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

Abstract

Using semiquantitative reverse transcription-polymerase chain reaction assays, we investigated the expression of variant messenger RNAs relative to wild-type estrogen receptor (ER)-α messenger RNA in normal breast tissues and their adjacent matched breast tumor tissues. Higher ER variant truncated after sequences encoding exon 2 of the wild-type ER-α (ERC4) messenger RNA and a lower exon 3 deleted ER-α variant (ERD3) messenger RNA relative expression in the tumor compartment were observed in the ER-positive/PR-positive and the ER-positive subsets, respectively. A significantly higher relative expression of exon 5 deleted ER-α varient (ERD5) messenger RNA was observed in tumor components overall. These data demonstrate that changes in the relative expression of ER-α variant messenger RNAs occur between adjacent normal and neoplastic breast tissues. We suggest that these changes might be involved in the mechanisms that underlie breast tumorigenesis. Estrogen receptor (ER)-α and ER-β are believed to mediate the action of estradiol in target tissues. Several ER-α and ER-β variant messenger RNAs have been identified in both normal and neoplastic human tissues. Most of these variants contain a deletion of one or more exons of the wild-type (WT) ER messenger RNAs. The putative proteins that are encoded by these variant messenger RNAs would therefore be missing some functional domains of the WT receptors, and might interfere with WT-ER signaling pathways. The detection of ER-α variants in both normal and neoplastic human breast tissues raised the question of their possible role in breast tumorigenesis. We have previously reported an increased relative expression of exon 5 deleted ER-α variant (ERD5) messenger RNA and of another ER-α variant truncated of all sequences following the exon 2 of the WT ER-α (ERC4) messenger RNA in breast tumor samples versus independent normal breast tissues. In contrast, a decreased relative expression of exon 3 deleted ER-α variant (ERD3) messenger RNA in tumor tissues and cancer cell lines versus independent normal reduction mammoplasty samples has recently been reported. These data were obtained in tissues from different individuals and possible 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 matched adjacent primary breast tumor tissues. Eighteen cases were selected from the Manitoba Breast Tumor Bank, which had well separated and histopathologically characterized normal and adjacent neoplastic components. All tumors were classified as primary invasive ductal carcinomas. Six tumors were ER-negative/progesterone receptor (PR)-negative, nine were ER-positive/PR-positive, two were ER-positive/PR-negative, and one was ER-negative/PR-positive, as measured by ligand-binding assay. For each specimen, total RNA was extracted from frozen normal and tumor tissue sections and was reverse transcribed. The expressions of ERC4, ERD3 and ERD5 messenger RNAs relative to WT ER-α messenger RNA were investigated by previously validated semiquantitative reverse transcription polymerase chain reaction (PCR) assays performed using three different sets of primers. = 0.019, Wilcoxon signed-rank test). ). =0.035, Wilcoxon signed-rank test). A statistically significant higher ERC4 messenger RNA expression was found in ER-positive/PR-positive tumors as compared with matched normal breast tissues. ERC4 variant messenger RNA has previously been demonstrated to be more highly expressed in ER-positive tumors that showed poor as opposed to tumors that showed good prognostic characteristics. Interestingly, we also have reported similar levels of expression of ERC4 messenger RNA in primary breast tumors and their concurrent axillary lymph node metastases. Taken together, these data suggest that the putative role of the ERC4 variant might be important at different phases of breast tumorigenesis and tumor progression; alteration of ERC4 messenger RNA expression and resulting modifications in ER signaling pathway probably occur before breast cancer cells acquire the ability to metastasize. Transient expression assays revealed that the protein encoded by ERC4 messenger RNA was unable to activate the transcription of an estrogen-responsive element-reporter gene or to modulate the wild-type ER protein activity. The biologic significance of the changes observed in ERC4 messenger RNA expression during breast tumorigenesis remains to be determined. , who showed a decreased relative expression of ERD3 messenger RNA in neoplastic breast tissues compared with independent reduction mammoplasty and breast tumor. Transfection experiments showed that the activation of the transcription of the pS2 gene by estrogen was drastically reduced in the presence of increased ERD3 expression. The authors hypothesized that the reduction in ERD3 expression could be a prerequisite for breast carcinogenesis to proceed. We observed a significantly higher relative expression of ERD5 messenger RNA in breast tumor components compared with matched adjacent normal breast tissue. These data confirm our previous observations performed on unmatched normal and neoplastic human breast tissues. Upregulated expression of this variant has already been reported in ER-negative/PR-positive tumors, as compared with ER-positive/PR-positive tumors, suggesting a possible correlation between ERD5 messenger RNA expression and breast tumor progression. Even though it has been suggested that ERD5 could be related to the acquisition of insensitivity to antiestrogen treatment (ie tamoxifen), accumulating data refute a general role for ERD5 in hormone-resistant tumors. Only ER-positive pS2-positive tamoxifen-resistant tumors have been shown to express significantly higher levels of ERD5 messenger RNA, as compared with control tumors. Taken together, these data suggest that the exact biologic significance of ERD5 variant expression during breast tumorigenesis and breast cancer progression, if any, remains unclear. In conclusion, we have shown that the relative expressions of ERC4 and ERD5 variant messenger RNAs were increased in human breast tumor tissue, as compared with normal adjacent tissue, whereas the expression of ERD3 variant messenger RNA was decreased in breast tumor tissues. These results suggest that the expressions of several ER-α variant messenger RNAs are deregulated during human breast tumorigenesis. Further studies are needed to determine whether these changes are transposed at the protein level. Furthermore, the putative role of ER-α variants in the mechanisms that underlie breast tumorigenesis remains to be determined.

