Drastic Down-regulation of Kru¨ppel-Like Factor 4 Expression Is … · Drastic Down-regulation of...

Drastic Down-regulation of Kruppel-Like Factor 4 Expression Is

Critical in Human Gastric Cancer Development and Progression

Daoyan Wei,1Weida Gong,

2Masashi Kanai,

1Christian Schlunk,

1Liwei Wang,

1

James C. Yao,1Tsung-Teh Wu,

3Suyun Huang,

2,4and Keping Xie

1,4

Departments of 1Gastrointestinal Medical Oncology, 2Neurosurgery, 3Pathology, and 4Cancer Biology, University of TexasM.D. Anderson Cancer Center, Houston, Texas

Abstract

Kruppel-like factor 4 (KLF4) is highly expressed in epithelialtissues such as the gut and skin. However, the role of KLF4 inhuman gastric cancer development and progression isunknown. Here we show that KLF4 protein expression wasdecreased or lost in primary tumors and, in particular, lymphnode metastases when compared with that in normal gastricmucosa. Moreover, loss of KLF4 expression in the primarytumors was significantly associated with poor survival, andalso an independent prognostic marker in a multivariateanalysis. Consistently, most human gastric cancer cell linesexhibited loss of or a substantial decrease in KLF4 expressionat both RNA and protein levels. Enforced restoration of KLF4expression resulted in marked cell growth inhibition in vitroand significantly attenuated tumor growth and total abroga-tion of metastasis in an orthotopic animal model of gastriccancer. Mechanism studies indicated that promoter hyper-methylation and hemizygous deletion contributed to thedown-regulation of KLF4 expression and the induction ofapoptosis contributed to the antitumor activity of KLF4.Collectively, our data provide first clinical and casual evidenceand potential mechanism that the alteration of KLF4expression plays a critical role in gastric cancer developmentand progression. (Cancer Res 2005; 65(7): 2746-54)

Introduction

Although the incidence of gastric cancer declined in the Westfrom the 1940s to the 1980s, it remains a major public healthproblem throughout the world (1). In Asia and parts of SouthAmerica in particular, it is the most common epithelial malignancyand leading cause of cancer-related deaths. In fact, gastric cancerremains the second most frequently diagnosed malignancyworldwide and cause of 12% of all cancer-related deaths each year(1, 2). Advances in treatment of this disease are likely to come froma fuller understanding of its biology and behavior.The aggressive nature of human metastatic gastric carcinoma

is related to a number of molecular abnormalities, includingmicrosatellite instability, inactivation of various tumor suppressorgenes, activation of various oncogenes, and reactivation of telome-rase (3–6). These abnormalities affect the downstream signaltransduction pathways involved in the control of cell growth anddifferentiation and confer a tremendous survival and growthadvantage to gastric cancer cells (4–6). Although gastric cancer

may harbor multiple molecular alterations, they may not bespecific for gastric cancers (5–10). Thus, the identified abnormal-ities may represent only the pathogenesis of gastric cancer, andthey have not been identified as a specific sequence of changesleading to gastric carcinoma (11, 12). Therefore, the role of geneticchanges such as altered tumor suppressor genes in gastric cancerdevelopment and progression remains unclear.Kruppel-like factor 4 (KLF4; formerly GKLF) is a zinc-finger

transcription factor and its mRNA expression is found primarilyin the post-mitotic, terminally differentiated epithelial cellsin organs such as skin, lungs, gastrointestinal tract, and severalothers (13, 14). In cell culture, KLF4 expression is associated withgrowth arrest (13). Expression of this factor can be increased byserum deprivation, contact inhibition, and DNA damage (13, 15).Conversely, KLF4 expression seems to decrease in early intestinaladenomas, colonic adenomas and colonic adenocarcinomas,esophageal cancer, bladder cancer, prostate cancer, and benignprostate hypertrophy of mice and/or patients with hereditary andsporadic tumors (16–20). Moreover, forced overexpression of KLF4due to either transient or stable transfection inhibits cellproliferation and growth of colon, bladder, and esophageal cancer(13, 21). However, KLF4 expression has been shown to increase inprimary breast ductal carcinoma (22) and oral squamouscarcinoma (19). Although these findings are inconsistent, theysuggest that KLF4 has an important function in tumor develop-ment and progression, molecular basis of which requires furtherelucidation. Moreover, it remains unknown whether and, if so, howKLF4 contributes to the development and progression of gastriccancer.In the present study, we investigated the level of KLF4 expression

in human gastric cancer cells and tissues and the effect of itsalteration on gastric cancer biology and clinical outcome. We foundthat KLF4 expression was substantially decreased or lost in humangastric cancer, particularly in metastatic lymph nodes. Further-more, decreased or lost KLF4 expression was directly correlatedwith poor patient survival. Consistently, restoration of or anincrease in KLF4 expression significantly inhibited gastric cancergrowth in vitro and gastric tumor formation in vivo . This antitumoractivity was due to the induction of apoptosis. Therefore, our datasuggest that genetically and epigenetically inactivated KLF4 is amolecular marker for poor prognosis and directly contribute togastric cancer development and progression.

Materials and Methods

Human tissue specimens and immunohistochemistry. We used 86

human gastric cancer tissue specimens as described previously (23). Patientcharacteristics were summarized in Table 1. We also included in this study

51 lymph node metastasis specimens and 60 normal gastric tissue specimens

obtained from patients without gastric cancer. Sections (5-Am thick) of

Requests for reprints: Keping Xie, Department of Gastrointestinal MedicalOncology, University of Texas M.D. Anderson Cancer Center, Unit 426, 1515 HolcombeBoulevard, Houston, TX 77030. Phone: 713-792-2828; Fax: 713-745-1163; E-mail:[email protected].

I2005 American Association for Cancer Research.

Cancer Res 2005; 65: (7). April 1, 2005 2746 www.aacrjournals.org

Research Article

Research. on April 9, 2020. © 2005 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

formalin-fixed, paraffin-embedded tumor specimens were prepared andprocessed as described previously (23). Standard immunostaining proce-

dure was done using a rabbit polyclonal antibody against human KLF4

(clone H180, 1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA).

