Characterization of Estrogen Receptor Variant mRNAs from Human Breast Cancers
Helmut
Dotzlaw,
Moussa
Alkhalaf,
and Leigh
Department of Biochemistry and Molecular University of Manitoba Winnipeg, Manitoba, Canada R3E OW3
C. Murphy*
Biology
they do not prove conclusively that mutant or variant ERs exist. Furthermore, these data may also be explained by extra-receptor abnormalities of other factors necessary for normal ER function. On the other hand, at least four separate groups of investigators, using different recombinant DNA techniques, have now reported variant forms of ER mRNA molecules in human breast cancer biopsy samples. Garcia et al. (4, 5) have identified point mutations in the A/B domain of the human ER. This mutant ER was subsequently found in normal uterine tissue and is a genetic polymorphism (referred to as the B-variant ER gene) rather than a tumor-specific mutation. Interestingly, this mutation is associated with low levels of estrogen binding, and women who are heterozygous for the B-variant gene have a higher proportion of spontaneous abortion than those who have two normal ER alleles (6). Fuqua et al. (7) have identified mutant ER mRNAs in some human breast cancer biopsies using reverse transcription and amplification by polymerase chain reaction technology. ER mRNAs which are deleted in exon 5 or exon 7 have been reported. Interestingly, the mutant ER mRNA occurs in the presence of the wild-type ER transcript, although in some cases the mutant is of similar or greater abundance than the wild type. Furthermore, some of these mutant ER mRNAs can be found in normal uterine tissue (8). Grahame et al. (9) have isolated mutant ER cDNA clones from the T-47D,, human breast cancer cell line, which is resistant to antiestrogens. Abnormalities in these mutant ER cDNAs include frame-shift mutations predicting an ER protein truncated just beyond the second zinc finger or truncated near the end of exon 5, as well as an in-frame deletion of the nuclear localization signal. In this laboratory, we have identified abnormally sized ER mRNAs by Northern analysis (lo), and most importantly, the data demonstrated the high abundance relative to normal ER mRNA of these transcripts in some breast cancer biopsy samples. Furthermore, differential hybridization using probes to either the 5’- or 3’-translated regions of the normal ER mRNA suggest that the variant-sized transcripts are missing a substantial portion of the E/F coding region which contains the ligand binding domain of the normal ER protein. cDNA probes representing various regions of the 3’-untrans-
The cDNAs for variant estrogen receptor (ER) mRNAs previously identified in human breast cancer biopsy samples have been cloned and characterized. Some of these cDNAs are unique to a tumor sample (e.g. clones 24 and 5), while others are present in multiple breast tumor samples (e.g. clone 4). The 5’ ends of the variant cDNAs are essentially identical to sequences present in exons 1, 2, and 3 of the normal ER mRNA. However, at points which mark either the exon S/intron or exon B/intron boundaries, the variant cDNA sequences diverge and are unrelated to the normal ER mRNA. The unique sequences of clones 24 and 5 are unknown, and the unique sequence of clone 4 is related to the long interspersed repetitive LINE-1 sequences. The variant mRNAs contain open reading frames which could encode proteins containing known functional domains of the normal ER but missing others. In particular, the hormone binding domain of the normal ER is always missing. Furthermore, some of the variant transcripts may encode other unique proteins. In transient expression assays the proteins encoded by the variant ER mRNAs are unable to activate transcription of an estrogen-responsive reporter gene; neither are they able to modulate the ability of normal ER proteins to activate transcription. (Molecular Endocrinology 6: 773-785, 1992)
INTRODUCTION
The presence of abnormal estrogen receptor (ER) proteins in some human breast tumors has been suggested previously from subcellular distribution and nuclear translocation experiments (1, 2). Moreover, studies using immunohistochemical staining techniques have added support to the presence of ER proteins which display functional abnormalities such as inability to bind to the nucleus when charged with ligand, or receptor proteins which interact with the nucleus in the absence of the ligand (3). Although these data are suggestive, 0s88-&309/92/0773-0785$03.00/0 Molecular Endomology Copynght 0 1992 by The Endcmne
Soctety
773 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 21 November 2015. at 02:18 For personal use only. No other uses without permission. . All rights reserved.