Introduction:

Estrogen receptor (ER)-α and ER-β are believed to mediate the action of estradiol in target tissues. Several ER-α and ER-β variant messenger RNAs have been identified in both normal and neoplastic human tissues. Most of these variants contain a deletion of one or more exons of the wild-type (WT) ER messenger RNAs. The putative proteins that are encoded by these variant messenger RNAs would therefore be missing some functional domains of the WT receptors, and might interfere with WT-ER signaling pathways. The detection of ER-α variants in both normal and neoplastic human breast tissues raised the question of their possible role in breast tumorigenesis.

We have previously reported an increased relative expression of exon 5 deleted ER-α variant (ERD5) messenger RNA and of another ER-α variant truncated of all sequences following the exon 2 of the WT ER-α (ERC4) messenger RNA in breast tumor samples versus independent normal breast tissues. In contrast, a decreased relative expression of exon 3 deleted ER-α variant (ERD3) messenger RNA in tumor tissues and cancer cell lines versus independent normal reduction mammoplasty samples has recently been reported. These data were obtained in tissues from different individuals and possible interindividual differences cannot be excluded.

Aims:

The goal of this study was to investigate the expressions of ERC4, ERD5 and ERD3 variant messenger RNAs in normal breast tissues and their matched adjacent primary breast tumor tissues.

Materials and methods:

Eighteen cases were selected from the Manitoba Breast Tumor Bank, which had well separated and histopathologically characterized normal and adjacent neoplastic components. All tumors were classified as primary invasive ductal carcinomas. Six tumors were ER-negative/progesterone receptor (PR)-negative, nine were ER-positive/PR-positive, two were ER-positive/PR-negative, and one was ER-negative/PR-positive, as measured by ligand-binding assay. For each specimen, total RNA was extracted from frozen normal and tumor tissue sections and was reverse transcribed. The expressions of ERC4, ERD3 and ERD5 messenger RNAs relative to WT ER-α messenger RNA were investigated by previously validated semiquantitative reverse transcription polymerase chain reaction (PCR) assays performed using three different sets of primers.

Results:

= 0.019, Wilcoxon signed-rank test).

).

=0.035, Wilcoxon signed-rank test).

Discussion:

A statistically significant higher ERC4 messenger RNA expression was found in ER-positive/PR-positive tumors as compared with matched normal breast tissues. ERC4 variant messenger RNA has previously been demonstrated to be more highly expressed in ER-positive tumors that showed poor as opposed to tumors that showed good prognostic characteristics. Interestingly, we also have reported similar levels of expression of ERC4 messenger RNA in primary breast tumors and their concurrent axillary lymph node metastases. Taken together, these data suggest that the putative role of the ERC4 variant might be important at different phases of breast tumorigenesis and tumor progression; alteration of ERC4 messenger RNA expression and resulting modifications in ER signaling pathway probably occur before breast cancer cells acquire the ability to metastasize. Transient expression assays revealed that the protein encoded by ERC4 messenger RNA was unable to activate the transcription of an estrogen-responsive element-reporter gene or to modulate the wild-type ER protein activity. The biologic significance of the changes observed in ERC4 messenger RNA expression during breast tumorigenesis remains to be determined.

