Novel alternatively spliced variant with a deletion of 52 BP in exon 6 of the progesterone receptor gene is observed frequently in breast cancer tissues†
Article first published online: 5 MAR 2003
Copyright © 2003 Wiley-Liss, Inc.
International Journal of Cancer
Volume 105, Issue 2, pages 182–185, 10 June 2003
How to Cite
Hisatomi, H., Kohno, N., Wakita, K., Nagao, K., Hirata, H., Hikiji, K. and Harada, S. (2003), Novel alternatively spliced variant with a deletion of 52 BP in exon 6 of the progesterone receptor gene is observed frequently in breast cancer tissues. Int. J. Cancer, 105: 182–185. doi: 10.1002/ijc.11050
The sequences reported in this study have been deposited in the GenBank database (accession numbers AB085843, AB085844 andAB085845).
- Issue published online: 31 MAR 2003
- Article first published online: 5 MAR 2003
- Manuscript Accepted: 4 JAN 2003
- Manuscript Revised: 4 NOV 2002
- Manuscript Received: 21 AUG 2002
- progesterone receptor;
- alternative splicing;
- breast cancer
The human progesterone receptor (PR) is a ligand-activated nuclear transcription factor that mediates progesterone action in target tissues. We found a novel alternatively spliced variant (ASV) of the PR mRNA in breast cancer tissues. The deleted transcript was characterized by an out-of-frame deletion of 52 bp in exon 6 (PR delta6/2 ASV). The PR delta6/2 ASV mRNA results in a partial defect in the region of the ligand-binding domain of the hormone receptor, where conserved residues are missing from the core of the protein. To clarify the clinical significance of the PR delta6/2 ASV, we investigated the expression of this ASV in noncancerous and cancerous tissues from patients with breast cancer using RT-PCR. The novel PR delta6/2 mRNA was detected in 24 of 39 (61.5%) cancerous tissues and in 3 of 39 (7.7%) noncancerous tissues from patients with breast cancer. PR delta6/2 ASV mRNA was expressed more frequently in breast cancer tissues than in noncancerous tissues (p < 0.0001), which suggests a possible relationship between the expression of PR delta6/2 and breast cancer. Our observations may provide a novel strategy for the genetic diagnosis of breast cancer. © 2003 Wiley-Liss, Inc.
The progesterone receptor (PR) serves as a marker for hormone dependence and prognosis in patients with breast cancer, because functional PRs are required to mediate the antiproliferative effects of progestin therapies.1, 2, 3 To date, several alternatively spliced variants (ASVs) of the PR transcript have been reported.4, 5, 6 Alternative splicing is a common gene control mechanism, and up to a third of human genes are spliced alternatively.7, 8 Several ASVs have been associated with human diseases.7, 8, 9, 10, 11 Richer et al.4 identified 4 ASVs of the PR gene: PR delta2 lacking exon 2, PR delta4 lacking exon 4, PR delta6 lacking exon 6 and PR delta5+6 lacking both exons 5 and 6. Furthermore PR delta4/2 lacking 126 base pairs (bp) in exon 4, PR delta3 lacking exon 3 and PR delta2+3 lacking both exons 2 and 3 were identified.5, 12 We subsequently identified a transcript lacking both exons 4 and 6 (PR delta4+6).6 The proteins encoded by PR delta2, delta2+3, delta6, and delta4+6 were not accompanied by a frame shift mutation compared to the full length isoform, and neither were the proteins encoded by PR delta3, delta4, delta4/2 and delta5+6.
We have identified a novel ASV in which a sequence of 52 bp is deleted from exon 6 of the PR gene (PR delta6/2). The expression of this PR ASV in cancerous and noncancerous tissues is unknown. We investigated the occurrence of the 52-bp deletion in exon 6 of the PR gene in cancerous and noncancerous tissues from breast cancer patients.
MATERIAL AND METHODS
Both cancerous and noncancerous tissues were obtained during surgical resection of breast tissues from 39 unrelated Japanese patients with breast cancer (female, 53.5 ± 13 yr of age) (Table I). Noncancerous tissue at a distance of over 5 cm from the edge of the breast cancer nodule was obtained from each patient. Samples were frozen in liquid nitrogen immediately after surgical resection and stored at −80°C until analysis. Written informed consent was obtained from each patient. Total RNA was extracted from 10 mg tissues and the MCF7 cell line (a breast cancer derived cell line) using the RNAzol B reagent (Sawady, Shinjuku, Japan) according to the manufacturer's instructions. The final RNA preparation was resuspended in diethylpyrocarbonate-treated water and quantified by absorbance analysis at 260 nm. Complementary DNA (cDNA) was prepared by incubating DNase-treated total RNA (1.0 μg) with M-MLV transcriptase (Invitrogen, Carlsbad, CA) in the presence of random primers.
|Number||Age (yr)||Grade||Node metastasis|
The primer set for the amplification of PR mRNA was designed according to GenBank NM_000926, using a forward primer in the exon 5 region: 5′-TGA CCA GAT AAC TCT CAT TCA GTA TTC TTG G-3′, and a reverse primer in the exon 7 region: 5′-TTT GAC TTC GTA GCC CTT CCA AAG-3′. The reaction parameters were 95°C for 30 sec, 60°C for 40 sec, and 72°C for 30 sec for 45 cycles, followed by a 10 min extension at 72°C using AmpliTaq Gold DNA polymerase (Perkin-Elmer Applied Biosystems, Foster City, CA). The PCR products were confirmed by 3.0% agarose gel electrophoresis in TBE buffer (Fig. 1a). The PCR products of PR were purified using a High Pure PCR Product Purification Kit (Roche Molecular Biochemical Diagnostics, Indianapolis, IN), cloned into a pCR2.1 vector (Invitrogen) and then sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction Kit with an ABI PRISM 3100 Genetic Analyzer (Perkin-Elmer Applied Biosystems). Finally, the sequences were compared to the full-length PR mRNA sequence (Fig. 1b).