Vol6 No. 5
MOL ENDO. 1992 774
lated domain of the normal ER mRNA also failed to detect the variant-sized transcripts (Murphy, L. C., and H. Dotzlaw, unpublisheddata). This suggestedthat the smaller sized transcripts were also missing the 3’untranslated region of the normal ER mRNA. Other groups have not identified abnormal ER mRNAs by Northern analysis, and our preliminary data suggest that the variant ER mRNAs which we have identified are quite different to those described by other groups. More importantly, the demonstration of these abnormally sized ER transcripts by Northern analysis suggested their presencein high abundance.Therefore, in order to fully understandthe nature of these variant ER mRNAs we have cloned and characterized the correspondingcDNAs.
RESULTS Isolation of Variant ER cDNA Primaryscreeningof the cDNA library, prepared from a human breast cancer biopsy which showed a high abundance of abnormally sized ER-like mRNA (lo), usedthe OR8-J fragment (Fig. 1). Twenty-nine positive clones were isolated and plaque purified. Duplicate nitrocellulosefilter lifts were taken of each clone. One of these filters was hybridized again with the OR8-J fragment, and the other was hybridized with the more 3’ cDNA probe OR8-0 fragment (Fig. 1). Two patterns of hybridization were found. The first showed both OR-8/J
probes hybridized equally well and was consistent with these clonesbeing normal ER cDNA. The second identified clones in which the OR8-J but not the OR8-0 cDNA fragment hybridized and were good candidates for variant ER cDNAs. Twenty-five out of the 29 exhibited the variant pattern. Interestingly, in a library prepared from the human breast cancer cell line, T-47D-5 (11) RNA, when subjectedto similarscreening,revealed that two clonesout of eight were of the variant pattern. The lower proportion of variant ER cDNA clones in the T-47D-5 library is consistent with there being little or no variant ER mRNA detectable by Northern blotting in this cell line (12). All cloneswere in pBluescript SK, and approximately 200 nucleotides were initially sequenced from the 5’ and the 3’ ends using TI and T, primers. Only clones were chosen which were of sufficient length [r2 kilobasepairs(kbp)] to ensure that they might represent closeto full-length cDNAs. This preliminarysequencing revealed that all the cloneswere polyadenylateddownstreamfrom an authentic polyadenylationsignal.So by this criterion at least, the variant cDNAs represented authentic mRNA molecules.The 5’ ends of every clone were found to have identical sequencesto that of the 5’ end of the normal ER mRNA, while the 3’ ends, determinedby the presenceof a poly(A) tail, were found to have sequencestotally unrelated to the normal ER sequence. Interestingly, there were multiple variant mRNAs, sincethe 3’ ends of someof the clones were not only different from that of the normal ER but were also different from those of other variant cDNAs.
OR-S/O
6323 bp
Riboombe
1
Clone 5 mRNA
Fig. 1. Schematic Comparison of the Normal Human ER cDNA Nucleotide Sequence with That of the Variant Clones 24 and 5 The human ER mRNA consists of eight exons as shown. The initiator (AUG) and the termination (UGA) codons are shown, as are the functional domains (A-F, 13) of the coding region. A convenient HindIll restriction site (shown by the vertical black bar) in the 5’ end of the E domain was used to divide the OR-8 cDNA into the two fragments J and 0 (10). These cDNA probes were used to differentially screen the human breast cancer biospy cDNA libraries. Sequence identity between the normal human ER and the variant clones is shown in the open boxes. Sequence identity between clone 24 and clone 5 is shown by the shaded boxes. The unique sequence of clone 24 is shown by the checked box, and the unique sequence of clone 5 is shown by the cross-hatched box. The open reading frames of both the human ER and clone 24 are shown by the positions of the initiator codon (ATG) and the termination codons (TGA and TAA, respectively). Also shown are: riboprobe 1, used in the RNAase protection assay shown in Fig. 3A; probe 2, used in the Northern and Southern blot analysis of clone 24; and probe 3, used in the Northern and Southern blot analysis of clone 5.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 21 November 2015. at 02:18 For personal use only. No other uses without permission. . All rights reserved.
