Helmut Dotzlaw

Estrogen receptor-β mRNA variants in human and murine tissues

Abstract Estrogen receptor (ER)- β mRNA splice variants have been identified in human breast tumors as well as normal human and mouse ovarian, uterine and mammary tissues. In both species transcripts deleted in exons 5 or 6, or 5+6 have been characterized by RT-PCR followed by cloning and sequencing. In mouse tissues an ER- β transcript containing 54 nucleotides inserted in frame between exons 5 and 6 was identified. Interestingly, no equivalent of the mouse inserted transcript was detected in any of the four human tissues analyzed.

Lumican and decorin are differentially expressed in human breast carcinoma.

Previous studies have shown that lumican is expressed and increased in the stroma of breast tumours. Lumican expression has now been examined relative to other members of the small leucine‐rich proteoglycan gene family in normal and neoplastic breast tissues, to begin to determine its role in breast tumour progression. Western blot study showed that lumican protein is highly abundant relative to decorin, while biglycan and fibromodulin are only detected occasionally in breast tissues (n=15 cases). Further analysis of lumican and decorin expression performed in matched normal and tumour tissues by in situ hybridization showed that both mRNAs were expressed by similar fibroblast‐like cells adjacent to epithelium. However, lumican mRNA expression was significantly increased in tumours (n=34, p<0.0001), while decorin mRNA was decreased (p=0.0002) in neoplastic relative to adjacent normal stroma. This was accompanied by a significant increase in lumican protein (n=12, p=0.0122), but not decorin. Further evidence of altered lumican expression in breast cancer was manifested by discordance between lumican mRNA and protein localization in some regions of tumours but not in adjacent morphologically normal tissues. It is concluded that lumican is the most abundant of these proteoglycans in breast tumours and that lumican and decorin are inversely regulated in association with breast tumourigenesis. Copyright

Estrogen Receptor Variants and Mutations

There is a large and increasing body of experimental and clinical data supporting the existence or variant estrogen receptor (ER) proteins in both normal and neoplastic estrogen target tissues including human breast. Therefore, future examination of ER signal transduction and/or measurement of ER protein must take into account variant ER expression. The functions of variant ER proteins, either physiological or pathological, remain unclear, although a role(s) for some ER variants in breast tumorigenesis and breast cancer progression would be consistent with the accumulated data. Possible tissue specific expression leads to the speculation that ER variants may have a role in tissue specific estrogen action. The following review focuses on the current knowledge available in the scientific literature with respect to the type and characteristics of estrogen receptor variants and mutations that have been identified to occur naturally in tissues and cell lines.

Mammaglobin, a potential marker of breast cancer nodal metastasis

The Mammaglobin gene, a breast‐specific member of the uteroglobin gene family, has been previously identified as being overexpressed in some breast tumours, but the cellular origin and relationship to tumour progression are unknown. Using a subtractive hybridization approach, mammaglobin mRNA has also been found to be overexpressed in the in situ compared to the invasive element within an individual breast tumour. Further study by in situ hybridization performed in 13 breast tumours, selected to include normal, in situ, and invasive primary tumour elements, and in most cases axillary lymph node metastases, revealed that mammaglobin expression occurs in all elements, is restricted to epithelial cells, and is significantly increased in tumour cells compared with normal cells ( p< 0·04). Analysis of mammaglobin expression within 20 independent primary breast tumours and their corresponding axillary lymph nodes revealed that all 13 lymph nodes positive and none of the seven nodes negative for metastatic breast carcinoma by histology were mammaglobin‐positive by reverse transcription‐polymerase chain reaction (RT‐PCR) ( p=0·0001). These results suggest that mammaglobin could be a marker of axillary lymph node breast metastases. Copyright

The Pathophysiological Role of Estrogen Receptor Variants in Human Breast Cancer

The accumulated evidence supports the expression of estrogen receptor variants at both the mRNA and protein levels. The relative level of expression of some estrogen receptor variant mRNAs and possibly progesterone receptor variant mRNAs is altered during breast tumorigenesis and breast cancer progression. The altered expression of estrogen receptor variants may effect estrogen signal transduction as well as the interpretation of assays where the estimation of estrogen receptor levels is used as a guide to treatment strategies and prognosis.