A positive reaction was indicated by a reddish-brown precipitate in thenuclei and cytoplasm. Depending on the percentage of positive cells and

staining intensity, KLF4 staining was classified into three groups: negative,

weak, and strong expression as described previously (23).Cell lines and culture conditions. Human gastric cancer cell lines AGS,

HTB103, HTB135, N87, SNU-1, and TMK1 were purchased from the

American Type Culture Collection (Manassas, VA) and SK-GT5 was

obtained from Dr. Gary K. Schwartz (Memorial Sloan-Kettering CancerCenter, New York, NY). All of the cell lines were maintained in plastic flasks

in minimal essential medium supplemented with 10% fetal bovine serum,

sodium pyruvate, nonessential amino acids, L-glutamine, and a vitamin

solution (CMEM, Flow Laboratories, Rockville, MD).Generation of recombinant adenovirus and conditions of adenovi-

rus transduction. The full-length KLF4 cDNA in pcDNA3.1 vector was

kindly provided by Dr. Chichuan Tseng (Boston University School ofMedicine; ref. 24). The two PCR primers (5V-gataaggtaccatggatt-acaaggatgacgacgataagggggctgtcagcgacgcgctgctc-3V and 5V-taatgcggcc-gcttaaaaatgcctcttcatgtgtaagg-3V) were used to generate FLAG-KLF4

sequence. The recombinant adenovirus containing KLF4 (Ad-KLF4) andenhanced green fluorescent protein (EGFP; Ad-EGFP) were generated with

the use of the Adeasy Adenoviral Vector System (Stratagene, La Jolla, CA)

and purified by double CsCl gradient centrifugation to achieve a titer of

approximately 1010 plaque forming units/mL.Western blot analysis. Fresh gastric cancer and corresponding

noncancerous gastric tissues were obtained from patients who underwent

gastrectomy at M.D. Anderson Cancer Center. The cancerous and

noncancerous portions were macroscopically identified and excised byexperienced pathologists and further confirmed by histopathologic

examination. Additionally, whole cell lysates were prepared from humannormal and gastric cancer tissue specimens or cell cultures. Four paired

normal gastric and gastric tumor tissue specimens were selected from the

patients with known expression levels of KLF4 as confirmed by

immunostaining as well as a similar percentage of tumor epithelial cellsrelative to stromal cells. Standard Western blotting was done with the H-180

polyclonal rabbit antibody against human KLF4 (Santa Cruz Biotechnology).

For detection of recombinant KLF4, anti-FLAG antibody (Sigma Co., StLouis, MO) was used. Protein sample loading was monitored by incubating

the same membrane filter with a glyceraldehyde-3-phosphate dehydroge-

nase (GAPDH) antibody (25).

Cell proliferation assay. N87 and SK-GT5 cells were seeded at 4 � 105

cells per well in 6-well culture plates. Twelve hours later, the cells were

incubated for 2 hours at 37jC in serum-free medium or serum-free medium

with Ad-KLF4 or Ad-EGFP at a multiplicity of infection (MOI) of 20. After

being washed with serum-free medium, the transduced cells werereplenished with DMEM and incubated for 1 to 4 days. The cell numbers

were counted daily via the trypan blue exclusion method with a

hemocytometer.DNA ladder assay. N87 and SK-GT5 cells were seeded at 1� 106 cells per

well in 100-mm culture plates. Twelve hours later, the cells were incubated

for 2 hours at 37jC in serum-free medium with Ad-KLF4 at MOI of 0, 10,

or 20, and the total MOI in each group was adjusted with Ad-EGFP to equalof 20. After being washed with a serum-free medium, the transduced cells

were replenished with complete minimal essential medium and incubated

for 36 hours. Next, DNA was isolated and a DNA ladder assay was done

with Apopladder Ex kit (Takara Biochemicals, Shiga, Japan) according tothe manufacturer’s instructions. Extracted DNA was then subjected to

electrophoresis on a 1.5% agarose gel and detected by Sybergreen staining.

Detection of apoptosis in situ. N87 and SK-GT5 cells were seeded at 0.5

to 1 � 105 cells per well in eight-chamber culture slides. Twelve hours later,the cells were infected with Ad-KLF4 or Ad-EGFP at a MOI of 20. Thirty-six

Table 1. Patient characteristics and KLF4 expression

Characteristic Total (n = 86) KLF4 staining P

Negative (%), n = 27 Weak (%), n = 47 Strong (%), n = 12

Sex

Men 56 20 (35.7) 29 (51.7) 7 (12.5) 0.487Women 30 7 (23.3) 18 (60.0) 5 (16.7)

Age (years)

Mean (SD) 61.8 (14.0) 57.4 (15.0) 62.6 (13.5) 68.4 (11.3) 0.463

Pathology typePapillary 12 1 (8.3) 9 (75.0) 2 (16.7) 0.358

Tubular 28 7 (25.0) 15 (53.6) 6 (21.4)

Diffuse 8 2 (25.0) 4 (50.0) 2 (25.0)

Mucinous 5 2 (40.0) 2 (40.0) 1 (20.0)Signet ring 21 9 (42.9) 11 (52.3) 1 (4.8)

Mixed 12 6 (50.0) 6 (50.0) 0 (0.0)

StageI 14 0 (0.0) 12 (85.7) 2 (14.3) 0.005

II 28 5 (17.9) 17 (60.7) 6 (21.4)

III 30 15 (50.0) 11 (36.7) 4 (13.3)

IV 14 7 (50.0) 7 (50.0) 0 (0.0)Residual disease

R0 69 16 (23.2) 41 (59.4) 12 (17.4) 0.003

R1, R2 17 11 (64.7) 6 (35.3) 0 (0.0)

Lauren’s classificationIntestinal 53 14 (26.4) 30 (56.6) 9 (16.9) 0.351

Diffuse 33 13 (39.4) 17 (51.5) 3 (9.1)

NOTE: Pearson’s m2 test was done to determine the statistical significance of the relationship of KLF4 expression with various variables.

Kruppel-Like Factor 4 and Gastric Cancer

www.aacrjournals.org 2747 Cancer Res 2005; 65: (7). April 1, 2005



hours after infection, the cells were processed and apoptotic cells weredetected in situ with the In situ Cell Death Detection Kit (Roche Applied

Science, Indianapolis, IN) according to the manufacture’s instruction. A

positive control slide known to express the target antigen and a negative

control slide without the terminal deoxynucleotidyl transferase–mediateddUTP-biotin nick-end labeling (TUNEL) reaction mixture added were

included for each staining procedure. Brown staining of nuclei was

interpreted as positive immunoreactivity.

Northern blot analysis. Total RNA was extracted using TRIzol reagent

(Invitrogen, San Diego, CA), and standard Northern blotting was done as

described previously (25). For detecting KLF4, [32P]-dCTP-labeled KLF4

cDNA probe was applied, and [32P]-dCTP-labeled GAPDH cDNA probe was

used to monitor RNA sample loading.

