Altered expression of estrogen receptor-α variant messenger RNAs between adjacent normal breast and breast tumor tissues

Estrogen receptor (ER)-α and ER-β are believed tomediate the action of estradiol in target tissues. Several ER-α andER-β variant messenger RNAs have been identified in both normal andneoplastic human tissues. Most of these variants contain a deletion of one ormore exons of the wild-type (WT) ER messenger RNAs. The putative proteins thatare encoded by these variant messenger RNAs would therefore be missing somefunctional domains of the WT receptors, and might interfere with WT-ERsignaling pathways. The detection of ER-α variants in both normal andneoplastic human breast tissues raised the question of their possible role inbreast tumorigenesis. We have previously reported an increased relative expression ofexon 5 deleted ER-α variant (ERD5) messenger RNA and of another ER-αvariant truncated of all sequences following the exon 2 of the WT ER-α(ERC4) messenger RNA in breast tumor samples versus independent normal breasttissues. In contrast, a decreased relative expression of exon 3 deletedER-α variant (ERD3) messenger RNA in tumor tissues and cancer cell linesversus independent normal reduction mammoplasty samples has recently beenreported. These data were obtained in tissues from different individuals andpossible interindividual differences cannot be excluded. The goal of this study was to investigate the expressions of ERC4,ERD5 and ERD3 variant messenger RNAs in normal breast tissues and their matchedadjacent primary breast tumor tissues. Eighteen cases were selected from the Manitoba Breast Tumor Bank,which had well separated and histopathologically characterized normal andadjacent neoplastic components. All tumors were classified as primary invasiveductal carcinomas. Six tumors were ER-negative/progesterone receptor(PR)-negative, nine were ER-positive/PR-positive, two wereER-positive/PR-negative, and one was ER-negative/PR-positive, as measured byligand-binding assay. For each specimen, total RNA was extracted from frozennormal and tumor tissue sections and was reverse transcribed. The expressionsof ERC4, ERD3 and ERD5 messenger RNAs relative to WT ER-α messenger RNAwere investigated by previously validated semiquantitative reversetranscription polymerase chain reaction (PCR) assays performed using threedifferent sets of primers. As shown Figure 1a , two PCR products wereobtained that corresponded to WT ER and ERC4 messenger RNAs. For each case, themean of the ratios obtained in at least three independent PCR experiments isshown for both normal and tumor compartments (Fig 1b ). Astatistically higher ERC4 messenger RNA relative expression was found in theneoplastic components of ER-positive/PR-positive tumors, as compared withmatched adjacent normal tissues ( n = 9; P = 0.019, Wilcoxonsigned-rank test). Two PCR products were obtained that corresponded to WT ER and ERD3messenger RNAs (Fig 2a ). A significantly higherexpression of ERD3 messenger RNA was observed in the normal compared with theadjacent neoplastic components of ER-positive subset ( n =8; P =0.023, Wilcoxon signed-rank test; Fig 2b ). Two PCR products were obtained that corresponded to WT ER and ERD5complementary DNAs (Fig 3a ). As shown in Figure 3b , a statistically significant higher relativeexpression of ERD5 messenger RNA was observed in tumor components when thisexpression was measurable in both normal and adjacent tumor tissues ( n =15; P =0.035, Wilcoxon signed-rank test). A statistically significant higher ERC4 messenger RNA expressionwas found in ER-positive/PR-positive tumors as compared with matched normalbreast tissues. ERC4 variant messenger RNA has previously been demonstrated tobe more highly expressed in ER-positive tumors that showed poor as opposed totumors that showed good prognostic characteristics. Interestingly, we also havereported similar levels of expression of ERC4 messenger RNA in primary breasttumors and their concurrent axillary lymph node metastases. Taken together,these data suggest that the putative role of the ERC4 variant might beimportant at different phases of breast tumorigenesis and tumor progression;alteration of ERC4 messenger RNA expression and resulting modifications in ERsignaling pathway probably occur before breast cancer cells acquire the abilityto metastasize. Transient expression assays revealed that the protein encodedby ERC4 messenger RNA was unable to activate the transcription of anestrogen-responsive element-reporter gene or to modulate the wild-type ERprotein activity. The biologic significance of the changes observed in ERC4messenger RNA expression during breast tumorigenesis remains to bedetermined. A higher relative expression of ERD3 messenger RNA in the normalbreast tissue components compared with adjacent neoplastic tissue was found inthe ER-positive subgroup. These data are in agreement with the recently published report ofErenburg et al , who showed a decreased relative expression of ERD3messenger RNA in neoplastic breast tissues compared with independent reductionmammoplasty and breast tumor. Transfection experiments showed that theactivation of the transcription of the pS2 gene by estrogen was drasticallyreduced in the presence of increased ERD3 expression. The authors hypothesizedthat the reduction in ERD3 expression could be a prerequisite for breastcarcinogenesis to proceed. We observed a significantly higher relative expression of ERD5messenger RNA in breast tumor components compared with matched adjacent normalbreast tissue. These data confirm our previous observations performed onunmatched normal and neoplastic human breast tissues. Upregulated expression ofthis variant has already been reported in ER-negative/PR-positive tumors, ascompared with ER-positive/PR-positive tumors, suggesting a possible correlationbetween ERD5 messenger RNA expression and breast tumor progression. Even thoughit has been suggested that ERD5 could be related to the acquisition ofinsensitivity to antiestrogen treatment (ie tamoxifen), accumulating datarefute a general role for ERD5 in hormone-resistant tumors. Only ER-positivepS2-positive tamoxifen-resistant tumors have been shown to expresssignificantly higher levels of ERD5 messenger RNA, as compared with controltumors. Taken together, these data suggest that the exact biologic significanceof ERD5 variant expression during breast tumorigenesis and breast cancerprogression, if any, remains unclear. In conclusion, we have shown that the relative expressions of ERC4and ERD5 variant messenger RNAs were increased in human breast tumor tissue, ascompared with normal adjacent tissue, whereas the expression of ERD3 variantmessenger RNA was decreased in breast tumor tissues. These results suggest thatthe expressions of several ER-α variant messenger RNAs are deregulatedduring human breast tumorigenesis. Further studies are needed to determinewhether these changes are transposed at the protein level. Furthermore, theputative role of ER-α variants in the mechanisms that underlie breasttumorigenesis remains to be determined.

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