Promoter Analysis and Chromosomal Mapping of Human EBAG9 Gene

doi:10.1006/bbrc.2000.2920

Biochemical and Biophysical Research Communications

Volume 273, Issue 2, 5 July 2000, Pages 654-660

https://doi.org/10.1006/bbrc.2000.2920 Get rights and content

Abstract

The human EBAG9 was previously identified as an estrogen responsive gene using CpG-genomic binding site cloning (Watanate et al., (1998) Mol. Cell. Biol. 18: 442–449). Recently it was revealed that the EBAG9 is identical with RCAS1 which is a cancer cell surface antigen implicated in immune escape. Here, we isolated and analyzed the 5′-flanking region of human EBAG9 gene. We determined transcription initiation site, which has a homology with an initiator element YYCAYYYY, and found that TATA motif was absent. Deletion analysis of the 5′-flanking region using MCF-7 breast cancer cells indicated that the sequences −86 to −36 containing the ERE had the basal level of promoter activity and the upstream GC-rich region positively regulated the activity. EBAG9 promoter luciferase reporters containing the ERE could respond to estrogen, and electrophoretic mobility shift assay showed that ERα bound to the ERE. Moreover, fluorescent in situ hybridization analysis has shown that the human EBAG9 gene is located at chromosome 8q23 which is frequently amplified in tumors. These findings suggest that the human EBAG9 might be involved in carcinogenesis as an estrogen responsive gene.

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Cited by (45)