Southern blot analysis. DNA samples were isolated using the QIAampDNA Mini Kit (Qiagen, Valencia, CA). Extraction of genomic DNA from cell

lines was conducted according to standard procedures. Southern blot

analysis was done using 8 Ag of genomic DNA digested with EcoRI or EcoRI

plus NcoI. A full-length KLF4 cDNA was used as the probe.Methylation-specific PCR. Methylation-specific PCR was done using

genomic DNA, whichwasmodifiedwith bisulfite according tomanufacturer’s

instruction (EN DNA Methylation Kit, Orange, CA). For detecting unmethy-

lated DNA, the forward primer was 5V-gtttttatattaatgaggtaggtgaggtg-3V andthe reverse primer was 5V-aaacaaaaacaaaaaaatcaaaaaca-3V, which were

designed to amplify a 118-bp sequence between nucleotides �156 and �39

relative to the translation initiation site of the human KLF4 exon 1 region. For

detecting methylated DNA, the forward primer was 5V-tttatattaatgaggtaggt-gaggc-3Vand the reverse primer was 5V-gaaaacaaaaaaatcaaaaacgac-3V, whichwere designed to amplify a 111-bp sequence between nucleotides �153 and

�43 relative to the translation initiation site of the human KLF4 exon 1region. The negative control was water. The positive control for the

methylation-specific reaction was normal human genomic DNA from

Promega (Madison, WI), which was treated in vitro with SssI methylase

(New England Biolabs, Beverly, MA). Each PCR reaction of 50 AL consistedof 40 ng DNA and 200 nmol/L each of the forward and reverse primers and

0.5 AL HotStart Taq enzyme (Qiagen). Each PCR reaction was hot started at

95jC for 15 minutes and then amplified for 35 cycles (94jC for 30 seconds,

54jC for 30 seconds, and 72jC for 30 seconds). PCR products were visualizedon a 2% agarose gel stained with ethidium bromide. The products were also

directly sequenced to determine the locations of CpG methylation.

Animals. Female athymic BALB/c nude mice were purchased from

Jackson Laboratory (Bar Harbor, ME). The mice were housed in laminar

flow cabinets under specific pathogen-free conditions and used when they

were 8 weeks old. The animals were maintained in facilities approved by the

Association for Assessment and Accreditation of Laboratory Animal Care in

accordance with the current regulations and standards of the U.S.

Department of Agriculture, Department of Health and Human Services,

and NIH.

Tumor growth and metastasis. To prepare tumor cells for inoculation,

cells in the exponential growth phase were harvested via brief exposure to a

0.25% trypsin/0.02% EDTA solution (w/v). Cell viability was determined

using trypan blue exclusion, and only single-cell suspensions that were >95%

viable were used. Tumor cells (1 � 106 cells per mouse) were then injected

into either subcutis or stomach wall of nude mice. The animals were killed

60 days after the tumor cell injection or when they had become moribund.

Next, the primary gastric tumors were harvested and weighed. Regional

lymph nodes (at least five for each mouse) were collected and examined for

tumor metastasis by histopathology. Metastasis was expressed as percentage

incidence using the following formula: metastasis incidence (%) = [mice with

metastasis / total mice used] � 100; where metastasis were regional and/or

distant. In addition, each mouse’s liver was fixed in Bouin’s solution for

24 hours to differentiate the neoplastic lesions from the organ parenchyma;

metastases on the surface of liver were counted (double blinded) with the aid

of a dissecting microscope as described previously (25).

Statistics. The two-tailed m2 test was done to determine the significanceof the difference between the covariates. The Kaplan-Meier method was

used to calculate survival durations, and the log-rank test was used to

compare the cumulative survival durations in the patient groups.

Furthermore, the Cox proportional hazards model was used to computemultivariate hazards ratios for the study variables; the level of KLF4

expression, age, sex, Lauren’s histology type, stage (American Joint

Committee on Cancer), and completeness of surgical resection (R0 versus

R1 and R2) were included in the model. The SPSS software program (version11.05; SPSS, Inc., Chicago, IL) was used for the analyses. For in vitro and

in vivo studies, each experiment was done independently at least twice with

similar results; one representative experiment was presented. The

significance of the in vitro data was determined using Student’s t test(two tailed), whereas that of the in vivo data was determined using the two-

tailed Mann-Whitney U test. In all of the tests, a P < 0.05 was defined as

statistically significant.

Results

Distinct Kruppel-like factor 4 expression in human normalgastric and gastric tumor tissue. To determine the effect of KLF4expression on gastric cancer development and progression, we didimmunohistochemical staining of paraffin-embedded normalgastric tissues and gastric cancer tissue specimens with anantibody against KLF4 protein. We found KLF4 expression in thecytoplasm and nuclei in most of the cells in the specimensobtained from patients who did not have cancer. We observedstrong positive staining in the cytoplasm and nuclei of cellslocalized predominantly in the glandular epithelium (glandulardifferentiation; Fig. 1A) but decreased KLF4 expression in the cellslocated near the neck region of gastric mucous and near the gastricpit ( foveolar differentiation). In sharp contrast, KLF4 expressionwas significantly decreased or lost in the cytoplasm and nuclei ofvarious types of gastric cancer cells (Fig. 1B). Western blot analysiswas used to further examine the expression of KLF4 in four pairedhuman normal gastric and tumor tissue specimens (Fig. 1F and G).As reported previously (24), we observed two bands for KLF4 inWestern blot analysis, and the band shown (Fig. 1F) was thepredominant and higher molecular weight band. Consistent withthe level of KLF4 protein expression determined via immunostain-ing, Western blot analysis showed that normal gastric tissuespecimens had a significantly higher level of KLF4 expression thandid gastric tumor tissue specimens. These results indicated thatKLF4 is commonly expressed in human normal gastric cells butrarely expressed in human gastric cancer.Kruppel-like factor 4 expression in normal human gastric

mucosa, gastric tumor tissue, and metastatic lymph nodes. Wesystematically analyzed KLF4 expression in 86 primary gastrictumor, 51 lymph node metastasis, and 60 normal human gastrictissue specimens. In the primary tumor tissue specimens, the levelof KLF4 expression was strong, weak, and negative in 12 (14%), 47(54.7%), and 27 (31.4%) of the cases, respectively (Table 1). Amongthe three KLF4 expression categories, we found no significantdifferences in distribution according to sex, tumor pathologictypes, and Lauren’s histology classification. However, we didobserve a significant difference in the distribution of the patientsaccording to residual disease status (P = 0.003), and significantly,patients showed a clearly progressive loss of KLF4 expression fromstage I to stage IV according to the American Joint Committee onCancer staging (P = 0.005), suggesting that loss of KLF4 expressioncontributed to gastric cancer progression.When we compared the KLF4 expression in normal gastric

mucosa, primary tumors, and metastatic lymph nodes, we foundsignificantly lower expression in both the primary tumors andmetastatic lymph nodes than in the normal mucosa (P < 0.0001).Moreover, the expression of KLF4 was even lower in themetastatic lymph nodes than in the primary tumors (P < 0.05;