Variant
ERs in Human
Breast
775
Cancer
Nucleotide
”
AGCCT *.**.
Lys ‘-G, MG
1'
TCTGC **,*.
CCTGC *****
GGGGA *****
CRCGG ***.*
TCTGC *****
ACCCT ***t,
GCCCC *,***
Ile 1le *rq Girl Girl Phe Pro Girl lx” Thr RTC ATA AGG CAG CAG T T T CCT CAG TTR *cc
GGG *cc
CGA GGR CAG TCA Ax
Ttr *cc
CGGCC *****
.ACIx:‘\ **.**
*rg *rg Ix” PG.3 AGR CTC
TMGC ACCGG ACGGC CRTTC CACCT
.'I, GCRGR GCGCA CATCT ATGGG CCTCG AGACR GGCCR cnccc
TTGGC T C T T T
.-'+ ATCCT GAGCT CTGTC TGCAG GCCTG GCAGA AGTCT GTGCC TAGAG GGMT :, . ATGGA AGAGC T T T R T GRCGG CCAGG GCCCC TCTCT CAGGA CTCCT GmG .‘-’
AGAGG T T G T C cws.v,
GCCCA AGACG AGTTG GCTCT GCTGC TTTX,
GGAAT
.G ^ CTGAT T T T G T ACCAC CCTGC T T T T A GGCAT R T T T T GTAX4 ATAGT CTTGG :-'
GCATC ATTGA MGGA
TTGCC T T G T G GCCTC TTGGA GGATC ACCRG G T T A T
' i CTGG,, C T G T T T T G C T GRGCG RWiCT CTGCT CTGXT RGTAT GCAGT AGACC .-
Helmut
Dotzlaw,
Moussa
Alkhalaf,
and Leigh
Department of Biochemistry and Molecular University of Manitoba Winnipeg, Manitoba, Canada R3E OW3
C. Murphy*
Biology
they do not prove conclusively that mutant or variant ERs exist. Furthermore, these data may also be explained by extra-receptor abnormalities of other factors necessary for normal ER function. On the other hand, at least four separate groups of investigators, using different recombinant DNA techniques, have now reported variant forms of ER mRNA molecules in human breast cancer biopsy samples. Garcia et al. (4, 5) have identified point mutations in the A/B domain of the human ER. This mutant ER was subsequently found in normal uterine tissue and is a genetic polymorphism (referred to as the B-variant ER gene) rather than a tumor-specific mutation. Interestingly, this mutation is associated with low levels of estrogen binding, and women who are heterozygous for the B-variant gene have a higher proportion of spontaneous abortion than those who have two normal ER alleles (6). Fuqua et al. (7) have identified mutant ER mRNAs in some human breast cancer biopsies using reverse transcription and amplification by polymerase chain reaction technology. ER mRNAs which are deleted in exon 5 or exon 7 have been reported. Interestingly, the mutant ER mRNA occurs in the presence of the wild-type ER transcript, although in some cases the mutant is of similar or greater abundance than the wild type. Furthermore, some of these mutant ER mRNAs can be found in normal uterine tissue (8). Grahame et al. (9) have isolated mutant ER cDNA clones from the T-47D,, human breast cancer cell line, which is resistant to antiestrogens. Abnormalities in these mutant ER cDNAs include frame-shift mutations predicting an ER protein truncated just beyond the second zinc finger or truncated near the end of exon 5, as well as an in-frame deletion of the nuclear localization signal. In this laboratory, we have identified abnormally sized ER mRNAs by Northern analysis (lo), and most importantly, the data demonstrated the high abundance relative to normal ER mRNA of these transcripts in some breast cancer biopsy samples. Furthermore, differential hybridization using probes to either the 5’- or 3’-translated regions of the normal ER mRNA suggest that the variant-sized transcripts are missing a substantial portion of the E/F coding region which contains the ligand binding domain of the normal ER protein. cDNA probes representing various regions of the 3’-untrans-
The cDNAs for variant estrogen receptor (ER) mRNAs previously identified in human breast cancer biopsy samples have been cloned and characterized. Some of these cDNAs are unique to a tumor sample (e.g. clones 24 and 5), while others are present in multiple breast tumor samples (e.g. clone 4). The 5’ ends of the variant cDNAs are essentially identical to sequences present in exons 1, 2, and 3 of the normal ER mRNA. However, at points which mark either the exon S/intron or exon B/intron boundaries, the variant cDNA sequences diverge and are unrelated to the normal ER mRNA. The unique sequences of clones 24 and 5 are unknown, and the unique sequence of clone 4 is related to the long interspersed repetitive LINE-1 sequences. The variant mRNAs contain open reading frames which could encode proteins containing known functional domains of the normal ER but missing others. In particular, the hormone binding domain of the normal ER is always missing. Furthermore, some of the variant transcripts may encode other unique proteins. In transient expression assays the proteins encoded by the variant ER mRNAs are unable to activate transcription of an estrogen-responsive reporter gene; neither are they able to modulate the ability of normal ER proteins to activate transcription. (Molecular Endocrinology 6: 773-785, 1992)
