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Association Between Expression of Transcription Factor Sp1 And Vol. 10, 4109–4117, June 15, 2004 Clinical Cancer Research 4109 Association between Expression of Transcription Factor Sp1 and Increased Vascular Endothelial Growth Factor Expression, Advanced Stage, and Poor Survival in Patients with Resected Gastric Cancer James C. Yao,1 Liwei Wang,1 Daoyan Wei,1 was not statistically significant. When Sp1 and VEGF ex- Weida Gong,2 Manal Hassan,1 Tsung-Teh Wu,3 pression, stage, completeness of resection, histology, and 4 1 1,5 patient age were entered in a Cox proportional hazards ؍ Paul Mansfield, Jaffer Ajani, and Keping Xie 1 2 model, strong Sp1 expression (P 0.021) and an advanced Departments of Gastrointestinal Medical Oncology, Neurosurgery, disease stage (P 0.001) were independently prognostic of 3Pathology, 4Surgical Oncology, and 5Cancer Biology, The University < of Texas M. D. Anderson Cancer Center, Houston, Texas poor survival. Given the importance of Sp1 in the expression of VEGF, our data suggest that dysregulated Sp1 expression and activation play important roles in VEGF overexpression ABSTRACT and, thus, gastric cancer development and progression. The biological and clinical behaviors of cancer are af- fected by multiple molecular pathways that are under the INTRODUCTION control of transcription factors. Improved understanding of Although the incidence of gastric cancer declined in the how transcription factors affect cancer biology may lead to West from the 1940s to the 1980s, it remains a major public improved ability to predict clinical outcome and discovery of health problem throughout the world (1). In Asia and parts of novel therapeutic strategies. We evaluated the relationship South America in particular, gastric cancer is the most common between Sp1 and vascular endothelial growth factor (VEGF) epithelial malignancy and leading cause of cancer-related expression, as well as their effect on survival in 86 cases of deaths. In fact, gastric cancer remains the second most fre- resected human gastric cancer. The degree of VEGF expres- quently diagnosed malignancy worldwide and cause of 12% of sion correlated highly with Sp1 expression (P < 0.01). Pa- all cancer-related deaths each year (1, 2). Advances in the tients with high Sp1 expression were 98 times more likely to treatment of this disease are likely to come from a fuller under- have high VEGF expression compared with those with neg- standing of its biology and behavior. The aggressive nature of ative Sp1 expression. Clinically, negative or weak Sp1 ex- human gastric carcinoma is related to mutations of various pression was associated with early stage (IA) in gastric oncogenes and tumor suppressor genes (3–5) and abnormalities cancer. Strong Sp1 expression was more frequently ob- in several growth factors and their receptors (4, 5). These ؍ served among patients with stage IB–IV disease (P 0.035). abnormalities affect the downstream signal transduction path- Similarly, whereas strong Sp1 expression was uncommonly ways involved in the control of cell growth and differentiation. observed among patients with N0 or N1 disease (19 and Specifically, these perturbations confer a tremendous survival 16%), N2/N3 gastric cancer was associated with strong Sp1 and growth advantage to gastric cancer cells. ؍ expression (48%; P 0.034). Strong Sp1 expression was The growth and metastasis of cancer depend on angiogen- also associated with inferior survival. The median survival esis, which is the formation of new blood vessels from a duration in patients who had a tumor with a negative, weak, preexisting network of capillaries (6). Of the numerous angio- ؍ and strong Sp1 expression was 44, 38, and 8 months (P genic factors discovered thus far, vascular endothelial growth 0.0075), respectively, whereas patients with strong VEGF factor (VEGF; Ref. 7), also known as vascular permeability expression had a shorter survival duration; the difference factor (8), has been identified as a key mediator of tumor angiogenesis. Previous studies have demonstrated significantly elevated expression of VEGF in biopsy specimens of human gastric cancer (9–11). The mechanism of VEGF expression and Received 11/21/03; revised 3/5/04; accepted 3/18/04. Grant support: Research Scholar Grant CSM-106640 from the Amer- its regulation in human gastric cancer are mostly unknown. ican Cancer Society and Grant 1R01-CA093829 and Cancer Center Increasing evidence suggests that VEGF expression is regulated Support Core Grant CA 16672-23 from the National Cancer Institute, by a plethora of external factors. Major stimulators of VEGF NIH (K. Xie). expression include hypoxia and acidosis (12, 13), hormones, The costs of publication