Cancer Research




Fig. 1C , D , and E ; Table 2), suggesting that loss of KLF4expression may also contribute to gastric cancer metastasis.Overall, there was a decrease in or loss of KLF4 expression,which correlated with tumor development and progression.Effect of Kruppel-like factor 4 expression on patient

survival. The median survival duration in patients who had atumor with negative, weak, or strong KLF4 expression was 378,

1,242, and 2,489 days,respectively. Thus, decreased KLF4 expressionwas associated with an inferior survival duration (P = 0.0002).According to Kaplan-Meier plots of overall survival in patients withgastric cancers, the survival for 12 patients who had a tumor withstrong KLF4 expression was significantly longer than that for the 47patients with weak KLF4 expression and the 27 patients withnegative KLF4 expression (P < 0.001; Fig. 1H). Other variables that

Figure 1. KLF4 protein expression and localization innormal gastric mucosa, primary gastric cancer, and lymphnode metastasis and its association with patient survival.Tissue sections were prepared from formalin-fixed,paraffin-embedded specimens of normal human gastrictissue (A) and gastric cancer tissue (B ). KLF4 proteinstaining was performed with an antibody against KLF4protein. The photographs were taken at a magnification of50�. Note that the majority of the tumor cells (T ) exhibiteda loss of KLF4 expression in sharp contrast to the residualnormal glandular epithelial cells (N ). In addition, thephotographs representative of strong (C ) and negative (D )KLF4 protein expressions in primary gastric cancer andnegative KLF4 expression in a metastatic lymph node (E)were taken at a magnification of 400�. Whole cell proteinextracts were further prepared from four-paired normalgastric (N) and gastric tumor tissue (T ) specimens. Theexpression level of KLF4 protein was determined by Westernblot analysis. Equal protein sample loading was monitored byhybridizing the same membrane filter with an anti-GAPDHantibody. The level of KLF4 protein expression wassignificantly decreased in tumor tissue when compared withthat in normal tissue (F and G). For Kaplan-Meier plots ofoverall survival in patients with gastric cancers (H ), thesurvival for 12 patients who had a tumor with strong KLF4expression was significantly longer than that for the 47patients with weak KLF4 expression and the 27 patientswith negative KLF4 expression (P < 0.001).

Table 2. KLF4 expression levels in different tissue specimens

Specimens Total KLF4 staining P

Negative (%) Weak (%) Strong (%)

Normal 60 0 (0) 12 (20.0) 48 (80.0)

Primary tumor 86 27 (31.4) 47 (54.7) 12 (14.0) <0.0001*

Lymph node metastases 51 26 (51.0) 24 (47.1) 1 (2.0) <0.0001c, 0.015

b

NOTE: Pearson’s two-tailed m2 test was done with the SPSS software program to determine the statistical significance of the level of expression of KLF4

in different tissue specimens.

*Primary tumor versus noncancerous tissue.cLymph node metastasis versus noncancerous tissue.bPrimary tumor versus lymph node metastasis.





affected survival in univariate analyses included disease stages (P <0.001) and completeness of resection (P = 0.0002). The patients’ ageat diagnosis (as a continuous variable in Cox proportional hazardsanalysis), sex, and Lauren’s classification did not have a statisticallysignificant effect on survival.Next, the patients’ level of KLF4 expression, disease stage,

completeness of resection, Lauren’s histology type, age, and sexwere entered in a Cox proportional hazards model for multivariateanalysis. When the effect of covariates was adjusted, the loss ofKLF4 expression was an independent predictor of poor survival(P = 0.017). The odds ratio in the group with negative KLF4expression (4.86; 95% confidence interval, 1.614-14.640) and weakKLF4 expression (3.83; 95% confidence interval, 1.38-10.640) werestatistically significantly higher (P < 0.01) than that in the groupwith strong KLF4 expression (reference). In addition, the advancedstage (P < 0.01) was also independent predictors of poor survival inthis Cox proportional hazards model of multivariate survival

analysis. However, patients’ gender or age at diagnosis, complete-ness of resection, and Lauren’s histology type had no statisticallysignificant effect on survival in the multivariate analyses.In vitro cell growth suppression by restoration of Kruppel-

like factor 4 expression. To examine the biological activities ofthe KLF4 gene in gastric cancer cells, we first examined theexpression of KLF4 in various human gastric cancer cell lines at themRNA level via Northern blot analysis (Fig. 2A) and protein levelvia Western blot analysis (Fig. 2B). Normal gastric mucosaspecimens were included as references for KLF4 expression. Theexpression of KLF4 was substantially decreased in all seven gastriccancer cell lines compared with that in normal gastric mucosacells. N87 and SK-GT5 gastric cancer cell lines, which express KLF4at low levels, were chosen for restoration of KLF4 expression viatransduction of adenoviral KLF4 . At both mRNA (Fig. 2C) andprotein levels (Fig. 2D), KLF4 was dose-dependently expressed inthe tumor cells. The increased KLF4 expression was consistent with

Figure 2. KLF4 expression in human gastric cancer cell lines and effects of restored KLF4 expression on tumor cell growth in vitro and in vivo. A panel of gastriccancer cell lines was incubated for 18 hours in a medium. Cellular RNA and total protein lysates were harvested from the cultures and normal human gastric mucosatissues (lanes 1 and 2) were included as controls. The tumor cell lines were AGS (lane 3), HTB103 (lane 4), HTB135 (lane 5), SK-GT5 (lane 6), N87 (lane 7), SNU-1(lane 8), and TMK-1 (lane 9). A, KLF4 RNA expression was measured by Northern blot analysis; B, KLF4 protein expression was measured by Western blot analysis.The relative KLF4 expression was expressed as K /G ratio (ratio between KLF4 and GAPDH). Note that both KLF4 RNA and protein expression were decreased inall human gastric cancer cell lines. For restoration of KLF4 expression and suppression of tumor growth in vitro, N87 and SK-GT5 cells were incubated for 18 hoursin medium alone (lane 1) or with control Ad-EGFP (lane 2) at an MOI of 30 or Ad-KLF4 (lanes 3-5) at an MOI of 10, 20, and 30. Ad-EGFP was used to adjust thetotal MOI equal to 30 MOI. Cellular RNA and total protein lysates were harvested from the cell cultures for KLF4 expression analysis for Northern blot analysis (C )and Western blot analysis (D). Tumor cells (E1 for N87 and E2 for SK-GT5) were plated into 60-mm dishes for 18 hours and incubated with an adenovirus for 2 hours.The cell numbers were determined by cell counting 1, 2, 3, and 4 days after adenoviral transduction at an MOI of 20. For inhibition of human gastric cancer growthby KLF4 in vivo, N87 and SK-GT5 cells transduced with control Ad-EGFP and Ad-KLF4, respectively, were injected s.c. (F1 for N87 and F2 for SK-GT5) into nude mice(1 � 106 per mouse) or the wall of the stomach (G1 for N87 and G2 for SK-GT5) of nude mice. Tumor sizes and metastasis were determined as described in Materialsand Methods. Note that the control NCI-N87 and SK-GT5 cells and NCI-N87 and SK-GT5 cells transduced with control Ad-EGFP produced larger tumors, whereasthose transduced with KLF4 only produced small tumors. *, P < 0.01, statistical significance in a comparison between the treated and respective untreated groups.One representative experiment of three with similar results.

