Ppel-Like Factor 4 Expression Contributes to Sp1overexpression

Human Cancer Biology

Loss of Kru« ppel-Like Factor 4 Expression Contributes to Sp1 Overexpression and Human Gastric Cancer Development and Progression Masashi Kanai,1Daoyan Wei,1Qiang Li,1ZhiliangJia,1JafferAjani,1Xiangdong Le,1 JamesYao,1andKepingXie1, 2

Abstract Purpose: Increasing evidence indicates that the transcription factor, Sp1, regulates the expression of multiple genes involved in tumor development and progression.We have recently reported that Sp1overexpression is directly correlated with the angiogenic potential of and poor prognosis for human gastric cancer. However, the underlying mechanisms that result in Sp1overexpression remain unclear. Experimental Design:The expression of Sp1and Kru« ppel-like factor 4 (KLF4), a potential tumor suppressor gene, in gastric cancer tissue was analyzed by immunohistochemistry and Western blot analysis. Alterations of Sp1and KLF4 expression were achieved by gene transfer and verified by Northern andWestern blot analyses. Furthermore, Sp1promoter activity assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay were done to identify the KLF4 binding sites on the Sp1promoter. Results: Mutually exclusive expression of Sp1and KLF4 was evident in gastric cancer and noncancerous tissue. Specifically, strong Sp1expression but loss of KLF4 expression was found in cancer tissue, whereas the adjacent noncancerous tissue showed negative Sp1expression but strong KLF4 expression. Enforced KLF4 expression repressed Sp1expression at the promoter activity, mRNA, and protein levels. Moreover, a region within the proximal Sp1promoter was identified to have overlapping KLF4- and Sp1-binding sites, to which KLF4 and Sp1compete for binding. Sp1positively regulated its own promoter, whereas KLF4 did the opposite. Conclusions: Our data suggests that disruption of KLF4-mediated negative regulation contributes to the molecular events of Sp1 overexpression and to the development and progression of human gastric cancer.

Although the incidence of gastric cancer declined in the west understanding of its biology and behavior. Although various from the 1940s to the 1980s, it remains a major public health genetic and molecular alterations have been found to be problem throughout the world (1). In Asia and parts of South associated with the malignant transformation of gastric cancer, America in particular, it is the most common epithelial they may represent only the pathogenesis of this disease, and malignancy and is a leading cause of cancer-related death. In they have not been identified as a specific sequence of changes fact, gastric cancer remains the fourth most frequently leading to gastric carcinoma (2, 3). Therefore, the role and diagnosed malignancy worldwide and is the cause of 12% of detailed mechanisms of genetic and epigenetic changes in all cancer-related deaths annually (1). Advances in the gastric cancer development and progression remain unclear. treatment of this disease are likely to come from a fuller Sp1 is a zinc finger transcription factor that is important to the transcription of many cellular and viral genes that contain GC boxes in their promoter. Additional transcription factors that are similar to Sp1 in their structural and transcriptional properties (Sp2, Sp3, and Sp4) have been cloned, thus forming Authors’ Affiliations: Departments of 1Gastrointestinal Medical Oncology and 2Cancer Biology, The University of Texas M.D. Anderson Cancer Center, the Sp1 multigene family (4). Although Sp1 has been perceived Houston,Texas as a basal transcription factor since its discovery, increasing Received 4/28/06; revised 7/2/06; accepted 7/20/06. evidence suggests that it regulates a variety of biological Grant support: Research Scholar grant CSM-106640 from the American Cancer functions, including cell survival, growth and differentiation, Society and grant1R01-CA093829 from the National Cancer Institute, NIH (K. Xie). and tumor development and progression (5). For example, Sp1 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance expression is increased in squamous cell carcinoma and with 18 U.S.C. Section 1734 solely to indicate this fact. colorectal cancer when compared with that in skin papillomas Requests for reprints: Keping Xie, Department of Gastrointestinal Medical and normal colorectal tissue, respectively (6, 7). Also, Oncology, Unit 426, The University of Texas M.D. Anderson Cancer Center, 1515 interference of Sp1 activity has been shown to suppress tumor Holcombe Boulevard, Houston,TX 77030. Phone: 713-792-2828; Fax: 713-745- 1163; E-mail: [email protected]. cell growth (8), and small interfering RNA (siRNA) duplexes of F 2006 American Association for Cancer Research. Sp1 mRNA block cell cycle progression or tumor formation in doi:10.1158/1078-0432.CCR-06-1034 athymic mice (9, 10). Furthermore, we have reported that Sp1

