated a significant reduction in sialyl 6-sulfo Lewisx, expression and cancer cell proliferation. To determine further supporting the role of DTDST in the induction of the direct effect of DTDST on sulfated glycan expression, sulfate glycan expression (Supplementary Fig. S4). we performed DTDST gene transfection experiments. Several During the experiments using the tet-off inducible con- experiments involving transient transfection of the DTDST struct, a significant growth suppression of the transfectant gene into HT29 cells could induce only a weak expression cells was observed by expressing DTDST through removal

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of doxycycline. Fig. 4D (top) shows the growth curves and bromodeoxyuridine (BrdUrd) incorporation assays of the transfectant cells with the tet-off promoter–driven DTDST expression vector. Significant growth suppression is evident when transcription of DTDST was induced by removal of doxycycline (DOX−). This was a reversible effect, and interruption of DTDST transcription by addition of doxycycline led to recovery of the growth of cancer cells (DOX+). This was a paradoxical phenomenon because control cells without DTDST expression vector showed moderate growth suppression in the presence of doxycycline (DOX+) rather than in its absence (DOX−), most probably because of the mild cytotoxicity of doxycycline.

Discussion

Previously, we showed that the expression of 6-sulfated carbohydrate determinants such as sialyl 6-sulfo Lewisx is significantly reduced in colon cancer cells compared with nonmalignant colonic epithelial cells, and we proposed the reduction in 6-sulfation associated with carcinogenesis to be the major mechanism leading to preferential expression of the nonsulfated form of sialyl Lewisx in cancer cells (10). The results of the present study indicated that the decreased transcription of the DTDST gene figures heavily in the reduction of 6-sulfated glycan expression in cancer cells. Transcription of the DTDST gene was most prominently decreased in colon cancer cells compared with nonmalignant colonic epithelial cells among the genes involved in the cellular sulfate metabolism. Results using an inhibitor of sulfate incorporation and the tet-off inducible DTDST expression vector clearly indicated that the reduction in DTDST transcription level leads to loss of sialyl 6-sulfo Lewisx and induction of cancer-associated expression of sialyl Lewisx.A generalized decrease of glycan sulfation, especially that of mucin glycoproteins, has long been known to associate with