Cancer Research




cell growth suppression in vitro as determined via cell counting(Fig. 2E). These data clearly showed that restoration of KLF4expression led to suppression of tumor cell growth.Inhibition of human gastric cancer growth and metastasis

by Kruppel-like factor 4 in vivo . To determine the effect of KLF4expression on tumor growth kinetics, N87 and SK-GT5 cells wereinjected s.c. into nude mice. As shown in Fig. 2F, control N87 andSK-GT5 or N87 and SK-GT5 cells transduced with control Ad-EGFPgrew progressively, whereas N87 and SK-GT5 cells transduced withKLF4 only grew slowly. To make them more biologically relevant,N87 and SK-GT5 cells were injected into the stomach wall of micein groups of 10 (an orthotopic animal model of gastric cancer). Thecontrol N87 and SK-GT5 cells and N87 and SK-GT5 cells transducedwith control Ad-EGFP produced larger tumors and metastasized toregional lymph nodes and the liver, whereas N87 and SK-GT5 cellstransduced with KLF4 only produced localized small tumors (Fig.2F and G). Therefore, enforced restoration of KLF4 expressionsuppressed human gastric cancer growth and metastasis.Induction of apoptosis by restored Kruppel-like factor 4

expression in gastric cancer cells. To further investigate themechanism by which KLF4 inhibited gastric cancer cell growth, westudied the effects of KLF4 expression on apoptosis and the cell cycle

via fluorescence-activated cell sorting (FACS) analysis. We foundthat increased expression of KLF4 induced apoptosis of both N87and SK-GT5 cells in a dose-dependent manner by FACS analysis(data not shown). These findings were confirmed using twoadditional assays for apoptosis: genomic DNA laddering (Fig. 3A)and TUNEL (Fig. 3B and C) assays. These results were consistentwith those of our in vitro cell counting assay, which showed that thecell number progressively decreased uponKLF4 transduction. Lastly,we did TUNEL assay on gastric cancer tissues with known negativeor positive KLF4 expression. Significantly decreased apoptosis wasdetected in tumor tissues with decreased or lost KLF4 expression(Fig. 3D1) compared with that in tumor tissues with positive KLF4expression (Fig. 3D2).Hemizygous deletion and DNA methylation of the exon 1

region of Kruppel-like factor 4 . To explore mechanisms for thedecrease or lost KLF4 expression in gastric cancer, we did Southernblot analysis of genomic DNA extracted from eight gastric cancercell lines. Based on the genomic structure of wild-type KLF4 locus,digestion of genomic DNA by EcoRI or EcoRI plus NcoI and thenprobe with full-length KLF4 cDNA will generate 11- or 3-kb band,respectively. As we expected, all cell lines exhibited a single 11-kbband when the genomic DNA was digested with EcoRI (Fig. 4A1).

Figure 3. KLF4 expression and apoptosis inductionin gastric cancer cells and tissues. N87 and SK-GT5cells were incubated for 36 hours with Ad-KLF4 atMOI of 0, 10, or 20. Ad-EGFP was used to adjustthe total MOI equal to 20. Genomic DNA ladderingwas determined (A ). Percentage apoptosis wasdetermined by TUNEL assay (B). Representativeapoptotic morphology of SK-GT5 cells wasphotographed after TUNEL assay (C). TUNEL assayalso was performed on gastric cancer tissues withknown negative (D1 ) or positive (D2 ) KLF4expression. Note that significantly decreasedapoptosis was detected in tumor tissues withdecreased or lost KLF4 expression as compared tothat in tumor tissues with positive KLF4 expression. *,P < 0.01, statistical significance in a comparisonbetween the treated and respective untreatedgroups. One representative experiment of three withsimilar results.





However, the intensities of KLF4 band signals from SK-GT5 andSNU-16 were reduced by 50% compared with those of other celllines, after normalization of DNA loading by calculating the ratiobetween KLF4 and GAPDH (K/G ratio, Fig. 4A2). Digestion withEcoRI plus NcoI produced a similar result (data not shown). Thesedata suggested that there were hemizygous deletions of the KLF4gene in SK-GT5 and SNU-16 cells.Moreover, because the promoter region of KLF4 contains typical

CpG islands (Fig. 4B1), we determined the DNA methylation statusby methylation-specific PCR using genomic DNA extracted fromsurgically resected gastric cancer specimens and matched normalgastric mucosa tissues as well as from the gastric cancer cell lines.Five gastric cancer cell lines (Fig. 4B2) and four of the five gastrictumors (Fig. 4B3) exhibited hypermethylation in the exon 1 regionof KLF4 , whereas none of the matched adjacent normal tissues hadhypermethylation in the same region (Fig. 4B3). These results werefurther confirmed by direct DNA sequencing of the PCR productsusing methylation-specific primers (data not shown).Finally, we determined whether blockade of gene hypermethy-

lation reactivates KLF4 expression in human gastric cancer cells.AGS, HTB103, N87, and SK-GT5 cell lines were incubated inmedium or medium containing 5-aza-2-deoxycytidine, an inhibitorof DNA methyltransferase, sodium butyrate (NaB), an inhibitor ofhistone deacetylase, or both. As shown in Fig. 4C , the treatmentsincreased KLF4 expression in all four cell lines compared with theircontrols. Therefore, promoter hypermethylation may contribute tothe reduced KLF4 expression in a subset of gastric cancer tissuesand gastric cancer cell lines.