www.aacrjournals.org 6395 Clin Cancer Res 2006;12(21)November 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Human Cancer Biology overexpression is directly correlated with the angiogenic Western blot analysis. Fresh gastric cancer and corresponding potential and poor prognosis of human gastric cancer (11, 12). noncancerous gastric tissue specimens were obtained from patients However, the underlying mechanisms that result in Sp1 who underwent gastrectomy at The University of Texas M.D. Anderson overexpression in tumors remain unknown. Although a Cancer Center. Both the cancerous and noncancerous specimens were number of studies have reported gene regulation by Sp1, few macroscopically identified and excised by experienced pathologists, and the presence or absence of cancer was further confirmed by have reported the transcriptional regulation of Sp1 itself. One histopathologic examination. Additionally, whole cell lysates were recent study showed that the Sp1 promoter contains a number prepared from gastric cancer tissue and adjacent noncancerous speci- of putative binding sites for several transcription factors, mens or cell cultures. Standard Western blotting was done with an including Sp1, Sp3, nuclear factor Y, activator protein 2, and anti-Sp1 antibody (PEP2; Santa Cruz Biotechnology, Santa Cruz, CA) CCAAT/enhancer-binding protein (13, 14). Because the Sp1 and anti-KLF4 antibody (H180; Santa Cruz Biotechnology). Two bands promoter contains putative Sp1 binding sites, Sp1 overexpres- were detected following KLF4 transfection, as reported previously sion may result from autotransactivation of its own promoter. (16, 19, 23), and the low molecular weight protein may result from a On the other hand, Kru¨ppel-like factor 4 (KLF4) is a zinc rapid proteolysis of KLF4 protein (30). Equal protein sample loading finger transcription factor that binds to a consensus sequence was monitored by incubating the same membrane filter with an 5¶-(G/A)(G/A)GG(C/T)G(C/T)-3¶. It is a member of the KLF anti– glyceraldehyde-3-phosphate dehydrogenase antibody (FL-335; family and is highly expressed in the gastrointestinal tract and Santa Cruz Biotechnology). The probe proteins were detected by using the enhanced chemiluminescence system (Amersham Life Sciences, other epithelial tissues (15). KLF4 can either activate or repress Piscataway, NJ) according to the manufacturer’s instructions. the transcription of several genes (16–19), and its alterations Sp1 promoter constructs, site-specific mutagenesis, and analysis of Sp1 lead to aberrant proliferation and differentiation in gastric and promoter activity. The full-length and minimal Sp1 promoter reporters colonic epithelium (20, 21). Additionally, accumulating in pGL3 luciferase constructs were used as described previously (13). clinical evidence shows that KLF4 is a potential tumor Site-specific mutagenesis of the minimal Sp1 promoter was done by suppressor. Reduced KLF4 expression has been reported in using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, various tumors including esophagus, stomach, colon, bladder, CA) according to the manufacturer’s instructions. The primers used lung, and adult T cell leukemia cells (22–27), and restored to generate the mutant vector were: sense, 5¶-gcagagaaggggcggttcctagg- ¶ ¶ aa ¶ KLF4 expression induces growth arrest in colon cancer cells ttgggcttg-3 and antisense, 5 -caagcccaacctagg ccgccccttctctgc-3 (the (28) or apoptosis in bladder, gastric, and leukemia cells mutated sites are shown in boldface). Mutation was confirmed by DNA sequencing. To examine the transcriptional regulation of the Sp1 (23, 24, 27). In fact, genetic or epigenetic alterations of the f KLF4 promoter by KLF4, SK-GT5 and N87 cells were seeded to 80% gene have been found in both gastric cancer and colo- confluence in six-well plates (in triplicate) and transiently transfected rectal cancer (23, 25). with 0.6 Ag of full-length or minimum Sp1 reporter plasmid and 0.3 Ag In the present study, we found mutually exclusive expression of effector expression