of this article were defrayed in part by the cytokines, and growth factors (14). Also, many tumor cells can payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to constitutively express VEGF in vitro without any apparent ex- indicate this fact. ternal stimulation (15). In fact, loss or inactivation of tumor Note: J. Yao and L. Wang contributed equally to this work. suppressor genes and activation of oncogenes are associated Requests for reprints: Keping Xie, Department of Gastrointestinal with VEGF overexpression (16, 17). VEGF promoter analyses Medical Oncology, Unit 426, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: have revealed several potential transcription factor-binding sites (713) 792-2013;Fax:(713) 745-1163;E-mail:[email protected]. such as hypoxia-inducible factor 1, activator protein 1, activator org. protein 2, Egr-1, Sp1, and many others (18–22), suggesting that Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. 4110 VEGF Expression by Sp1 in Human Gastric Cancer multiple signal transduction pathways may be involved in filter with an anti-␤-actin antibody (15). The probe proteins VEGF transcription regulation. A recent study by our group were detected using the Amersham-enhanced chemilumines- suggested that abnormal Sp1 activation augments the angio- cence system according to the manufacturer’s instructions. genic and metastatic capacity of tumor cells through overex- Immunohistochemistry. Sections (5-␮m thick) of for- pression of multiple Sp1 downstream genes, including VEGF malin-fixed, paraffin-embedded tumor specimens were deparaf- (15). Sp1 is a sequence-specific DNA-binding protein that is finized in xylene and rehydrated in graded alcohol. Antigen important in the transcription of many cellular and viral genes retrieval was performed with 0.05% saponin for 30 min at room that contain GC boxes in their promoter (23). Additional tran- temperature. Endogenous peroxidase was blocked using 3% scription factors (Sp2, Sp3, and Sp4) similar to Sp1 in their hydrogen peroxide in PBS for 12 min. The specimens were structural and transcriptional properties have been cloned, thus incubated for 20 min at room temperature with a protein- forming a Sp1 multigene family (24–30). Aberrant activities of blocking solution consisting of PBS (pH 7.5) containing 5% various members of the Sp1 family have been implicated in normal horse serum and 1% normal goat serum and then incu- tumor development and progression (15, 31–33), although the bated at 4°C in a 1:200 dilution of rabbit polyclonal antibody signaling mechanisms remain unknown. Because VEGF and against human Sp1 (clone PEP2) or 1:100 dilution of rabbit Sp1 markers predict patient survival, we asked whether the Sp1 polyclonal antibody against human VEGF (Santa Cruz Biotech- expression levels predict the VEGF expression levels given that nology). The samples were then rinsed and incubated for1hat Sp1 has been implicated as critical to VEGF regulation. room temperature with peroxidase-conjugated antirabbit IgG. In the present study, we examined the expression of and Next, the slides were rinsed with PBS and incubated for 5 min relationship between Sp1 and VEGF in the tissue of patients with 3,3Ј-diaminobenzidine (Research Genetics, Huntsville, with resected gastric cancer and their impact on survival dura- AL). The sections were washed three times with distilled water, tion. We found that elevated Sp1 activation and concomitant counterstained with Mayer’s hematoxylin (BioGenex Laborato- VEGF overexpression occurred in patients with human gastric ries, San Ramon, CA), and washed once each with distilled cancer and was inversely correlated with survival, suggesting water and PBS. Afterward, the slides were mounted using a that abnormally activated Sp1 expression represents a potential Universal Mount (Research Genetics) and examined using a molecular marker for poor prognosis and directly contributes to bright-field microscope. A positive reaction was indicated by a gastric cancer development and progression. reddish-brown precipitate in the nuclei (Sp1) or cytoplasm (VEGF; Refs. 34, 35). Depending on the percentage of positive cells and staining intensity, Sp1 and VEGF staining were clas- MATERIALS AND METHODS sified into three groups: negative; weak positive; and strong Human Tissue Specimens and Patient Information. positive. Specifically, the percentage of positive cells was di- We used human gastric cancer tissue specimens preserved in the vided into five grades (percentage scores): Ͻ10% (0), 10–25% Gastric Cancer Tissue Bank at and obtained information about (1), 26–50% (2), 51–75% (3), and Ͼ75% (4). The intensity of the patients from The University of Texas M. D. Anderson
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