Discussion

In the present study, we were the first to have investigated theexpression and potential role of KLF4 in human gastric cancerdevelopment and progression. First, we discovered the distinct KLF4expression patterns in normal gastric and gastric tumor tissues.Specifically, we found that KLF4 protein was expressed in thecytoplasm and nuclei of cells localized predominantly in theglandular epithelium (glandular differentiation), suggesting thatKLF4 plays an important role in the homeostasis and maintenanceof gastricmucosa. In contrast, we observed a substantially decreasedor lost KLF4 expression in both gastric tumor specimens and tumorcell lines. Second, restored expression of KLF4 significantly inhibitedgastric cancer growth in vitro and tumorigenicity in animal models.Third, mechanism study showed that promoter hypermethylationand hemizygous deletion were found in a subset of gastric cancertissues and cell lines and restoration of KLF4 expression inducedtypical apoptosis in gastric cancer cells. Finally, we observed aninverse correlation between decreased KLF4 expression and survival,and the expression of KLF4 was an independent prognostic factor topredict the outcome of patients. Therefore, we offered first clinicaland casual evidence and potential mechanism that the alteration ofKLF4 expression plays a critical role in gastric cancer developmentand progression and KLF4 pathway may be a potential target for thetreatment of gastric cancer.The mammalian gastrointestinal tract undergoes continuous

self-renewal throughout life via asymmetrical stem cell division,differentiation, and death, which is under tight regulation by an

Figure 4. Analysis of genetic and epigeneticchanges of KLF4 gene in gastric cancer. A,Southern blot analysis. A1, genomic DNAextracted from the indicated gastric cancer celllines was digested with EcoRI and probed withfull-length KLF4 cDNA, and the same membranewas striped and hybridized with GAPDH probe.A2, relative KLF4 signal after normalizationby GAPDH (K /G ratio). B, KLF4 promotermethylation analysis. B1, diagram of CpG sitesand CpG islands in the exon 1 region of KLF4 .Methylation-specific PCR was performed usingprimers specific for the unmethylated (U ) ormethylated (M) KLF4 exon 1 region in thegenomic DNA derived from gastric cell lines (B2)or gastric tumor tissue (T ) and the matchednormal gastric tissues (N , B3 ). In vitro , Sss Imethylase-treated genomic DNA was used as apositive control and H20 was used as a negativecontrol. C, reactivation of KLF4 expression. Cellswere cultured in the presence of AZA (1 Amol/L)for 3 days or NaB (4 mmol/L) for 20 hours orboth. Total RNA was extracted and KLF4expression was measured by Northern blot usingKLF4 cDNA as a probe. The same membranewas striped and hybridized with GAPDH probe(C1 ). The relative KLF4 signal was normalizedby GAPDH (C2 ).

Cancer Research




unknown molecular mechanism (26–29). Perturbation of thishomeostasis can lead to diseases such as cancer (30). Previousstudies identified a number of key factors in regulating prolifer-ation and differentiation of gut epithelial cells. These includemembers of several growth factor families, such as fibroblastgrowth factor (31, 32), epidermal growth factor (33–35), trans-forming growth factor-a (34, 35), sonic and Indian hedgehog (36),and platelet-derived growth factor-a (32). It has been suggestedthat the Wnt/h-catenin signaling pathway and its downstreammolecules, such as APC, Tcf-4, Fkh-6, Cdx-1, and Cdx-2, are vital fornormal gastrointestinal function, because their genetic changes atany stage seem to induce tumorigenesis (26, 27). However, theexact mechanism by which activities of these regulatory proteinsare orchestrated to control gastrointestinal epithelial cell prolifer-ation and differentiation has not been clearly defined.In the present study, the expression of KLF4 was predomi-

nantly found in the glandular epithelial cells in normal gastricmucosa but was substantially decreased or lost in gastric cancercells, whereas restoration of KLF4 expression resulted insignificant induction of apoptosis and tumor suppression. Thesefindings indicated that expression of this factor plays animportant role in the regulation of homeostasis and maintenanceof gastric mucosa. This notion has been supported by a numberof lines of evidences. For example, Garrett-Sinha et al. (13)reported that KLF4 mRNA is expressed at high levels in severaltypes of epithelial cells, including those in the epidermal layer ofthe skin, stomach, esophagus, and colon of newborn mice.Expression of KLF4 mRNA in epithelial cells is first detectedwhen they differentiate during embryonic development. Theexpression pattern suggests that this protein is involved interminal differentiation of the epithelial cells. More recently,Perreault et al. (37) showed that Foxl1 , a winged helixtranscription factor expressed in the mesenchyme of thegastrointestinal tract, plays an important role in regulatingepithelial cell proliferation and differentiation in the gastrointes-tinal tract. Foxl1�/� mice often have severe structural defects inthe epithelium of the stomach, duodenum, and jejunum, whichare the result of an increase in the number of proliferating cells,not a change in the rate of cell migration. For example, thenumber of bromodeoxyuridine-labeled cells in the stomachglands of Foxl1�/� mice has been shown to be 4.4-fold greaterthan that of Foxl1+/+ mice, whereas the level of nuclear h-cateninhas been shown to be 2-fold greater in Foxl1�/� mice than inFoxl1+/+ mice. These findings indicate that the Wnt/APC/h-cateninpathway is activated in Foxl1�/� mice (38). Moreover, there isevidence that KLF4 is a downstream target of the tumor suppressorgene APC (39), and that APC up-regulates KLF4 by inducing CDX2(40), which is a homeobox gene specifically expressed in theintestines that is important for the regulation of intestinal epithelialcell development and maintenance. In addition, we found that KLF4expression was decreased in gastric tissues displaying atrophy orintestinal metaplasia (data not shown). All of these findings showthat KLF4 plays an important role in the homeostasis andmaintenance of gastric mucosa. Because alterations such as gastricmucosa atrophy and intestinal metaplasia are commonly associatedwith a significantly elevated risk of gastric cancer (41). Our resultssuggest that loss of KLF4 expression might be an early event ingastric carcinogenesis.Currently, there is little evidence of the potential contribution

of altered KLF4 expression to gastric cancer development andprogression. In our study, we found that KLF4 expression in the

gastric cancer tissue was lost or significantly decreased whencompared with that in the normal gastric tissue. In addition,with progression from stage I to stage IV disease, we observed aprogressive decrease of KLF4 expression, with the lowestexpression occurring in metastatic lymph nodes. This loss ofKLF4 expression was associated with poor survival. Moreover,restoration of KLF4 expression in gastric cancer cells signifi-cantly inhibited cell growth in vitro and tumor formation in vivo .All of this clinical and experimental evidence strongly suggeststhat KLF4 functions as a tumor suppressor gene in humangastric cancer and that its alteration of expression plays animportant role in gastric cancer development and progression.However, the mechanism for drastically altered KLF4 expressionin gastric cancer is unknown. In the present study, we foundhemizygous deletions of KLF4 in two of eight gastric cancer celllines and apparent promoter hypermethylation in resectedgastric cancer tissues and gastric cell liens, indicating bothgenetic and epigenetic changes in KLF4 gene may cause thedecreased or lost KLF4 expression in gastric cancer. This notionis also supported by two recent reports. First, a high frequentloss of heterozygosity on chromosome 9q31 (where KLF4 islocated) was found in human gastric cancer (42). Second, loss ofheterozygosity, hypermethylation of the 5V-untranslated region,and several point mutations of KLF4 are associated with alteredKLF4 expression and function in colorectal cancer (16). On theother hand, loss of APC expression or h-catenin activation mayalso contribute to the down-regulation of KLF4 in gastric cancer(43–45), because KLF4 is a downstream target of the Wnt/APC/h-catenin pathway (39).Although little is known about the mechanisms by which KLF4