Figure 4. Effect of induction of DTDST transcription on sialyl 6-sulfo glycan expression and cell proliferation. A, human cultured colon cancer cells HT29 were transiently transfected by an expression vector for DTDST. Results of two experiments are shown. B to D, human cultured colon cancer cells HT29 were stably transfected with tet-off inducible expression vector for DTDST. B, RT-PCR analysis of induction of DTDST transcription by removal of doxycycline (DOX−). C, flow cytometric analyses of expression of sialyl Lewisx glycan defined by CSLEX-1 antibody, sialyl 6-sulfo Lewisx glycan defined by G72 antibody, and pan 6-sulfo antigen defined by KN412 antibody. Induction of DTDST transcription by removal of doxycycline (DOX−) confers expression of 6-sulfated glycans defined by G72 and KN412 antibodies, whereas diminished DTDST transcription by addition of doxycycline (DOX+) induces expression of sialyl Lewisx accompanied by loss of 6-sulfated glycans. D, growth curve and BrdUrd incorporation of cells stably transfected with tet-off inducible expression vector for DTDST (HT29/DTDST). Induction of DTDST transcription by removal of doxycycline (closed circles or closed columns, DOX−) markedly suppressed cell proliferation compared with cells with diminished DTDST transcription (open circles or open columns, DOX+). In contrast, control HT29 cells (bottom) showed modestly increased proliferation in the absence of doxycycline (closed triangles or closed columns, DOX−) compared with cells cultured with doxycycline (open triangles or open columns, DOX+). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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malignant transformation of colonic epithelial cells (12–14). why earlier trials conducted by other researchers and our- Decreased sulfation of the sialyl Lewisx determinant, which is selves (4, 16, 17) only partially succeeded in identifying the carried mainly by mucin glycoproteins, is in line with this no- genes responsible for the cancer-associated expression of tion. Sulfate deprivation does not uniformly affect the sulfat- sialyl Lewisx based on a positive correlation of sialyl Lewisx ed glycan expression. Some sulfated glycolipids such as expression with the transcription level of some particular sulfatides are reported to rather increase in the advanced glycosyltransferase gene. stage of cancers (15). It is well known that sulfate deprivation Epigenetic silencing of the DTDST gene led to diminished x using NaClO3 strongly affects the expression of chondroitin sialyl 6-sulfo Lewis expression and to induction of sialyl sulfates, but it exerts much less effect on heparan sulfates, Lewisx expression. The physiologic function of the sialyl Lewisx and all candidate enzymes involved in sulfation of sialyl glycan was shown to serve as a ligand for selectins (44, 45). 6-sulfo Lewisx belong to the same gene family as chondroitin Interaction of sialyl Lewisx on cancer cells with endothelial 6-O-sulfotransferases. The Km value of the synthetic enzymes selectins was earlier shown to facilitate tumor angiogenesis for PAPS, as well as other multiple factors, must be involved (11) and hematogenous metastasis (2, 8, 9). Although this in the effect of sulfate deprivation, which is uneven depend- may provide an advantage to cancer cells, sialyl 6-sulfo ing on the molecular species. Lewisx is also capable of serving as a ligand for all three Although the possibility that reduced transcription of the members of the selectin family (46). Surprisingly, the present I-GlcNAc6ST also partly contributes to the loss of sialyl 6-sulfo study revealed that silencing the DTDST gene facilitates cell Lewisx expression cannot be completely ruled out, the signifi- proliferation. Thus, the reduction of DTDST transcription cant levels of other 6ST isoenzymes such as GlcNAc6ST-1 confers a definite growth advantage on cancer cells. and HEC-GlcNAc6ST in cancer cells, which were shown to Whether or not the sialyl 6-sulfo Lewisx or the sialyl Lewisx be sufficient for sialyl 6-sulfo Lewisx synthesis, would detract determinant per se is directly involved in regulation of cell from the contribution of I-GlcNAc6ST. For instance, although proliferation is not fully clear at this moment. Although some Colo201 cells lacked I-GlcNAc6ST, the level of HEC- earlier studies suggested an association between sialyl Lewisx GlcNAc6ST was sufficient to synthesize sialyl 6-sulfo Lewisx expression and cell proliferation (47, 48), the reduction of (Fig. 2). HT29 cells, which also lacked I-GlcNAc6ST, ex- DTDST transcription will cause a generalized decrease in pressed enough HEC-GlcNAc6ST transcripts to confer sialyl the sulfation reaction of not only sialyl Lewisx but also vari- 6-sulfo Lewisx expression when DTDST was supplied by the ous other molecules such as proteoglycans. Some of such sul- tet-off inducible vector (Fig. 4). The possibility that reduced fated molecules may well play a key role in regulating the transcription of the PAPSS2 and PAPST2 genes may also con- proliferative activity of cancer cells. For instance, loss of tribute to the loss of sialyl 6-sulfo Lewisx expression in cancer 6-O-sulfation at GlcNAc of heparan sulfate, which is selective- cells cannot be entirely set aside. However, the observed ly affected by sodium chlorate treatments (41), is known to change in the mRNA levels was not prominent, and would facilitate Wnt signaling (49, 50). In such a case, the loss of be compensated by the increase of PAPSS1 transcription in sialyl 6-sulfo Lewisx and the acquisition of sialyl Lewisx would the case of PAPSS2. be phenomena coincidental with DTDST silencing and serve We previously proposed that the increased expression of as markers for highly proliferative cancer cells. the sialyl Lewisa determinant, another cancer-associated glycan, is due to the reduction of disialyl Lewisa expression. The Disclosure of Potential Conflicts of Interest disialyl Lewisa determinant was preferentially expressed in nonmalignant epithelial cells, and the epigenetic silencing No potential conflicts of interest were disclosed. of the gene for the enzyme responsible for transfer of the sec- ond sialic acid residue on malignant transformation con- Grant Support ferred the induction of cancer-associated sialyl Lewisa expression (9, 43). The situation is similar also for sialyl Grants-in-aid from the Ministry of Education, Culture, Sports, Science and x DTDST Technology, Japan (21590324 and on priority areas 17015051); grants-in-aid for Lewis in that the reduction of transcription is also the Third-Term Comprehensive Ten-Year Strategy for Cancer Control from the mostly due to epigenetic silencing of the gene. These findings Ministry of Health and Welfare, Japan; and grants from the Uehara Memorial Foundation, the Nagono Medical Foundation, and the Life Science Foundation collectively imply that normal epithelial cells generally ex- of Japan (R. Kannagi). press highly complex glycans, and partial impairment of their The costs of publication of this article were defrayed in part by the payment synthetic pathway by epigenetic silencing of synthetic genes of page charges. This article must therefore be hereby marked advertisement in on malignant transformation induces the expression of accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cancer-associated glycans, which usually have less complex Received 07/01/2009; revised 02/16/2010; accepted 03/04/2010; published structures (incomplete synthesis; ref. 9). This well explains OnlineFirst 05/11/2010.

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Epigenetic Silencing of the Sulfate Transporter Gene DTDST Induces Sialyl Lewis x Expression and Accelerates Proliferation of Colon Cancer Cells

Akiko Yusa, Keiko Miyazaki, Naoko Kimura, et al.

Cancer Res Published OnlineFirst May 11, 2010.

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