, who showed a decreased relative expression of ERD3 messenger RNA in neoplastic breast tissues compared with independent reduction mammoplasty and breast tumor. Transfection experiments showed that the activation of the transcription of the pS2 gene by estrogen was drastically reduced in the presence of increased ERD3 expression. The authors hypothesized that the reduction in ERD3 expression could be a prerequisite for breast carcinogenesis to proceed.

We observed a significantly higher relative expression of ERD5 messenger RNA in breast tumor components compared with matched adjacent normal breast tissue. These data confirm our previous observations performed on unmatched normal and neoplastic human breast tissues. Upregulated expression of this variant has already been reported in ER-negative/PR-positive tumors, as compared with ER-positive/PR-positive tumors, suggesting a possible correlation between ERD5 messenger RNA expression and breast tumor progression. Even though it has been suggested that ERD5 could be related to the acquisition of insensitivity to antiestrogen treatment (ie tamoxifen), accumulating data refute a general role for ERD5 in hormone-resistant tumors. Only ER-positive pS2-positive tamoxifen-resistant tumors have been shown to express significantly higher levels of ERD5 messenger RNA, as compared with control tumors. Taken together, these data suggest that the exact biologic significance of ERD5 variant expression during breast tumorigenesis and breast cancer progression, if any, remains unclear.

In conclusion, we have shown that the relative expressions of ERC4 and ERD5 variant messenger RNAs were increased in human breast tumor tissue, as compared with normal adjacent tissue, whereas the expression of ERD3 variant messenger RNA was decreased in breast tumor tissues. These results suggest that the expressions of several ER-α variant messenger RNAs are deregulated during human breast tumorigenesis. Further studies are needed to determine whether these changes are transposed at the protein level. Furthermore, the putative role of ER-α variants in the mechanisms that underlie breast tumorigenesis remains to be determined.

Introduction

].

].

] recently reported a decreased relative expression of ERD3 messenger RNA in tumor tissues and cancer cell lines versus independent normal reduction mammoplasty samples. Those data, which suggested that alteration in ERD5, ERD3 and clone 4 messenger RNA expression might occur during breast tumorigenesis, were obtained in tissues from different individuals, and possible interindividual differences cannot be excluded.

In order to clarify this issue, we investigated the expression of these three variant messenger RNAs in normal breast tissues and their matched adjacent primary breast tumor tissues.

Human breast tissues and reverse transcription

In order to investigate the expressions of ERC4, ERD3 and ERD5 messenger RNA relative to WT-ER messenger RNA within matched normal and breast tumor tissues, eighteen cases were selected in the National Cancer Institute of Canada Manitoba Breast Tumor Bank (Winnipeg, Manitoba, Canada), which had well separated and histopathologically characterized normal and adjacent neoplastic components. The Tumor Bank, which serves as a national Tumor Bank and is funded by the National Cancer Institute of Canada, has been reviewed and received approval from the Ethics Review Committee, Faculty of Medicine, University of Manitoba.

]. Briefly, each specimen had been rapidly frozen as soon as possible after surgical removal. A portion of the frozen tissue block was processed to create a paraffin-embedded tissue block that was matched and oriented relative to the remaining frozen block. These paraffin blocks provide high quality histologic sections, which are used for pathologic interpretation and assessment, and are mirror images of the frozen sections used for RNA extractions.

].

Triple primer polymerase chain reaction

].

]. PCR products migrating with the apparent size of 149 and 536 base pairs were shown to correspond to WT-ER and ERC4 complementary DNAs, respectively.

Polymerase chain reaction

].

P] dCTP (3000 Ci/mmol), 4ng/μ l of each primer of the primer set considered (ERD3 or ERD5 primer set) and 0.3 unit of Taq DNA polymerase. Each cycle consisted of 30s at 94°C, 30s at 60°C and 30s at 72°C. PCR products were then separated on 6% polyacrylamide gels containing 7mol/l urea (polyacrylamide gel electrophoresis). Following electrophoresis, the gels were dried and autoradiographed. For each PCR, two PCR products were obtained, which were identified by subcloning and sequencing. PCR products migrating with the apparent size of 354 and 483 base pairs, using ERD3 and ERD5 primer set, respectively, were shown to correspond to WT-ER complementary DNA. PCR products migrating with the apparent size of 237 and 344 base pairs, using ERD3 and ERD5 primer set, were shown to correspond to ERD3 and ERD5 complementary DNAs, respectively.

