Abstract
The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) has been identified as a primary estrogen-responsive gene in human breast cancer MCF7 cells. A high expression of EBAG9 has been observed in invasive breast cancer and advanced prostate cancer, suggesting a tumor-promoting role of the protein in malignancies. Here we show that intratumoral (i.t.) administration of small interfering RNA against EBAG9 exerted overt regression of tumors following s.c. implantation of murine renal cell carcinoma (RCC) Renca cells. Overexpression of EBAG9 did not promote the proliferation of culture Renca cells; however, the inoculated Renca cells harboring EBAG9 (Renca-EBAG9) in BALB/c mice grew faster and developed larger tumors compared with Renca cells expressing vector alone (Renca-vector). After renal subcapsular implantation, Renca-EBAG9 tumors significantly enlarged compared with Renca-vector tumors in BALB/c mice, whereas both Renca-EBAG9 and Renca-vector tumors were developed with similar volumes in BALB/c nude mice. No apparent difference was observed in specific cytotoxic T-cell responses against Renca-EBAG9 and Renca-vector cells; nonetheless, the number of infiltrating CD8+ T lymphocytes was decreased in Renca-EBAG9 subcapsular tumors. Furthermore, immunohistochemical study of EBAG9 in 78 human RCC specimens showed that intense and diffuse cytoplasmic immunostaining was observed in 87% of the cases and positive EBAG9 immunoreactivity was closely correlated with poor prognosis of the patients. Multivariate analysis revealed that high EBAG9 expression was an independent prognostic predictor for disease-specific survival (P = 0.0485). Our results suggest that EBAG9 is a crucial regulator of tumor progression and a potential prognostic marker for RCC.
Highlights
Estrogen receptor–binding fragment associated gene 9 (EBAG9) is an estrogen-responsive gene that we previously identified in MCF-7 human breast carcinoma cell line using a CpG-genomic binding site cloning method [1]
To determine the role of EBAG9 in tumor growth of renal cancer cells, we investigated the effects of synthesized small interfering RNA (siRNA) duplexes targeting EBAG9 on s.c. tumor models of Renca cells implanted in syngeneic BALB/c mice
Intratumoral injection of EBAG9 siRNA reduced the protein levels of endogenous EBAG9 compared with the levels of EBAG9 in parental Renca cells or in the Renca tumor treated with control scrambled siRNA duplexes (Fig. 1A)
Summary
Estrogen receptor–binding fragment associated gene 9 (EBAG9) is an estrogen-responsive gene that we previously identified in MCF-7 human breast carcinoma cell line using a CpG-genomic binding site cloning method [1]. EBAG9 protein, whose molecular size is 32 kDa by Western blot analysis, is expressed in estrogen. The protein expression of EBAG9 is estrogen inducible, as it has been shown in ovariectomized mice treated with 17h-estradiol administration [2]. The physiologic function of EBAG9 has not been well defined, yet the molecule may be implicated in cancer pathophysiology, with several lines of evidence of the protein expression in malignancies, including breast [3], ovarian [4], prostate [5], and hepatocellular carcinomas [6].
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