plasmids as indicated in each experiment by using of Sp1 and KLF4 in human gastric cancer. Enforced KLF4 LipofectAMINE (Invitrogen) according to the manufacturer’s instruc- expression repressed Sp1 expression at the promoter activity, tions. The reporter luciferase activity was determined 48 hours later mRNA, and protein levels. A region within the proximal Sp1 with a luciferase assay kit (Promega, Madison, WI). Promoter activity promoter was identified to have overlapping KLF4- and was normalized according to the protein concentration as described Sp1-binding sites, to which KLF4 and Sp1 compete for binding. previously (13). Sp1 positively regulated its own promoter, whereas KLF4 did Electrophoretic mobility shift assay. SK-GT5, N87, and FG cells were seeded to f80% confluence in 15 cm culture dishes and transduced the opposite. Collectively, our data suggests that disruption of with Ad-KLF4 or Ad-EGFP at a MOI of 10. Twenty-four hours later, the KLF4-mediated negative regulation contributes to the molecular cells were collected and nuclear extracts were prepared as previously events of Sp1 overexpression and to the development and described (31). Electrophoretic mobility shift assays (EMSA) were done progression of human gastric cancer. by using the Gel Shift Assay System (Promega) according to the manufacturer’s instructions. Briefly, the oligonucleotides as indicated in each experiments were annealed and 5¶-end-labeled with [32P]ATP by Materials and Methods using T4 polynucleotide kinase (Amersham Life Sciences) according to standard procedures. The nuclear extracts (10 Ag each) were preincu- Cell lines and culture conditions. The human gastric cancer cell lines, bated in binding buffer for 10 minutes at room temperature; this was AGS, HTB103, HTB135, N87, SNU-1, and TMK1, and FG human followed by the addition of the [32P]ATP-labeled double-strand pancreatic cancer cell line, were purchased from the American Type oligonucleotide and a second incubation at room temperature for Culture Collection (Manassas, VA), and the human gastric cancer cell 25 minutes. For the supershift assay, specific Sp1 (PEP2), KLF4 (H180, line, SK-GT5, was obtained from Dr. Gary K. Schwartz (Memorial Sloan- sc-20691 X), and FLAG (M2; Sigma Aldrich, St. Louis, MO) antibodies Kettering Cancer Center). All of the cell lines were maintained in plastic were added with a final concentration of 0.1 Ag/AL and incubated for flasks in minimal essential medium supplemented with 10% fetal bovine 15 minutes at room temperature before the addition of a labeled probe. serum, sodium pyruvate, nonessential amino acids, L-glutamine, and a Samples were then loaded on a 4.5% polyacrylamide gel, and vitamin solution (Flow Laboratories, Rockville, MD) in 5% CO2 at 37jC. electrophoresis was done at 4jC for 4 hours at 200 V. The gel was Northern blot analysis. SK-GT5 and N87 cells were seeded to f80% then dried for 40 minutes at 80jC and exposed to Kodak film (Eastman confluence in 10 cm culture dishes and transfected with a recombinant Kodak, Rochester, NY) at À70jC. adenovirus containing KLF4 (Ad-KLF4) or a recombinant adenovirus Generation of recombinant adenoviruses and transduction of tumor containing enhanced green fluorescent protein (Ad-EGFP) at a cells. The full-length KLF4 and nucleus localization–deficient mutant multiplicity of infection (MOI) of 10. Twenty-four hours later, total KLF4 in pcDNA3 plasmid were provided by Dr. Chichuan Tseng. RNA was extracted by using TRIzol reagent (Invitrogen, San Diego, CA). Adenoviral vectors (Ad-KLF4 and Ad-EGFP) were generated with the use Standard Northern blotting was done using 10 Ag of RNA and KLF4 and of the AdEasy Adenoviral Vector System (Stratagene). The recombinant Sp1 cDNA probes. Equal RNA loading was monitored by hybridizing adenoviruses were expanded in HEK293 cells and purified by double the same membrane with a human 36B4 cDNA probe as described CsCL gradient centrifugation to achieve a titer of f1010 plaque-forming previously (29). units/mL as previously described (23).

