Hypermethylation can selectively silence multiple promoters of steroid receptors in cancers

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Abstract

Multiple promoters and differential splicing of 5′ upstream exons are often found in various nuclear receptor genes including steroid receptors. Three promoters control the expression of human estrogen receptor α (ERα) isoforms: ERα-A, ERα-B, and ERα-C, and two promoters control the expression of human progesterone receptor (PR) isoforms: PR-A and PR-B. The expression levels of these isoforms differ with respect to each other in certain target tissues. The role of these isoforms may differ in various types of cells and tissues. The ER and PR contain CpG islands in the 5′ upstream regions. One possible mechanism for changing the transcriptional status is methylation of CpG-enriched regions in these isoforms. We have investigated the expression and methylation status of the three different ERα promoters and the two different PR gene promoters by using methylation specific PCR (MSP) and direct DNA sequencing in several endometrial and prostate cancer cell lines and tissues. The results of these experiments suggest that ERα-A, ERα-B, and PR-A were expressed and ERα-C and PR-B were inactivated in endometrial cancers. To the contrary, ERα-A and ERα-B were inactivated and ERα-C, PR-A and PR-B were expressed in all prostate cancer. Treatment with demethylating agent (5-aza-2′-deoxycytidine) restored these gene expressions, suggesting that inactivation of this gene is through methylation. Our MSP and direct DNA sequencing showed that ERα-A, ERα-B, and PR-A genes were unmethylated and ERα-C and PR-B were methylated in endometrial cancers although ERα-A and ERα-B were methylated and ERα-C, PRA and PRB were unmethylated in prostate cancers. These reports clearly demonstrate that selective hypermethylation can selectively silence multiple promoters of steroid receptors in carcinogenesis.

Section snippets

Estrogen receptor isoforms

Estrogens exert their effects through two estrogen receptor (ER) types (α and β), which are members of the nuclear steroid–thyroid hormone receptor super-family (Evans, 1988, Mosselman et al., 1996). The receptors are expressed in a cell- and tissue-specific manner. The biological significance of ERα and ERβ however, ?is unclear at this moment. Several studies have shown that ERα regulation may be more complex than previously reported (Piva et al., 1993, Grandien, 1996). Three promoters, A, B,

Progesterone receptor isoforms

The progesterone receptor (PR) is a member of a closely related subgroup of nuclear receptors that includes the androgen, mineral corticoid, and glucocorticoid receptors. Within this subgroup, PR is unique since it occurs in target tissues as two distinct subtypes, PR-A and PR-B, of 94 and 114 kDa, respectively (Kastner et al., 1990, Giangrande et al., 1997) (Fig. 1). The PR-B isoform contains an N-terminal fragment of 164 amino acids, which is absent in the PRA isoform. Both isoforms arise by

Estrogens and endometrial cancer

A substantial and convincing body of experimental, clinical and epidemiological evidence indicates that hormones play a major role in the etiology of endometrial cancer (Miller and Langdon, 1997, Wormke et al., 2000, Elkas et al., 2000, Kaminsky and Spivack, 1999, Kounelis et al., 2000, Kristensen and Borresen-Dale, 2000). Biologically active steroids are produced and secreted in the endocrine organs such as ovary, testis, and adrenal cortex, are transported through circulation, and act on

Estrogens and prostate cancer

The prostate is under endocrine control during its growth and development (Cox and Crawford, 1995, Farnsworth, 1996, Brolin et al., 1992). Various steroid hormones act via intracellular receptors. Androgen receptors, ERs, and PRs have been identified in normal and prostate cancer tissues (Karr et al., 1979, Seitz and Wernert, 1987). Estrogens play a crucial role in female sexual development and in the regulation of the menstrual cycle, but they also have important effects on benign prostatic

Progesterones and endometrial cancer

Progesterones are involved in many regulatory processes in the endometrial epithelium (Cohen and Rahaman, 1995). Progesterones protect the endometrium against the hyperplastic effects of estradiol through PRs. Endometrial cancer is induced to hyper-estrogenism that is suppressed by progesterone (Cohen and Rahaman, 1995). This conclusion is based on studies of an association between endometrial cancer and polycystic ovarian disease as well as other hyper-estrogenic states. In these diseases, the

Progesterones and prostate cancer

The role of PR in the development and progression of prostate cancer is poorly understood (Widmark et al., 1995, Tilley et al., 1980). The consistent presence of progesterone receptors suggests that these receptors have some role in prostate cells (Srinivasan et al., 1995). Progesterones have been used in the treatment of prostate carcinoma at certain stages, although the exact mechanism of action remains to be resolved and the beneficial effect from a clinical point of view is controversial (

CpG islands in steroid receptor isoforms

The ER genes contain CG-enriched regions in 5′ upstream region (Keaveney et al., 1992, Piva et al., 1992) (Fig. 1). ERα gene methylation has been observed in several human cancers such as breast (Ottaviano et al., 1994, Tang et al., 1997), lung (Issa et al., 1996a), colorectal (Issa et al., 1994), and hematopoietic neoplasm (Issa et al., 1996b) and has been related to inactivation of ER gene expression. The PR gene also contains CpG islands in the 5′ upstream region (Kastner et al., 1990). CpG

CpG methylation and expression of estrogen receptor α isoforms in endometrial cancer

We have determined the expression and methylation status of ERα isoforms in endometrial cancer cell lines (Sasaki et al., 2001a). No ERα-C expression was found, although ERα-A and ERα-B expression were found in all endometrial cancer cell lines (Table 1). Only ERα-C was methylated in all of endometrial cell lines, whereas others were unmethylated. To investigate whether the expression of mRNAs for these steroid isoforms is inactivated by methylation of their promoters, we treated cells with the

CpG methylation and expression of estrogen receptor α isoforms in prostate cancer

In all prostate cancer cell lines, ERα-C was expressed but ERα-A and ERα-B were not expressed (Table 1) (Sasaki et al., 2002). In all prostate cell lines, the ERα-C promoter was unmethylated, whereas the ERα-A and ERα-B promoters were methylated. Treatment with 5-azaC restored the expression of ERα-A and ERα-B in all prostate cell lines. Thus, the expression of these steroid receptor isoforms was related to the methylation status of their corresponding promoters. The promoter for ERα-A and

CpG methylation and expression of progesterone receptor isoforms in endometrial and prostate cancer

No PR-B expression was found in all endometrial cancer cell lines, although PRA expression was observed (Table 1) (Sasaki et al., 2001b, Sasaki et al., 2002). Only PR-B was methylated in all of the cell lines, although PR-A was unmethylated. Treatment of cell lines with 5-azaC restored PR-B expression in all cancer cell lines. About 70% of endometrial cancer tissues were methylated for PR-B, whereas 100% of cancerous and normal endometrial samples were unmethylated for PR-A (Table 2).

Concluding remarks

The results of these experiments suggest that ERα-A and ERα-B were expressed and ERα-C and PR-B were inactivated in endometrial cancers. To the contrary, ERα-A and ERα-B were inactivated and ERα-C was expressed in prostate cancer. PR-A was expressed and PR-B was inactivated in endometrial cancers although they were both expressed in prostate cancer. Treatment with demethylating agent restored gene expression, suggesting that inactivation of these genes is through methylation. Our methylation

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