05-579 Sigma-AldrichAnti-Endostatin Antibody, clone 1837.46

The transcription factor RE1 silencing transcription factor (REST) is lost in approximately 20% of breast cancers. Although it is known that these RESTless tumors are highly aggressive and include all tumor subtypes, the underlying tumorigenic mechanisms remain unknown. In this study, we show that loss of REST results in upregulation of LIN28A, a known promoter of tumor development, in breast cancer cell lines and human breast tumors. We found that LIN28A was a direct transcriptional target of REST in cancer cells and that loss of REST resulted in increased LIN28A expression and enhanced tumor growth both in vitro and in vivo, effects that were dependent on heightened LIN28A expression. Tumors lacking REST expression were locally invasive, consistent with the increased lymph node involvement observed in human RESTless tumors. Clinically, human RESTless breast tumors also displayed significantly enhanced LIN28A expression when compared with non-RESTless tumors. Our findings therefore show a critical role for the REST-LIN28A axis in tumor aggression and suggest a causative relationship between REST loss and tumorigenicity in vivo.

Inhibition of angiogenesis has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in the large-scale production of the antiangiogenic proteins. This limitation may be resolved by in vivo delivery and expression of the antiangiogenic genes. We have constructed a recombinant adenovirus that expresses murine endostatin that is biologically active both in vitro, as determined in endothelial cell proliferation assays, and in vivo, by suppression of angiogenesis induced by vascular endothelial growth factor 165. Persistent high serum levels of endostatin (605-1740 ng/ml; mean, 936 ng/ml) were achieved after systemic administration of the vector to nude mice, which resulted in significant reduction of the growth rates and the volumes of JC breast carcinoma and Lewis lung carcinoma (P < 0.001 and P < 0.05, respectively). In addition, the endostatin vector treatment completely prevented the formation of pulmonary micrometastases in Lewis lung carcinoma (P = 0.0001). Immunohistochemical staining of the tumors demonstrated a decreased number of blood vessels in the treatment group versus the controls. In conclusion, the present study clearly demonstrates the potential of vector-mediated antiangiogenic gene therapy as a component in cancer therapy.

Endostatin, a carboxyl-terminal fragment of collagen XVIII, has been shown to regress tumors in mice. In this study, we have analyzed the mechanism of endostatin action on endothelial cells and nonendothelial cells. Endostatin treatment of cow pulmonary artery endothelial cells caused apoptosis, as demonstrated by three methods, annexin V-fluorescein isothiocyanate staining, caspase 3, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling assay. Moreover, addition of endostatin led to a marked reduction of the Bcl-2 and Bcl-XL anti-apoptotic protein, whereas Bax protein levels were unaffected. These effects were not seen in several nonendothelial cells. Collectively, these findings provide important mechanistic insight into endostatin action.