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Chromatin immunoprecipitation. SK-GT5 cells were seeded to Results f80% confluence in 10 cm culture dishes and transfected with Ad- KLF4 or Ad-EGFP at a MOI of 10. Twenty-four hours later, chromatin Mutually exclusive expression of Sp1 and KLF4 protein in immunoprecipitation was done by using the Chromatin Immunopre- human gastric cancer. Sections of paraffin-embedded gastric cipitation Assay Kit (Upstate Cell Signaling Solutions, Lake Placid, NY) according to the manufacturer’s instructions. Briefly, DNA cross- cancer tissue specimens were immunostained with antibodies binding proteins were cross-linked with DNA and lysed in SDS lysis against Sp1 and KLF4 protein (11, 23). After the analysis of Sp1 buffer. The lysate was sonicated to shear DNA to 200 to 500 bp. After and KLF4 expression scores in 86 primary gastric tumor tissue preclearing with a salmon sperm DNA/protein A agarose-50% slurry for specimens using previously published data (11, 23), we found 30 minutes at 4jC, chromatin samples were immunoprecipitated a significant inverse correlation between Sp1 expression and overnight with no antibody, an anti-Sp1 antibody (PEP2), an anti-KLF4 KLF4 expression (Fig. 1A; P = 0.006). Moreover, we found antibody (H180), and an anti-FLAG antibody (M2). The region KLF4 expression only in residual noncancerous epithelial cells between À224 and À53 bp of the Sp1 promoter was amplified by (Fig. 1B1), but strong Sp1 expression in the majority of the ¶ ¶ using the following primers: sense, 5 -caggcacgcaacttagtc-3 ,and tumor epithelial cells (Fig. 1B2). In most of the gastric cancer ¶ ¶ antisense, 5 -gtaaggaggagggagcag-3 . The PCR products were separated specimens, KLF4 expression was significantly decreased or lost on a 1.5% agarose gel, stained with ethidium bromide, and visualized (Fig. 1B3), whereas Sp1 overexpression was strikingly evident under UV light. siRNA. RNA interference was done by using the TranSilent Human (Fig. 1B4). To further confirm these findings, we did Western Sp1 siRNA (Panomics, Inc., Redwood, CA) or Human KLF4 siRNA blot analyses to examine the expression of KLF4 in two paired (Ambion, Austin, TX) following the manufacturer’s instructions. Briefly, human gastric tumor and adjacent noncancerous gastric tissue SK-GT5 cells were seeded to f80% confluence in six-well plates in specimens, two tumor tissue specimens exhibiting strong Sp1 triplicate and transiently transfected with 2.5 Ag of a Sp1 or KLF4 siRNA expression, and two tumor tissue specimens exhibiting weak mix and 0.6 Ag of minimal Sp1 reporter plasmid with the use of Sp1 expression. As shown in Fig. 1C1, the loss of KLF4 LipofectAMINE 2000 (Invitrogen). The reporter luciferase activity was expression correlated with increased Sp1 expression. Addition- determined 48 hours later by using a luciferase assay kit (Promega). The ally, we determined the level of both Sp1 and KLF4 expression promoter activity was normalized according to the protein concentra- in various human gastric cancer cell lines. As compared with tion, and the efficacies of Sp1 and KLF4 siRNA, which were 90% and normal human gastric tissue, all of the gastric cancer cell lines 80%, respectively, were confirmed by Western blot analyses. had significantly reduced or lost KLF4 but exhibited Sp1 Statistical analysis. The two-tailed m2 test was done to determine overexpression (Fig. 1C2). These data suggested that the the significance of the difference between the expressions of KLF4 and Sp1 in human gastric cancer. Other statistical differences were evaluated expression of Sp1 and KLF4 was mutually exclusive. by using the two-tailed Student’s t test. Differences were considered Inhibition of Sp1 expression by enforced KLF4 overexpres- statistically significant at P < 0.05. All experiments were done at least sion. To determine whether KLF4 represses Sp1 expression, we twice, and one representative of at least two experiments with similar examined the effects of KLF4 overexpression on Sp1 expression results was presented. using Northern and Western blot analyses. We found that