Association of positive EBAG9 immunoreactivity with unfavorable prognosis in breast cancer patients treated with tamoxifen
2013, Clinical Breast Cancer
Citation Excerpt :
These findings suggest that EBAG9 functions as an estrogen-responsive gene in ER-positive breast cancer cells. However, promoter analysis demonstrated that the 5′-flanking region of the EBAG9 gene contains several transcription factor-binding sites in addition to a prototypic consensus estrogen-responsive element,20 suggesting that other transcriptional regulatory pathways also regulate EBAG9 expression in breast cancer cells. High-level EBAG9 expression mediated by factors other than ER might result in an unfavorable prognosis in breast cancer patients after tamoxifen treatment.
Breast cancer is primarily a hormone-dependent tumor that is regulated by the status of the estrogen and progesterone receptors. We previously identified EBAG9 as an estrogen-responsive gene in MCF-7 human breast carcinoma cells. Upregulation of EBAG9 expression has been observed in several malignant tumors such as advanced breast cancers, indicating that EBAG9 might contribute to tumor progression.
In the present study, we generated a monoclonal antibody against EBAG9, and then performed immunohistochemical analysis of EBAG9 expression in specimens obtained from breast cancer patients treated with tamoxifen as an adjuvant therapy.
EBAG9 immunoreactivity was detected in the cytoplasm of breast cancer cells and was significantly elevated in breast cancer samples from patients who relapsed during or after adjuvant tamoxifen treatment. Positive EBAG9 immunoreactivity was significantly correlated with poor patient prognosis.
These results suggest that EBAG9 expression in tumor regions is associated with an unfavorable prognosis in breast cancer patients treated with tamoxifen.
The effect of tumor-associated protein RCAS1 gene silencing on blood pressure and urinary protein excretion in pregnant mouse: A pilot study
2010, American Journal of Obstetrics and Gynecology
Citation Excerpt :
These observations strengthen our hypothesis about the possible relationship between RCAS1 gene silencing at the fetomaternal interface and preeclampsia. RCAS1 is identical with the estrogen-responsive protein EBAG9.7,10 Estrogen plays many roles in immunomodulation, but it mainly has antiinflammatory and vasoprotective effects.28
The level of tumor-associated receptor-binding cancer antigen that is expressed on SiSo cells (RCAS1) is decreased significantly in preeclamptic pregnancies. We hypothesized that RCAS1 protein gene silencing might affect blood pressure and proteinuria in pregnant mice.
On postcoital day 7.5, pregnant imprinting control region mice were subjected to the transfer of small interfering RNA (siRNA) against RCAS1 protein into the uterine cavity with the use of a hemagglutinating virus Japan envelope. Scramble siRNA was used as a negative control. Blood pressure and urine albumin/creatinine measurements were performed. The effect of the transferred siRNA was examined in uterine samples on postcoital day 8.5 with the use of Western blotting and immunohistochemistry analyses.
In the RCAS1 siRNA group, blood pressure significantly raised on postcoital days 9.5, 10.5, 11.5, and 15.5, whereas urine albumin/creatinine ratio was significantly increased on postcoital day 9.5
Our results suggest the importance of RCAS1 protein in the pathophysiologic condition of preeclampsia.
The human tumor-associated antigen RCAS1 in pregnancies complicated by pre-eclampsia
2008, Journal of Reproductive Immunology
Citation Excerpt :
The tumor-associated antigen RCAS1 was defined by its immunoreaction with the 22-1-1 monoclonal antibody (mAb), which was raised by immunization of mice with the human uterine cervical adenocarcinoma cell line SiSo (Sonoda et al., 1996). The gene product was termed ‘receptor-binding cancer antigen expressed on SiSo cells’ (RCAS1) and is identical with the estrogen-responsive protein EBAG9 (estrogen receptor-binding fragment-associated gene9) which is located on chromosome 8q23 (Nakashima et al., 1999; Ikeda et al., 2000). RCAS1 is a predominantly Golgi-localized membrane protein (Reimer et al., 2005).
The human tumor-associated antigen RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) is considered to play a role in the escape of tumor cells from immune surveillance and, at the same time, participates in the inhibition of the maternal immune response during pregnancy. The aim of our study was to investigate the expression of tumor-associated RCAS1 protein in the placenta and amniotic membranes and to assess and compare its concentration in amniotic fluid, maternal and cord blood sera in pregnancies complicated by pre-eclampsia. Samples were obtained from women with pre-eclampsia (N = 9), pre-eclampsia with IUGR (N = 4), normotensive IUGR (N = 7) and healthy term controls (N = 25) after delivery. Placentas were studied by immunohistochemistry, Western blot analysis and real-time (RT)-PCR. For assessment of RCAS1 protein concentrations in biological fluids, ELISA was performed. RCAS1 mRNA expression in the placentas of pre-eclamptic patients was significantly lower than in controls (p < 0.01). The maternal blood serum RCAS1 protein concentration in the pre-eclampsia cases was also significantly lower than in controls (p = 0.0207). The other study groups did not differ significantly. This study reveals the possible role of the RCAS1 protein in the development of pre-eclampsia through an immunological pathway.
Clinical significance of RCAS1 as a biomarker of uterine cancer
2006, Gynecologic Oncology
Expression of RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) is associated with prognosis of various malignancies including uterine cancer. Proteolytic cleavage of RCAS1 at extracellular domains (ectodomain shedding) yields soluble RCAS1. Although RCAS1 can induce apoptosis in normal peripheral lymphocytes, its biologic function in cancer patients is unclear. Here, we evaluated serum RCAS1 concentrations to clarify its biologic activity in uterine cancer.