INTRODUCTION
The presence of abnormal estrogen receptor (ER) proteins in some human breast tumors has been suggested previously from subcellular distribution and nuclear translocation experiments (1, 2). Moreover, studies using immunohistochemical staining techniques have added support to the presence of ER proteins which display functional abnormalities such as inability to bind to the nucleus when charged with ligand, or receptor proteins which interact with the nucleus in the absence of the ligand (3). Although these data are suggestive, 0s88-&309/92/0773-0785$03.00/0 Molecular Endomology Copynght 0 1992 by The Endcmne
Soctety
773 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 21 November 2015. at 02:18 For personal use only. No other uses without permission. . All rights reserved.
Vol6 No. 5
MOL ENDO. 1992 774
lated domain of the normal ER mRNA also failed to detect the variant-sized transcripts (Murphy, L. C., and H. Dotzlaw, unpublisheddata). This suggestedthat the smaller sized transcripts were also missing the 3’untranslated region of the normal ER mRNA. Other groups have not identified abnormal ER mRNAs by Northern analysis, and our preliminary data suggest that the variant ER mRNAs which we have identified are quite different to those described by other groups. More importantly, the demonstration of these abnormally sized ER transcripts by Northern analysis suggested their presencein high abundance.Therefore, in order to fully understandthe nature of these variant ER mRNAs we have cloned and characterized the correspondingcDNAs.
RESULTS Isolation of Variant ER cDNA Primaryscreeningof the cDNA library, prepared from a human breast cancer biopsy which showed a high abundance of abnormally sized ER-like mRNA (lo), usedthe OR8-J fragment (Fig. 1). Twenty-nine positive clones were isolated and plaque purified. Duplicate nitrocellulosefilter lifts were taken of each clone. One of these filters was hybridized again with the OR8-J fragment, and the other was hybridized with the more 3’ cDNA probe OR8-0 fragment (Fig. 1). Two patterns of hybridization were found. The first showed both OR-8/J
probes hybridized equally well and was consistent with these clonesbeing normal ER cDNA. The second identified clones in which the OR8-J but not the OR8-0 cDNA fragment hybridized and were good candidates for variant ER cDNAs. Twenty-five out of the 29 exhibited the variant pattern. Interestingly, in a library prepared from the human breast cancer cell line, T-47D-5 (11) RNA, when subjectedto similarscreening,revealed that two clonesout of eight were of the variant pattern. The lower proportion of variant ER cDNA clones in the T-47D-5 library is consistent with there being little or no variant ER mRNA detectable by Northern blotting in this cell line (12). All cloneswere in pBluescript SK, and approximately 200 nucleotides were initially sequenced from the 5’ and the 3’ ends using TI and T, primers. Only clones were chosen which were of sufficient length [r2 kilobasepairs(kbp)] to ensure that they might represent closeto full-length cDNAs. This preliminarysequencing revealed that all the cloneswere polyadenylateddownstreamfrom an authentic polyadenylationsignal.So by this criterion at least, the variant cDNAs represented authentic mRNA molecules.The 5’ ends of every clone were found to have identical sequencesto that of the 5’ end of the normal ER mRNA, while the 3’ ends, determinedby the presenceof a poly(A) tail, were found to have sequencestotally unrelated to the normal ER sequence. Interestingly, there were multiple variant mRNAs, sincethe 3’ ends of someof the clones were not only different from that of the normal ER but were also different from those of other variant cDNAs.