Fig. 1. Mutually exclusive expression of Sp1and KLF4 protein in noncancerous gastric mucosa and gastric cancer tissue and cells. Tissue sections were prepared from formalin-fixed, paraffin-embedded specimens of noncancerous human gastric and gastric cancer tissue. KLF4 and Sp1 protein staining was done with specific antibodies against Sp1and KLF4 protein. A, inverse relationship between KLF4 and Sp1expression was determined by Pearson’s m2 test (P =0.006).B, KLF4 (B1 and B3)andSp1(B2 and B4); magnification, Â50. Of note is that the majority of the tumor cells (T) exhibited loss of KLF4 expression in sharp contrast with the residual normal glandular epithelial cells (N).Whole cell lysates were prepared from two paired gastric cancer tissue specimens and normal gastrictissue specimens, two tumor tissue specimens exhibiting strong Sp1expression, and two tumor tissue specimens exhibiting weak Sp1expression (C1) or from various human gastric cancer cell lines as indicated and normal human gastrictissue ( C2). The level of Sp1and KLF4 expression was determined by using Western blot analysis. Equal protein sample loading was monitored by hybridizing the same membrane with an anti ^ glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH).

www.aacrjournals.org 6397 Clin Cancer Res 2006;12(21)November 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Human Cancer Biology restored KLF4 expression repressed Sp1 expression at both the EMSA. Both Sp1 and KLF4 bound to the same oligonucleotide mRNA (Fig. 2A) and protein (Fig. 2B) levels in SK-GT5 and N87 probe as confirmed by supershifts (Fig. 3B, lanes 2 and 6 for cells. Transduction with Ad-EGFP did not affect the KLF4 and Sp1 and lanes 3, 4, 7, and 8 for KLF4), and restored KLF4 Sp1 expression, suggesting that the repressed Sp1 expression expression repressed Sp1 binding (Fig. 3B, lane 1 versus lane 5). was not mediated by adenoviral transduction. To determine Mutant oligonucleotide probes completely abolished both Sp1 the mechanisms by which KLF4 inhibits Sp1 expression, we and KLF4 binding (Fig. 3B, lanes 9 and 10). These data examined the effect of KLF4 on Sp1 promoter activity using a suggested that KLF4 and Sp1 competitively bind to an full-length Sp1 promoter-driven pGL3 luciferase reporter. In overlapping region of the Sp1 promoter. both SK-GT5 and N87 cells, cotransfection of a KLF4 expression Next, we sought to determine whether Sp1 and KLF4 bind to vector inhibited Sp1 promoter activity, whereas cotransfection the same region of the Sp1 promoter in vivo using a chromatin of a mutant KLF4 expression vector, which lacked the nuclear immunoprecipitation assay. The schematic structure of the localization signal (32), did not inhibit but rather slightly proximal Sp1 promoter and the nucleotide positions and increased Sp1 promoter activity (Fig. 2C). A previous study sequences of the PCR forward and reverse primers are shown in reported that a minimal Sp1 promoter (À217 bp upstream of Fig. 4A. For chromatin immunoprecipitation assays, SK-GT5 the transcription initiation site) contains binding sites for cells were transduced for 24 hours with Ad-KLF4 or Ad-EGFP at various transcription factors, including Sp1, nuclear factor Y, a MOI of 10 and chromatin fragments were immunoprecipi- activator protein 2, and CCAAT/enhancer-binding protein tated overnight with or without specific anti-FLAG antibody. As (13, 14). We then tested the effect of KLF4 expression on this compared with EGFP-transduced cells, restored KLF4 expres- minimal Sp1 promoter luciferase reporter. Consistent with the sion clearly bound to the target sequence of the Sp1 promoter results of the use of the full-length Sp1 promoter construct, (Fig. 4B, lane 6). To determine whether overexpression of KLF4 KLF4 inhibited the minimal Sp1 promoter activity, but mutant affects Sp1 binding to its own promoter, chromatin fragments KLF4 did not (Fig. 2D). These results suggested that the were immunoprecipitated overnight with or without specific minimal Sp1 promoter contains KLF4-responsive element(s) anti-Sp1, Sp, and KLF4 antibodies. Increased KLF4 binding that mediate transcriptional repression of Sp1 gene expression. clearly reduced the binding of Sp1 and Sp3 to the same region Identification of a KLF4-binding site within the minimal Sp1 (Fig. 4C, lane 3 versus lane 7 for Sp3; lane 4 versus lane 8 for promoter. Initially, we used a software program to identify Sp1). The experiment was repeated thrice and the relative KLF4-binding sites on the Sp1 promoter and found a putative bindings of Sp1 and Sp3 (band intensity relative to that of KLF4-binding site overlapping a proximal Sp1-binding site input) were calculated and their reductions were significant (Fig. 3A). To confirm that KLF4 and Sp1 bind to this sequence, (P < 0.01). These data supported the idea that KLF4 and Sp1 we generated an oligonucleotide probe (Fig. 3A). For all in vitro bind to the same region of the Sp1 promoter in vivo in a binding assays, N87 (Fig. 3B1), SK-GT5 (Fig. 3B2), and FG cells competitive manner. (Fig. 3B3) were transduced for 24 hours with Ad-KLF4 or Ad- Antagonizing effects of Sp1 and KLF4 on Sp1 promoter. To EGFP at a MOI of 10. The nuclear proteins were extracted for determine whether Sp1 and KLF4 have antagonizing effects on