Via ELISA, we measured serum RCAS1 concentrations in samples from 54 healthy blood donors and 113 patients—63 with cervical cancer and 50 with endometrial cancer. We also counted the peripheral lymphocyte number. We correlated via statistical means the RCAS1 values with patients' clinicopathologic variables. We assessed inhibition of growth of K562 cells, which express the putative RCAS1 receptor, via WST-1 assay of serum samples to clarify RCAS1's biologic activity.
Uterine cancer patients had significantly higher serum RCAS1 concentrations than did healthy blood donors (P < 0.05). Patients with adenocarcinoma had significantly higher RCAS1 concentrations than did those with squamous cell carcinoma (P = 0.0340). RCAS1 values were also significantly associated with response to treatment (P < 0.001). FasL and TNF-α serum concentrations were not significantly different for the different groups, however. The WST-1 assay showed that patients' serum induced K562 cell growth inhibition, but this effect partially recovered after immunodepletion of RCAS1. Peripheral lymphocyte number and serum RCAS1 concentration were inversely related (P = 0.0310).
RCAS1 may be a biomarker of uterine cancer because of its potential to predict results of uterine cancer treatment and inhibit growth of immune cells.
Invasive potency related to RCAS1 expression in uterine cervical cancer
2005, Gynecologic Oncology
RCAS1 expression is significantly associated with clinical prognosis in various human cancers, which suggests that RCAS1 may be involved in acquisition of malignant phenotypes. To investigate the relationship between RCAS1 and one such characteristic, tumor invasiveness, we examined RCAS1 expression in cervical neoplasms ranging from the precancerous state to invasive cancer.
RCAS1 expression was studied retrospectively via immunohistochemical methods. Samples consisted of biopsy tissue from 90 patients with intraepithelial neoplasia and resected tumor tissue from 154 patients with invasive cancer. Statistical analysis was done to correlate RCAS1 expression and clinicopathologic variables in patients with a depth of cancer cell invasion into stromal tissue of >5 mm.
RCAS1 expression was detected in patients with intraepithelial cancer and invasive cancer but not in patients with dysplasia. The occurrence and degree of RCAS1 expression increased with the depth of invasion. In patients with invasive cancer, RCAS1 overexpression was significantly correlated with invasion of the lymph-vascular space, lymph node metastasis in two or more sites, and tumor volume; RCAS1 expression was not associated with histologic subtype. Overall survival rates for patients with RCAS1 overexpression were significantly shorter than those for patients without RCAS1 overexpression. In connective tissue surrounding tumor cells, the number of cells expressing vimentin significantly decreased in relation to RCAS1 expression level. Moreover, significant associations between expression levels of RCAS1 and those of MMP-1 and laminin-5 were found.
RCAS1 may contribute to acquisition of malignant uterine cervical phenotypic characteristics including invasion, metastasis, and tumor growth via connective tissue remodeling.
Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1
2005, Cell
Citation Excerpt :
In addition, a number of recent strategies including gene expression profiling on microarrays have identified potential ER target genes in human breast cancer cells and only a few cis-elements targeted directly by ER have been identified to date. For example, estrogen responsive elements (ERE) have been identified within the 1 kb 5′-proximal region of the estrogen-regulated genes TFF-1 (pS2), EBAG9, and Cathepsin D (Augereau et al., 1994; Berry et al., 1989; Ikeda et al., 2000), and the proximal promoters of target genes that lack EREs, including c-Myc and IGF-I, contain AP-1 and Sp-1 sites that appear essential for transcription in in vitro reporter assays (Dubik and Shiu, 1992; Umayahara et al., 1994). Few, if any regulatory elements at significant distances from the mRNA start sites of target genes have been shown to be directly targeted by ER, and computation approaches to identify novel ER binding domains have focused primarily on gene proximal regions (Bajic and Seah, 2003; Bourdeau et al., 2004).
Estrogen plays an essential physiologic role in reproduction and a pathologic one in breast cancer. The completion of the human genome has allowed the identification of the expressed regions of protein-coding genes; however, little is known concerning the organization of their cis-regulatory elements. We have mapped the association of the estrogen receptor (ER) with the complete nonrepetitive sequence of human chromosomes 21 and 22 by combining chromatin immunoprecipitation (ChIP) with tiled microarrays. ER binds selectively to a limited number of sites, the majority of which are distant from the transcription start sites of regulated genes. The unbiased sequence interrogation of the genuine chromatin binding sites suggests that direct ER binding requires the presence of Forkhead factor binding in close proximity. Furthermore, knockdown of FoxA1 expression blocks the association of ER with chromatin and estrogen-induced gene expression demonstrating the necessity of FoxA1 in mediating an estrogen response in breast cancer cells.

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¹: To whom correspondence should be addressed at Department of Biochemistry, Saitama Medical School, 38 Morohongo, Moroyama, Iruma, Saitama 350-0495, Japan. Fax: 81-492-94-9751.

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Regular ArticlePromoter Analysis and Chromosomal Mapping of Human EBAG9 Gene

Abstract

J. Steroid. Biochem. Mol. Biol.

Trends Endocrinol. Metab.

FEBS Lett.

Virology

Biochem. Biophys. Res. Commun.

Am. J. Pathol.

Estrogen receptor null mice: what have we learned and where will they lead us?

Endocr. Rev.

Steroid hormones and risk of breast cancer

Cancer

Hormonal carcinogenesis

Carcinogenesis

Isolation of estrogen-responsive genes with a CpG island library

Mol. Cell. Biol.

Inhibition of cell growth and induction of apoptotic cell death by the human tumor-associated antigen RCAS1

Nat. Med.

A novel tumor-associated antigen expressed in human uterine and ovarian carcinomas

Cancer

Regular Article
Promoter Analysis and Chromosomal Mapping of Human EBAG9 Gene