Loss of Kru « ppel-Like Factor 4 Expression Contributes to Sp 1 Overexpression and Human Gastric Cancer Development and Progression

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 correlatedwith the angiogenic potential of and poor prognosis for human gastric cancer. However, the underlying mechanisms that result in Sp1overexpression remain unclear. Experimental Design:The expressionof Sp1andKru« ppel-like factor 4 (KLF4), apotential tumor suppressor gene, in gastric cancer tissue was analyzed by immunohistochemistry andWestern blot analysis. Alterations of Sp1and KLF4 expressionwere 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 KLF4and 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 Sp1overexpression and to the development and progression of human gastric cancer. Although the incidence of gastric cancer declined in the west from the 1940s to the 1980s, it remains a major public health problem throughout the world (1). In Asia and parts of South America in particular, it is the most common epithelial malignancy and is a leading cause of cancer-related death. In fact, gastric cancer remains the fourth most frequently diagnosed malignancy worldwide and is the cause of 12% of all cancer-related deaths annually (1). Advances in the treatment of this disease are likely to come from a fuller understanding of its biology and behavior. Although various genetic and molecular alterations have been found to be associated with the malignant transformation of gastric cancer, they may represent only the pathogenesis of this disease, and they have not been identified as a specific sequence of changes leading to gastric carcinoma (2, 3). Therefore, the role and detailed mechanisms of genetic and epigenetic changes in gastric cancer development and progression remain unclear. 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 the Sp1 multigene family (4). Although Sp1 has been perceived as a basal transcription factor since its discovery, increasing evidence suggests that it regulates a variety of biological functions, including cell survival, growth and differentiation, and tumor development and progression (5). For example, Sp1 expression is increased in squamous cell carcinoma and colorectal cancer when compared with that in skin papillomas and normal colorectal tissue, respectively (6, 7). Also, interference of Sp1 activity has been shown to suppress tumor cell growth (8), and small interfering RNA (siRNA) duplexes of Sp1 mRNA block cell cycle progression or tumor formation in athymic mice (9, 10). Furthermore, we have reported that Sp1 Human Cancer Biology Authors’ Affiliations: Departments of Gastrointestinal Medical Oncology and Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston,Texas Received 4/28/06; revised 7/2/06; accepted 7/20/06. Grant support: Research Scholar grant CSM-106640 from theAmerican Cancer Society and grant1R01-CA093829 from the National Cancer Institute, NIH (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 accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Keping Xie, Department of Gastrointestinal Medical Oncology, Unit 426,The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,TX 77030. Phone: 713-792-2828; Fax: 713-7451163; E-mail: kepxie@mail.mdanderson.org. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-1034 www.aacrjournals.org Clin Cancer Res 2006;12(21) November1, 2006 6395 Cancer Research. on January 13, 2018. © 2006 American Association for clincancerres.aacrjournals.org Downloaded from overexpression is directly correlated with the angiogenic potential and poor prognosis of human gastric cancer (11, 12). However, the underlying mechanisms that result in Sp1 overexpression in tumors remain unknown. Although a number of studies have reported gene regulation by Sp1, few have reported the transcriptional regulation of Sp1 itself. One recent study showed that the Sp1 promoter contains a number of putative binding sites for several transcription factors, including Sp1, Sp3, nuclear factor Y, activator protein 2, and CCAAT/enhancer-binding protein (13, 14). Because the Sp1 promoter contains putative Sp1 binding sites, Sp1 overexpression may result from autotransactivation of its own promoter. On the other hand, Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that binds to a consensus sequence 5¶-(G/A)(G/A)GG(C/T)G(C/T)-3¶. It is a member of the KLF family and is highly expressed in the gastrointestinal tract and other epithelial tissues (15). KLF4 can either activate or repress the transcription of several genes (16–19), and its alterations lead to aberrant proliferation and differentiation in gastric and colonic epithelium (20, 21). Additionally, accumulating clinical evidence shows that KLF4 is a potential tumor suppressor. Reduced KLF4 expression has been reported in various tumors including esophagus, stomach, colon, bladder, lung, and adult T cell leukemia cells (22–27), and restored KLF4 expression induces growth arrest in colon cancer cells (28) or apoptosis in bladder, gastric, and leukemia cells (23, 24, 27). In fact, genetic or epigenetic alterations of the KLF4 gene have been found in both gastric cancer and colorectal cancer (23, 25). In the present study, we found mutually exclusive expression of Sp1 and KLF4 in human gastric cancer. Enforced KLF4 expression repressed Sp1 expression at the promoter activity, mRNA, and protein levels. A region within the proximal Sp1 promoter was identified to have overlapping KLF4and Sp1-binding sites, to which KLF4 and Sp1 compete for binding. Sp1 positively regulated its own promoter, whereas KLF4 did the opposite. Collectively, 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. Materials andMethods Cell lines and culture conditions. The human gastric cancer cell lines, AGS, HTB103, HTB135, N87, SNU-1, and TMK1, and FG human pancreatic cancer cell line, were purchased from the American Type Culture Collection (Manassas, VA), and the human gastric cancer cell line, SK-GT5, was obtained from Dr. Gary K. Schwartz (Memorial SloanKettering Cancer Center). All of the cell lines were maintained in plastic flasks inminimal essentialmedium supplementedwith 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine, and a vitamin solution (Flow Laboratories, Rockville, MD) in 5%CO2 at 37jC. Northern blot analysis. SK-GT5 and N87 cells were seeded to f80% confluence in 10 cm culture dishes and transfected with a recombinant adenovirus containing KLF4 (Ad-KLF4) or a recombinant adenovirus containing enhanced green fluorescent protein (Ad-EGFP) at a multiplicity of infection (MOI) of 10. Twenty-four hours later, total RNA was extracted by using TRIzol reagent (Invitrogen, San Diego, CA). Standard Northern blotting was done using 10 Ag of RNA and KLF4 and Sp1 cDNA probes. Equal RNA loading was monitored by hybridizing the same membrane with a human 36B4 cDNA probe as described previously (29). Western blot analysis. Fresh gastric cancer and corresponding noncancerous gastric tissue specimens were obtained from patients who underwent gastrectomy at The University of Texas M.D. Anderson Cancer Center. Both the cancerous and noncancerous specimens were macroscopically identified and excised by experienced pathologists, and the presence or absence of cancer was further confirmed by histopathologic examination. Additionally, whole cell lysates were prepared from gastric cancer tissue and adjacent noncancerous specimens or cell cultures. Standard Western blotting was done with an anti-Sp1 antibody (PEP2; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-KLF4 antibody (H180; Santa Cruz Biotechnology). Two bands were detected following KLF4 transfection, as reported previously (16, 19, 23), and the low molecular weight protein may result from a rapid proteolysis of KLF4 protein (30). Equal protein sample loading was monitored by incubating the same membrane filter with an anti–glyceraldehyde-3-phosphate dehydrogenase antibody (FL-335; Santa Cruz Biotechnology). The probe proteins were detected by using the enhanced chemiluminescence system (Amersham Life Sciences, Piscataway, NJ) according to the manufacturer’s instructions. Sp1 promoter constructs, site-specific mutagenesis, and analysis of Sp1 promoter activity. The full-length and minimal Sp1 promoter reporters in pGL3 luciferase constructs were used as described previously (13). Site-specific mutagenesis o