Fig. 2. Inhibition of Sp1expression by KLF4 overexpression. SK-GT5 and N87 cells were seeded to f80% confluence in 60 mm culture dishes and transduced with Ad-KLF4 orAd-EGFP for 24 hours at a MOI of 10. A, total RNAs were extracted, and the level of Sp1and KLF4 expression was determined by using Northern blot analysis. Equal mRNA sample loading was monitored by hybridizing the same membrane with a 36B4 probe. B, whole cell lysates were prepared, and the level of Sp1and KLF4 expression was determined by using Western blot analysis. Equal protein sample loading was monitored by hybridizing the same membrane with an anti ^ glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH). To determine the effect of KLF4 on Sp1reporter activity, the full-length Sp1promoter reporter (C)orminimalSp1 promoter reporter (D) was transfected into SK-GT5 and N87 cells with KLF4, mutant KLF4, or a control (pcDNA3) expression vector. Luciferase activity was determined 48 hours later and normalized according to the protein concentration. The Sp1 promoter activity was expressed as the fold change. Transfections were done in triplicate. *, P < 0.01, statistically significant compared with the respective control groups.

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Fig. 3. Binding of KLF4 to the Sp1promoter in vitro. A, schematic representation of the Sp1promoter sequences used as probes in EMSA. B, for all of the binding assays, N87 (B1), SK-GT5 (B2)andFG(B3) cells were seeded to f80% confluence in150 mm culture dishes and transduced with Ad-EGFP (lanes 1-4 and 9)orAd-KLF4(lanes 5-8 and 10 )ataMOIof10. Nuclear protein was extracted 24 hours later, and EMSA was done using both wild-type (Wt; lanes 1-8)and mutant (Mut; lanes 9 and 10) probes. For supershift assays, specific antibodies against Sp1 (lanes 2 and 6), KLF4 (lanes 3 and 7), or FL AG (lanes 4 and 8)were added and incubated for 25 minutes at room temperature before the addition of a labeled probe. Sp1 (3)andKLF4(., /) supershifted bands.

the Sp1 promoter activity, we generated a minimal Sp1 Sp1 overexpression but significantly reduced or lost KLF4 promoter reporter containing the same mutation applied to expression. Restored expression of KLF4 inhibited Sp1 expres- EMSA probe (Fig. 5A). Mutation of the KLF4-binding site sion at both the mRNA and protein level in human gastric increased the minimal Sp1 promoter activity, suggesting that cancer cell lines (23). Furthermore, we located the KLF4 this site was important for the negative regulation of Sp1 binding site within the proximal Sp1 promoter sequence that promoter activity and made SK-GT5 cells less sensitive to the overlaps the Sp1 binding site and competition with Sp1, which repressive effect of KLF4 restoration (Fig. 5B). Finally, we tested is a positive regulator of its own transcription. These findings the effect of interference with endogenous Sp1 expression on suggestthatlossofKLF4expressioncontributestoSp1 the minimal Sp1 promoter activity using a siRNA. Treatment overexpression in human gastric cancer. with Sp1 siRNA significantly knocked down the endogenous Tumor development and progression require cell growth, Sp1 protein expression (Fig. 5C, inset), which was accompanied apoptosis resistance, cell invasion, and angiogenesis. Increasing by a reduced minimal Sp1 promoter activity (Fig. 5C). In evidence shows that Sp1 regulates multiple genes that are contrast, KLF4 knockdown increased Sp1 expression (Fig. 5C, important for tumor development and progression (33). These inset), which was consistent with increased Sp1 promoter genes include epidermal growth factor receptor (34), hepato- activity (Fig. 5C). These data suggested that endogenous Sp1 is cyte growth factor receptor (35), transforming growth factor-a positively involved in Sp1 promoter activity, whereas KLF4 does (36), and thymidine kinase (37), which are key to cell the opposite. proliferation and growth; Bcl-2 (38), Bcl-x (39), and survivin (40), which are key to cell survival and apoptosis resistance; Discussion matrix metalloproteinase-2 (41, 42) and urokinase-type plas- minogen activator (43), which are key to cell migration and In this study, we found mutually exclusive expression of invasion; and basic fibroblast growth factor and vascular KLF4 and Sp1 in human gastric cancer and noncancerous endothelial growth factor (44–47), which are key to angio- tissues. This observation is consistent with our previous reports genesis. Consistent with these findings, we recently reported showing that the majority of human gastric cancer cases exhibit that Sp1 overexpression is associated with vascular endothelial