Quantitation and statistical analysis

) in any of the four repetitions performed. Only cases that had detectable levels in at least three of the replicates in both their normal and tumor compartments were included in the statistical analysis. The significance of the differences in the relative levels of expression of ERC4, ERD3 and ERD5 messenger RNAs between matched normal and tumor components was determined using the Wilcoxon signed-rank test.

Relative expression of ERC4 messenger RNA in matched normal and

			breast tumor tissues

].

= 0.019, Wilcoxon signed-rank test).

Relative expression of ERD3 messenger RNA in matched normal and

			breast tumor tissues

] have previously shown that the coamplification of WT-ER and an exon-deleted ER-α variant complemetary DNA resulted in the amplification of two PCR products, the relative signal intensity of which provided a previously validated measurement of exon-deleted ER-α variant expression.

= 0.023, Wilcoxon signed-rank test).

Relative expression of ERD5 messenger RNA in matched normal and

			breast tumor tissues

= 0.035, Wilcoxon signed-rank test).

Discussion

].

]. Triple-primer PCR assay applied to the detection of ERC4 messenger RNA in 18 matched normal and tumor breast tissues gave a measurable value of expression in 36 out of the 36 samples studied. This contrasts with the detection of 30 out of 36 and 33 out of 36 obtained using ERD3-specific and ERD5-specific primers, respectively. These differences in sensitivity probably result from different primer set efficiencies under our experimental conditions.

] obtained by comparing ERC4 messenger RNA expression between independent normal reduction mammoplasty samples and a group of ER-positive/PR-positive breast tumors. Even though a higher ERC4 messenger RNA relative expression was observed in the tumor component of 12 out of 18 cases, this difference did not reach statistical significance. This absence of statistically significant differences might result from the low number of matched cases studied or from the different biology of ER-negative cases. Further studies are needed to clarify this issue and to draw any conclusion regarding the expression of ERC4 messenger RNA in ER-negative samples.

]. The biologic significance of the changes observed in ERC4 messenger RNA expression during breast tumorigenesis and tumor progression therefore remains unclear.

invasiveness, as compared with control cells. These data led the authors to hypothesize that the reduction of ERD3 expression could be a prerequisite for breast carcinogenesis to proceed. They suggested that if high levels of ERD3 could attenuate estrogenic effects in normal breast tissue, low levels might lead to an excessive and unregulated mitogenic action of estrogen.

]. Taken together, these data suggest that the exact biologic significance of ERD5 variant expression during breast tumorigenesis and breast cancer progression, if any, remains unclear.

] recently reported a study performed on 15 cases. They observed an apparent difference in ER variant messenger RNA expression between adjacent normal and tumor samples. That study was performed using a less sensitive PCR approach, however, because PCR products were stained using ethidium bromide, and no attempt was made to quantify ER variant messenger RNA expression relative to WT-ER messenger RNA expression.

], suggest that the expressions of several ER-α variant messenger RNAs are deregulated during human breast tumorigenesis. Further studies are needed to determine whether these changes are transposed at the protein level. Only the use of specific antibodies that are able to recognize specifically the different ER variant proteins putatively encoded by these variant messenger RNAs will allow this issue the be addressed. Furthermore, the putative role of ER-α variants in the mechanisms that underlie breast tumorigenesis remain to be determined.

Acknowledgement

This work was supported by a grant from the US Army Medical Research and Materiel Command (DAMD17-95-1-5015). The Manitoba Breast Tumor Bank is supported by funds from the National Cancer Institute of Canada (NCIC). EL is a recipient of an US Army Medical Research and Materiel Command Postdoctoral Fellowship (DAMD17-96-1-6174), PHW is a Medical Research Council of Canada (MRC) Clinician-Scientist, and LCM is an MRC Scientist.

Figures and Tables

<0.05. M, molecular weight marker (fx174 Haelll digest, Gibco BRL, Grand Island, New York, NY).

<0.05. M, molecular weight marker.

<0.05. m, molecular weight marker.

Keywords

• breast cancer
• estrogen receptor
• tumorigenesis
• variant messenger RNA