Transcription Factor Sp1 Correlates With Development And Targeting Antiangiogenic Therapy In Gastric Cancer

BACKGROUD AND AIMS The development, progression and biologicalbehaviors of cancer are affected by mutiple molecular pathways that are underthe control of transcription factors. The transcription factor Sp1 regulates theexpression of multiple genes including cell growth,apoptosis and angiogenesis.However, it remains unclear whether its expression correlates with themolecular mechanism of development and angiogenesis in human gastriccancer, whether targeting Sp1 has a therapeutic benefit for gastric cancer.Improved understanding of how Sp1 affect the development and angiogenicphenotypes of gastric cancer and led to discovery of novel target therapeuticstrategy,therefore the following three specific aims were proposed:1)investigate the expression of Sp1 relationship with the development andprognosis;2)evaluate the expression of endothelial nitric oxide synthase and itsrelationship with the angiogenic phenotype and the expression of thetranscription factor Sp1;3)determine whether transcription factor Sp1 is aneffective target of antiangiogenic therapy for human gastric cancer.MATERIALS AND METHODS We used human gastric cancer tissuespecimens preserved in the Gastric Cancer Tissue Bank, and information aboutthe patients was obtained from what was recorded in its comprehensive database at The University of Texas M.D. Anderson Cancer Center. Primarygastric cancer in these patients was diagnosed and treated at M. D. AndersonCancer Center from 1985 to 1998. The patients had well-documented clinicalhistories and follow-up information. None of them underwent preoperativechemotherapy and/or radiation therapy. Eighty-six patients with primarygastric cancer were randomly selected for this study. All of the patients hadundergone gastrectomy with lymph node dissection. All of the patients alsowere observed at M.D. Anderson through the end of 1999. The medianfollow-up period for the 86 patients was 25.7 months. At the end of 1999, 30patients were still alive, whereas 56 patients had died. Fiftythree lymph nodemetastasis specimens and 57 normal gastric tissue specimens obtained frompatients without gastric cancer were also included in this study. Usingimmunohistochemistry, we investigated Sp1 expression patterns in 86 cases ofgastric cancer having various clinicopathologic characteristics, 57 cases ofnormal gastric tissue specimens, and 53 cases of lymph node metastases. Theactivity of Sp1 in the tumor and normal tissue was detected by electrophoreticmobility shift analysis(EMSA). The expression of transcription factor Sp1 oftumor and clinical characteristics of patients were statistically analyzed using aCox proportional hazard model, the effect of independent factor on prognosiswas determined. The NOSⅢexpression level and tumor microvessel density(MVD) status were determined via immunohistochemistry, using archivedtissue specimens from 86 resected gastric cancer cases, these findings were then correlated with Sp1 expression and clinicopathological features.Simultaneously, treatment with mithramycin A(MTT), a Sp1 inhibitor,suppressed the expression of Sp1 and its downstream target genes in both cellculture and tumors growing in nude mice. Treatment with bevacizumab(BVZ),a neutralizing antibody against VEGF, suppressed human gastric cancergrowth in nude mice. The expression of Sp1 was determined byimmunohistochemistry.RESULTS Strong Sp1 expression was detected in tumor cells, whereas no orvery weak Sp1 expression was detected in stromal cells and normal glandularcells surrounding or within the tumors. Simultaneously, Sp1 protein wasexpressed predominantly in the nuclei of cells located in the mucous neckregion, whereas Sp1 expression was not detected either in the cells locatedtoward the gastric pit (foveolar differentiation) or cells of the glandularepithelium (glandular differentiation).The median survival duration in patientswho had a tumor with negative, weak, and strong Sp1 expression was 43, 37,and 8 months, respectively(P＜0.01). NOSⅢprotein expression wassignificantly higher in both primary tumors and lymph node metastases than innormal gastric mucosa. In primary tumors, NOSⅢexpression correlatedhighly with Sp1 expression(P=0.001) and MVD status(P=0.001). Patients withstrong Sp1 expression were more likely to have strong NOSⅢexpression (15times) and a high MVD (7 times) than were those with negative Sp1expression. In univariate survival analyses, strong NOSⅢexpression, strong Sp1 expression, and a high MVD were associated with worse survival. In aCox proportional hazards model, only strong NOSⅢand Sp1 expression andadvanced disease stage were independently prognostic of poor survival. Invitro, MIT inhibited Sp1 expression and the expression of its downstreamtarget molecules in human gastric cancer cells, whereas BVZ did notsignificantly inhibit tumor growth but upregulated Sp1 expression. In mousemodels, MIT treatment substantially inhibits Sp1 expression and reduced MVDin tumor tissues. However, the drastic inhibition of Sp1 in tumor wasaccompanied by no discernible inhibition of Sp1 expression in normal tissue,suggesting that MIT selectively Sp1 expression in tumor cells. Moreover,Western blot analyses showed that MIT treatment inhibited the expression ofVEGF, EGFR and PDFG; which was consistent with drastic inhibition of Sp1expression. And also, the inhibition is concentration-dependant. Combinationof BVZ and MIT has synergistic antitumor effects than the use of BVZ or MITalone did without an increase in toxicity. Furthermore, the incidence of tumorgrowth in the stomach and of metastasis in the liver and/or other organs wassignificantly lower in the mice that received BVZ and MIT.CONCLUTIONS Strong Sp1 expression was detected in tumor tissues. Sp1expression is a significant predictor of survival in human gastric cancer. Theexpression of NOSⅢis related to MVD status and the expression of Sp1.Although the status of MVD is not the independently prognostic of survival,the expression of NOSⅢmay represent a potential molecular marker for poor prognosis. BVZ treatment targeting VEGF leads to resistance of gastric cancerto BVZ in part via positive feedback activation of the transcription factor Sp1and subsequent overexpression of Sp1's downstream target genes. Combineduse of low doses of BVZ and MIT had a synergistic antiangiogenic effect inhuman gastric cancer. Sp1 inhibitors can inhibit the expression of itsdownstream genes which related to angiogenesis by inhibiting Sp1 expression.Transcription factor Sp1 is an effective target of antiangiogenic therapy forhuman gastric cancer.