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Fig. 6. Schematic model of Sp1regulation by KLF4.A, innormal gastrictissue, Sp1 positively regulates Sp1transcription, whereas KLF4 negatively regulates Sp1 Fig. 4. Binding of KLF4 to the Sp1promoter in vivo. A, schematic structure of the transcription through competition with Sp1for Sp1-binding sites on the Sp1promoter. proximal Sp1promoter.The nucleotide positions and sequences of PCR forward and A proper KLF4/Sp1ratio maintains a properlevelof Sp1expression and function. reverse primers are shown. For the chromatin immunoprecipitation assay, SK-GT5 B, intumor tissue, geneticandepigeneticalterations may leadto reducedorlost KLF4 cells were seeded to f80% confluence in 10 cm culture dishes and transduced for expression, and consequently incur enhanced positive autoregulation of Sp1 24 hours with Ad-KLF4 orAd-EGFP at a MOI of 10. B, chromatin fragments were transcriptionby Sp1protein. Sp1overexpressionpromotes tumor development and immunoprecipitated overnight without (lanes 2 and 5)orwithananti-FLAG progression through the alterationof the expressionof its downstream target genes. antibody (lanes 3 and 6). Lanes 1and 4, control input of chromatin fragments. The PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light. C, similarly, chromatin fragments were On the other hand, accumulating evidence indicates that immunoprecipitated overnight with no antibody (lanes 2 and 7), an anti-Sp3 antibody (lanes 3 and 8), an anti-Sp1antibody (lanes 4 and 9), or an anti-KLF4 KLF4 is a putative tumor suppressor gene. Reduced and/or lost antibody (lanes 5 and 10). Lanes 1and 6, control input of chromatin fragments.The KLF4 expression has been reported in various tumors including PCR products were visualized. All experiments were done at least twice, and one of esophagus, stomach, colon, bladder, lung, and adult T cell two or three representative experiments was shown. leukemia (22–27). We recently reported that genetic or epigenetic loss of KLF4 expression in human gastric cancer growth factor expression, advanced stage, and poor prognosis and SK-GT5 cells used in the present study carry hemizygous in patients with resected gastric cancer (11, 12), and increased deletion of the KLF4 gene (23). Functionally, KLF4 up-regulates angiogenic potential of human pancreatic cancer (48). Thus, differentiation marker genes such as keratin 4 or intestine Sp1 may play a central regulatory role in many such pathways alkaline phosphotase (16, 17), whereas KLF4 down-regulates of tumor development and progression and may be an positive cell cycle–regulatory genes such as cyclin D1 and attractive target for cancer treatment. In fact, we recently ornithine decarboxylase (18, 19). Interestingly, several reports showed that the cyclooxygenase-2 inhibitor, celecoxib, inhibits have indicated that KLF4 represses the expression of several angiogenesis and metastasis of human pancreatic cancer in part genes by competing with Sp1 (49, 50). However, whether KLF4 by suppression of Sp1 activity (31). is involved in Sp1 regulation and tumor development and progression in gastric cancer is unknown. The Sp1 promoter contains several putative Sp1-binding sites, suggesting that Sp1 regulates its own expression (13). This notion of positive autoregulation is substantiated by our results, showing that knocking down Sp1 expression with a siRNA significantly decreased Sp1 promoter activity. Conversely, a putative KLF4-binding site within the proximal Sp1 promoter sequence overlaps the proximal Sp1-binding site, to which region both Sp1 and KLF4 compete for binding as shown by in vitro and in vivo functional analyses. Moreover, Sp1 promoter activity was increased upon inactivation of the KLF4- binding site. Therefore, this region plays an important role in the positive and negative regulation of Sp1 by Sp1 and KLF4, respectively. However, this mutation did not completely abrogate the inhibitory effects of KLF4, suggesting that KLF4 may interfere with other Sp1 binding sites, Sp1 protein stability, or other mechanisms which require further Fig. 5. Mutational analysis of the KLF4-binding site and repression of the Sp1 promoter by knocking down Sp1expression. A, schematic structure of the proximal investigation. Sp1promoter. The nucleotide sequences of the overlapping Sp1- and KLF4-binding Several previous studies showed that Sp1 activity is regulated sites were shown in both wild-type (Wt) and mutant (Mut)form.B, wild-type by posttranslational modification, degradation, and interaction and mutant Sp1promoter reporters were transfected into SK-GT5 cells with a KLF4 expression or control vector.The luciferase activity was determined 48 hours later with other transcription factors (5). The present study provided and normalized according to the protein concentration. The Sp1promoter activity a novel mechanism for the regulation of Sp1 expression, i.e., was expressed as the fold change. C, the Sp1promoter reporter was transfected into SK-GT5 cells with an Sp1siRNA vector mix, KLF4 siRNA vector mix, or control Sp1 expression itself is negatively regulated by KLF4. Physio- vector.The Sp1promoter activity was determined as described above, and the same logically, the balance between Sp1 and KLF4 expression may cell lysates were used forWestern blot analysis to confirm the efficacy of the Sp1 play a critical role in the homeostasis of gastrointestinal and KLF4 siRNA vectors (insets). All transfections were done in triplicate, and each experiment was repeated thrice. *, P < 0.01, statistically significant compared with mucosa. However, during gastric cancer development and respective control groups. progression, alteration of KLF4 expression may lead to aberrant