A point mutation in the human estrogen receptor gene is associated with the expression of an abnormal estrogen receptor mRNA containing a 69 novel nucleotide insertion

A novel ER-like mRNA containing a 69 nucleotideinsertion precisely between exon 5 and 6 sequenceswas previously identified in human breast cancer biopsysamples. Data are presented which suggest that the69 nucleotide sequence is normally present in intron5 of the human estrogen receptor gene. Theregion corresponding to and surrounding this 69 nucleotidesequence was cloned and the nucleotide sequence determined.Cloning and sequencing of the corresponding region ingenomic DNA isolated from a breast tumor expressingthe 69 nucleotide inserted ER mRNA, revealed anA→G point mutation immediately 3′ to the69 nucleotide sequence. This point mutation resulted inthe generation of a consensus splice donor site.A consensus splice acceptor site sequence is normallypresent immediately 5′ to the 69 nucleotide sequence.These data are consistent with the 69 nucleotidesequence being recognized as an exon by thesplicing machinery, and resulting in processing of amature ER mRNA containing the 69 nucleotide insert.

Estrogen regulation of nuclear matrix-intermediate filament proteins in human breast cancer cells

The tissue matrix consists of linkages and interactions of the nuclear matrix, cytoskeleton, and extracellular matrix. This system is a dynamic structural component of the cell that organizes and processes structural and functional information to maintain and coordinate cell function and gene expression. We have studied estrogen regulation of nuclear matrix associated proteins, including the intimately connected cytoskeletal intermediate filaments, in T‐47D5 human breast cancer cells. Three proteins (identified as cytokeratins 8, 18, and 19) present in the nuclear matrix‐intermediate filament fraction (NM‐IF) of cells grown in estrogen‐replete conditions were dramatically reduced when the cells were grown in acute (1 week) estrogen‐depleted conditions. Replacing estrogen in the medium of acute estrogen‐depleted cells restored expression of these proteins. T‐47D5 cells that are chronically depleted of estrogen (T5‐PRF) are estrogen‐nonresponsive in culture. These cells overexpressed these three proteins, compared to parent cells grown in the presence of estrogen. Treatment of the T5‐PRF cells with estrogen did not lead to further up‐regulation of these proteins. Treating T‐47D5 cells in estrogen‐replete conditions with the antiestrogens 4‐hydroxytamoxifen and ICI 164 384 (100 nM, 3 days) resulted in a significant reduction in these proteins, while no effect was seen in long‐term chronic estrogen‐depleted T‐47D5 cells. In conclusion, we have identified NM‐IF proteins (cytokeratins 8, 18, and 19) in human breast cancer cells that are estrogen regulated and may play a role in estrogen action in human breast cancer cells.

Oestrogen receptor variants and mutations in human breast cancer

Several oestrogen receptor variant and mutated mRNA species have been identified in human breast samples and cell lines. Over-expression and altered expression of some of these mRNAs have been correlated with breast tumourigenesis and progression. The following review focuses on the current knowledge available in the scientific literature with respect to the type and characteristics of oestrogen receptor variants and mutations that have been identified as occurring naturally in human breast tissues and cell lines.

Mechanisms of growth inhibition by antiestrogens and progestins in human breast and endometrial cancer cells.

Marked changes in both growth factor and proto-oncogene expression occur due to treatment of hormonally-responsive human cancers with progestins and antiestrogens. In human endometrial cancer cell lines the antiproliferative effects of progestins and antiestrogens in a particular cell line appear to be associated with similar effects on growth factor and/or proto-oncogene expression. This suggests that although these compounds initially interact with different steroid hormone receptors, the molecular mechanisms of their growth inhibition may be essentially similar. In the case of human breast cancer cell lines, however, the effects of progestins and antiestrogens on gene regulation are often different, suggesting that the molecular mechanisms of progestin and antiestrogen growth inhibition may be essentially dissimilar.

Investigation of the origin of variant, truncated estrogen receptor-like mRNAs identified in some human breast cancer biopsy samples

SummaryVariant, smaller sized, estrogen receptor-like mRNAs have been previously cloned and sequenced from human breast cancer biopsy samples. In this study we have found that one variant estrogen receptor-like mRNA, clone 24, which is expressed in only one tumor, consists of sequences from the normal ER gene which is found on chromosome 6 and sequences unrelated to the ER which are found normally on chromosome 12. A more commonly occurring variant estrogen receptor-like mRNA, clone 4, contains sequences from the normal ER gene and LINE-1 sequences. Similar but not identical LINE-1 sequences have been found in some of the intervening sequences of the normal ER gene. The generation of these transcripts may involve either alternative splicing or trans-splicing mechanisms.