may influence cancer development and progression, there are a fewlines of evidence indicating that altered KLF4 expression affectscell cycle (15, 17, 46–49). In the present study, we found thatrestoration of or an increase in KLF4 expression significantlyinduces apoptosis of gastric cancer cells in a dose-dependentmanner. Our results seem to be consistent with those of previousstudies on bladder cancer cells (17), colon cancer (24), andleukemia cells (50). However, the mechanism by which KLF4induces apoptosis is totally unknown and currently underinvestigation in our laboratory.In summary, we found that KLF4 was highly expressed in the

epithelium of normal gastric tissue, its expression was frequentlydecreased or lost in gastric cancer tissues and cancer cell lines,and restoration of KLF4 expression resulted in suppression ofgastric cancer growth in vitro and in vivo via induction ofapoptosis. These results show that KLF4 plays an important rolein the regulation of homeostasis and maintenance of gastricmucosa and functions as a tumor suppressor in gastriccarcinogenesis and progression and that KLF4 pathway is bothprognostic marker and potential therapeutic target for humangastric cancer treatment.

Acknowledgments

Received 10/7/2004; revised 1/3/2005; accepted 1/24/2005.Grant support: American Cancer Society research scholar grant CSM-106640 and

National Cancer Institute, NIH grant 1R01-CA093829 (K. Xie).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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Judy King for expert help in the preparation of this article, Don Norwoodfor editorial comments, and Chichuan Tseng (Boston University School of Medicine)for providing KLF4 in the pcDNA3.1 expression vector.





References1. Parkin DM, Pisani P, Ferlay J. Global cancer statistics.CA Cancer J Clin 1999;49:33–64.

2. Parkin DM, Pisani P, Ferlay J. Estimates of theworldwide incidence of 25 major cancers in 1990. IntJ Cancer 1999;80:827–41.

3. Tahara E. Molecular aspects of invasion and metasta-sis of stomach cancer. Verh Dtsch Ges Pathol 2000;84:43–9.

4. Chan AO, Luk JM, Hui WM, Lam SK. Molecular biologyof gastric carcinoma: from laboratory to bedside.J Gastroenterol Hepatol 1999;14:1150–60.

5. Fenoglio-Preiser CM, Wang J, Stemmermann GN,Noffsinger A. TP53 and gastric carcinoma: a review.Hum Mutat 2003;21:258–70.

6. Sud R, Wells D, Talbot IC, Delhanty JD. Geneticalterations in gastric cancers from British patients.Cancer Genet Cytogenet 2001;126:111–9.

7. Fricke E, Keller G, Becker I, et al. Relationship betweenE-cadherin gene mutation and p53 gene mutation, p53accumulation, Bcl-2 expression and Ki-67 staining indiffuse-type gastric carcinoma. Int J Cancer 2003;104:60–5.

8. Ascano JJ, Frierson H Jr, Moskaluk CA, et al.Inactivation of the E-cadherin gene in sporadicdiffuse-type gastric cancer. Mod Pathol 2001;14:942–9.

9. Kim KM, Kim MJ, Cho BK, Choi SW, Rhyu MG.Genetic evidence for the multi-step progression ofmixed glandular-neuroendocrine gastric carcinomas.Virchows Arch 2002;440:85–93.

10. Bae SI, Park JG, Kim YI, Kim WH. Genetic alterationsin gastric cancer cell lines and their original tissues. IntJ Cancer 2000;87:512–6.

11. El-Rifai W, Powell SM. Molecular biology of gastriccancer. Semin Radiat Oncol 2002;12:128–40.

12. Chan AO, Luk JM, Hui WM, Lam SK. Molecularbiology of gastric carcinoma: from laboratory tobedside. J Gastroenterol Hepatol 1999;14:1150–60.

13. Garrett-Sinha LA, Eberspaecher H, Seldin MF,de Crombrugghe B. A gene for a novel zinc-fingerprotein expressed in differentiated epithelial cells andtransiently in certain mesenchymal cells. J Biol Chem1996;271:31384–90.

14. Shields JM, Christy RJ, Yang VW. Identification andcharacterization of a gene encoding a gut-enrichedKruppel-like factor expressed during growth arrest.J Biol Chem 1996;271:20009–17.

15. Yoon HS, Yang VW. Requirement of Kruppel-likefactor 4 in preventing entry into mitosis following DNAdamage. J Biol Chem 2004;279:5035–41.

16. Zhao W, Hisamuddin IM, Nandan MO, Babbin BA,Lamb NE, Yang VW. Identification of Kruppel-likefactor 4 as a potential tumor suppressor gene incolorectal cancer. Oncogene 2004;23:395–402.

17. Ohnishi S, Ohnami S, Laub F, et al. Downregulationand growth inhibitory effect of epithelial-type Kruppel-like transcription factor KLF4, but not KLF5, inbladder cancer. Biochem Biophys Res Commun 2003;308:251–6.

18. Wang N, Liu ZH, Ding F, Wang XQ, Zhou CN, Wu M.Down-regulation of gut-enriched Kruppel-like factor

expression in esophageal cancer. World J Gastroenterol2002;8:966–70.

19. Foster KW, Ren S, Louro ID, et al. Oncogeneexpression cloning by retroviral transduction of adeno-virus E1A-immortalized rat kidney RK3E cells: trans-formation of a host with epithelial features by c-MYCand the zinc finger protein GKLF. Cell Growth Differ1999;10:423–34.

20. Luo A, Kong J, Hu G, et al. Discovery of Ca2+-relevantand differentiation-associated genes downregulated inesophageal squamous cell carcinoma using cDNAmicroarray. Oncogene 2004;23:1291–9.

21. Wang N, Liu ZH, Ding F, Wang XQ, Wu M. Effect ofexpression of gut-enriched Kruppel-like factor onesophageal squamous cancer cells. Sheng Wu HuaXue Yu Sheng Wu Wu Li Xue Bao (Shanghai)2003;35:55–8.

22. Foster KW, Frost AR, McKie-Bell P, et al. Increaseof GKLF messenger RNA and protein expressionduring progression of breast cancer. Cancer Res 2000;60:6488–95.

23. Wang L, Wei D, Huang S, et al. Transcription factorSp1 expression is a significant predictor of survival inhuman gastric cancer. Clin Cancer Res 2003;9:6371–80.

24. Chen ZY, Shie J, Tseng C. Up-regulation of gut-enriched Krupple-like factor by interferon-g in humancolon carcinoma cells. FEBS Lett 2000;477:67–72.