OR-S/O
6323 bp
Riboombe
1
Clone 5 mRNA
Fig. 1. Schematic Comparison of the Normal Human ER cDNA Nucleotide Sequence with That of the Variant Clones 24 and 5 The human ER mRNA consists of eight exons as shown. The initiator (AUG) and the termination (UGA) codons are shown, as are the functional domains (A-F, 13) of the coding region. A convenient HindIll restriction site (shown by the vertical black bar) in the 5’ end of the E domain was used to divide the OR-8 cDNA into the two fragments J and 0 (10). These cDNA probes were used to differentially screen the human breast cancer biospy cDNA libraries. Sequence identity between the normal human ER and the variant clones is shown in the open boxes. Sequence identity between clone 24 and clone 5 is shown by the shaded boxes. The unique sequence of clone 24 is shown by the checked box, and the unique sequence of clone 5 is shown by the cross-hatched box. The open reading frames of both the human ER and clone 24 are shown by the positions of the initiator codon (ATG) and the termination codons (TGA and TAA, respectively). Also shown are: riboprobe 1, used in the RNAase protection assay shown in Fig. 3A; probe 2, used in the Northern and Southern blot analysis of clone 24; and probe 3, used in the Northern and Southern blot analysis of clone 5.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 21 November 2015. at 02:18 For personal use only. No other uses without permission. . All rights reserved.
Variant
ERs in Human
Breast
775
Cancer
Nucleotide
”
AGCCT *.**.
Lys ‘-G, MG
1'
TCTGC **,*.
CCTGC *****
GGGGA *****
CRCGG ***.*
TCTGC *****
ACCCT ***t,
GCCCC *,***
Ile 1le *rq Girl Girl Phe Pro Girl lx” Thr RTC ATA AGG CAG CAG T T T CCT CAG TTR *cc
GGG *cc
CGA GGR CAG TCA Ax
Ttr *cc
CGGCC *****
.ACIx:‘\ **.**
*rg *rg Ix” PG.3 AGR CTC
TMGC ACCGG ACGGC CRTTC CACCT
.'I, GCRGR GCGCA CATCT ATGGG CCTCG AGACR GGCCR cnccc
TTGGC T C T T T
.-'+ ATCCT GAGCT CTGTC TGCAG GCCTG GCAGA AGTCT GTGCC TAGAG GGMT :, . ATGGA AGAGC T T T R T GRCGG CCAGG GCCCC TCTCT CAGGA CTCCT GmG .‘-’
AGAGG T T G T C cws.v,
GCCCA AGACG AGTTG GCTCT GCTGC TTTX,
GGAAT
.G ^ CTGAT T T T G T ACCAC CCTGC T T T T A GGCAT R T T T T GTAX4 ATAGT CTTGG :-'
GCATC ATTGA MGGA
TTGCC T T G T G GCCTC TTGGA GGATC ACCRG G T T A T
' i CTGG,, C T G T T T T G C T GRGCG RWiCT CTGCT CTGXT RGTAT GCAGT AGACC .-