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Sp1 expression. This notion is supported by the recent report of multiple genes key to the regulation of cell growth, apoptosis that conditional KLF4 knockout mice showed altered prolifer- resistance, angiogenesis, and cell invasion (Fig. 6B). Therefore, ation and differentiation and precancerous changes in the adult negative regulation of Sp1 expression by KLF4 may play an stomach (20). This mechanism may not be specific to gastric important role in the homeostasis of normal gastrointestinal cancer but rather a common mechanism in the progression of tissue, and loss of KLF4 expression and Sp1 overexpression may gastrointestinal cancer, because Sp1 overexpression and KLF4 contribute to tumor development and progression. Finally, down-regulation have also been reported in colorectal cancer because of the structural and functional similarity among (7, 25). members of the Sp1 family, KLF4 might also affect the In summary, we showed that KLF4 inhibited Sp1 expression expression and function of Sp2, Sp3, and Sp4, and the at the Sp1 promoter activity, mRNA, and protein levels, in part underlying mechanisms warrant further investigation (33, 47). through its binding site overlapping a Sp1-binding site. A proper Sp1/KLF4 ratio may control Sp1 transcription at the physiologic level (Fig. 6A). However, reduced or lost KLF4 Acknowledgments expression because of genetic or epigenetic changes in the tumor may trigger Sp1 overexpression because of attenuated We thank Judy King for expert help in the preparation of this manuscript, Don Norwood for editorial comments, ChichuanTseng (Boston University School negative regulation of KLF4 and augmented positive autoregu- of Medicine) for providing KLF4 in the pcDNA3.1expression vector, and Carlos J. lation by Sp1. The resulting Sp1 overexpression may promote Ciudad (University of Barcelona, Barcelona, Spain) for providing the Sp1promoter tumor development and progression by altering the expression construct.

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Masashi Kanai, Daoyan Wei, Qiang Li, et al.

Clin Cancer Res 2006;12:6395-6402.

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