25. Wei D, Wang L, He Y, Xiong HQ, Abbruzzese JL,Xie K. Celecoxib inhibits vascular endothelial growthfactor expression in and reduces angiogenesis andmetastasis of human pancreatic cancer via suppres-sion of Sp1 transcription factor activity. Cancer Res2004;64:2030–8.

26. Brittan M, Wright NA. Gastrointestinal stem cells.J Pathol 2002;197:492–509.

27. Brittan M, Wright NA. The gastrointestinal stem cell.Cell Prolif 2004;37:35–53.

28. Gordon JI, Hermiston ML. Differentiation and self-renewal in the mouse gastrointestinal epithelium. CurrOpin Cell Biol 1994;6:795–803.

29. Fenoglio-Preiser CM, Noffsinger AE, StemmermannGN, Lantz PE, Listrom MB, Rilke FO. Gastrointestinalpathology. 2nd ed. Philadelphia: Lippincott Williams &Wilkins; 1998.

30. Chen X, Whitney EM, Gao SY, Yang VW. Transcrip-tional profiling of Kruppel-like factor 4 reveals afunction in cell cycle regulation and epithelial differen-tiation. J Mol Biol 2003;326:665–77.

31. Terasaki T, Shimada K, Wakabayashi H, Tanaka M,Watanabe A. Study of the repairing process of gastriculcer using multivariate analysis of bFGF-positive cells,haemodynamics, PAS-positive mucus amount andglandular index in the gastric mucosa. J GastroenterolHepatol 1996;11:928–40.

32. Tarnawski A, Szabo IL, Husain SS, Soreghan B.Regeneration of gastric mucosa during ulcer healing istriggered by growth factors and signal transductionpathways. J Physiol Paris 2001;95:337–44.

33. Walker-Smith JA, Phillips AD, Walford N, et al.Intravenous epidermal growth factor/urogastroneincreases small-intestinal cell proliferation in congenitalmicrovillous atrophy. Lancet 1985;2:1239–40.

34. Jones MK, Tomikawa M, Mohajer B, Tarnawski AS.Gastrointestinal mucosal regeneration: role of growthfactors. Front Biosci 1999;4:D303–9.

35. Majumdar AP. Regulation of gastrointestinal muco-sal growth during aging. J Physiol Pharmacol 2003;54Suppl 4:143–54.

36. van den Brink GR, Hardwick JC, Nielsen C, et al.Sonic hedgehog expression correlates with fundic glanddifferentiation in the adult gastrointestinal tract. Gut2002;51:628–33.

37. Perreault N, Katz JP, Sackett SD, Kaestner KH. Foxl1controls the Wnt/h-catenin pathway by modulating theexpression of proteoglycans in the gut. J Biol Chem2001;276:43328–33.

38. Kaestner KH, Silberg DG, Traber PG, Schutz G. Themesenchymal winged helix transcription factor Fkh6 isrequired for the control of gastrointestinal proliferationand differentiation. Genes Dev 1997;11:1583–95.

39. Stone CD, Chen ZY, Tseng CC. Gut-enriched Kruppel-like factor regulates colonic cell growth through APC/h-catenin pathway. FEBS Lett 2002;530:147–52.

40. Dang DT, Mahatan CS, Dang LH, Agboola IA, YangVW. Expression of the gut-enriched Kruppel-like factor(Kruppel-like factor 4) gene in the human colon cancercell line RKO is dependent on CDX2. Oncogene2001;20:4884–90.

41. Uemura N, Okamoto S, Yamamoto S, et al. Heli-cobacter pylori infection and the development of gastriccancer. N Engl J Med 2001;345:784–9.

42. Kakinuma N, Kohu K, Sato M, et al. Candidateregions of tumor suppressor locus on chromosome9q31.1 in gastric cancer. Int J Cancer 2004;109:71–5.

43. Grace A, Butler D, Gallagher M, et al. APC geneexpression in gastric carcinoma: an immunohistochem-ical study. Appl Immunohistochem Mol Morphol 2002;10:221–4.

44. Fang DC, Luo YH, Yang SM, Li XA, Ling XL, Fang L.Mutation analysis of APC gene in gastric cancer withmicrosatellite instability. World J Gastroenterol 2002;8:787–91.

45. Ebert MP, Fei G, Kahmann S, et al. Increased h-catenin mRNA levels and mutational alterations of theAPC and h-catenin gene are present in intestinal-typegastric cancer. Carcinogenesis 2002;23:87–91.

46. Chen X, Johns DC, Geiman DE, et al. Kruppel-likefactor 4 (gut-enriched Kruppel-like factor) inhibits cellproliferation by blocking G1/S progression of the cellcycle. J Biol Chem 2001;276:30423–8.

47. Yoon HS, Chen X, Yang VW. Kruppel-like factor 4mediates p53-dependent G1/S cell cycle arrest inresponse to DNA damage. J Biol Chem 2003;278:2101–5.

48. Zhang W, Geiman DE, Shields JM, et al. The gut-enriched Kruppel-like factor (Kruppel-like factor 4)mediates the transactivating effect of p53 on thep21WAF1/Cip1 promoter. J Biol Chem 2000;275:18391–8.

49. Shie JL, Chen ZY, Fu M, Pestell RG, Tseng CC. Gut-enriched Kruppel-like factor represses cyclin D1promoter activity through Sp1 motif. Nucleic AcidsRes 2000;28:2969–76.

50. Yasunaga J, Taniguchi Y, Nosaka K, et al. Identifica-tion of aberrantly methylated genes in association withadult T-cell leukemia. Cancer Res 2004;64:6002–4.

Cancer Research




2005;65:2746-2754. Cancer Res Daoyan Wei, Weida Gong, Masashi Kanai, et al. ProgressionIs Critical in Human Gastric Cancer Development and Drastic Down-regulation of Krüppel-Like Factor 4 Expression

Updated version

http://cancerres.aacrjournals.org/content/65/7/2746

Access the most recent version of this article at:

Cited articles

http://cancerres.aacrjournals.org/content/65/7/2746.full#ref-list-1

This article cites 46 articles, 14 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/65/7/2746.full#related-urls

This article has been cited by 31 HighWire-hosted articles. Access the articles at:

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

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://cancerres.aacrjournals.org/content/65/7/2746To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/content/65/7/2746.full#ref-list-1

http://cancerres.aacrjournals.org/content/65/7/2746.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Drastic Down-regulation of Kru¨ppel-Like Factor 4 Expression Is … · Drastic Down-regulation of...

Documents

Transcript of Drastic Down-regulation of Kru¨ppel-Like Factor 4 Expression Is … · Drastic Down-regulation of...