Estimation of the PR delta6/2 transcript
The primers to amplify the mRNA of the full-length isoform and the novel ASV mRNA lacking 52 bp in exon 6 are shown in Table II. The PCR reaction was carried out for 45 cycles (95°C for 30 sec, 60°C for 40 sec, 72°C for 30 sec) using AmpliTaq Gold DNA polymerase.
|Forward primer||5′-GCA TGG TCC TTG GAG GTC GAA A-3′||Exon 3, bp 2067–2081, and exon 4, bp 2082–2087||487 bp|
|Reverse primer||5′-GAT GAT TCT TTC ATC CGC TGT TCA TTT A-3′||Exon 5, bp 2526–2532, and exon 6, bp 2533–2553|
|PR delta6/2 ASV|
|Forward primer||5′-GCA TGG TCC TTG GAG GTC GAA A-3′||Exon 3, bp 2067–2081, and exon 4, bp 2082–2087||487 bp|
|Reverse primer||5′-CTT GAC AAA CTC CTG TGG GAT TCA TTT A-3′||Exon 5, bp 2526–2532, and exon 6, bp 2585–2605|
The primer set for the amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control was designed according to GenBank M33197 as follows; exon 6: 5′-CTC TGC CCC CTC TGC TGA T-3′ and exon 8: 5′-GAG GGG GCC CTC CGA C-3′ (448 bp). The PCR reaction was carried out for 35 cycles (95°C for 30 sec, 60°C for 40 sec, 72°C for 30 sec).
The PCR products were confirmed by 3.0% agarose gel electrophoresis in TBE buffer. The PCR products of the full-length isoform of the PR gene and the PR delta6/2 ASV were sequenced directly using a BigDye Terminator Cycle Sequencing Ready Reaction Kit with an ABI PRISM 3100 Genetic Analyzer. The sequence was compared to the target mRNA sequence.
The amount of PR protein was measured using an ABBOTT PgR-EIA Monoclonal Kit (DAINABOT, Tokyo, Japan) following the manufacturer's instructions.
The PCR mixture presented 2 bands on electrophoresis. The sizes of the PCR products obtained were 281 bp (full-length isoform) and 229 bp (Fig. 1a). The sequence of the 229-bp product lacked 52 bp from exon 6 (Fig. 1b). Longer cDNA was sequenced based on 5′ and 3′ rapid amplification of cDNA ends (RACE) with long distance PCR. The protein encoded by the PR delta6/2 mRNA was 130 amino acids shorter than the full-length isoform, and the amino acid sequence of this ASV had changed due to a frame shift mutation.
ASV in patients with breast cancer
Each PCR product presented a single band on electrophoresis (Fig. 2). All the sequences of the PCR products were in complete agreement with the full-length isoform of the PR and PR delta6/2 mRNA sequences, respectively. These detection systems had a sensitivity of approximately 200 copies/μg total RNA. The positive control amplification for GAPDH mRNA afforded a single band of 448 bp and its message was positive in all samples.
The full-length isoform of the PR message was detected in both cancerous and noncancerous breast tissues (100%). The PR delta6/2 mRNA was detected at a higher frequency in cancerous tissues than in noncancerous tissues from patients with breast cancer (p < 0.0001) (Table III). In patients with PR delta6/2-positive noncancerous tissues, PR delta6/2 ASV also was present in the cancerous tissues.
|Cancerous||24 (61.5%)||15 (38.5%)|
|Non-cancerous||3 (7.7%)||36 (92.3%)|
In this study with limited numbers of patients, there were no statistical differences between the mRNA of the PR delta6/2 ASV and other clinicopathologic features, such as node-metastasis and the amount of the PR protein.
The novel ASV with a 52-bp deletion in exon 6 results in a partial defect in the region of the ligand-binding domain of the hormone receptors. Furthermore, a frame shift mutation causes nonsense mutations in this ASV, resulting in serious defects in the PR protein. Although the PR delta6/2 ASV will cause protein truncation, the protein of this ASV was not detectable by Western blot analysis (data not shown). The mRNA levels after 45 cycles of PCR were visible on an agarose gel, however, and the expression of PR delta6/2 mRNA was specific for breast cancer. The PR delta6/2 mRNA is predicted to introduce a novel peptide after premature termination. Usually, most mRNAs resulting in protein truncation, including the PR ASV mRNAs, are degraded efficiently by a pathway known as nonsense-mediated mRNA decay (NMD).13
Because the PR delta6/2 mRNA was detected frequently in breast cancer tissues, it is estimated that the splicing or NMD mechanism may be not complete in breast cancer cells. Further improved studies, however, are needed to clarify the mechanisms involved in this phenomenon. Carcinogenesis consists of multiple steps, and carcinoma development is associated with multigene alterations.14, 15, 16, 17, 18, 19 Inactivation or functional disorder of the splicing or NMD mechanism might be an important steps in carcinogenesis and cancer development. In addition, it is important for the diagnosis of breast cancer to know that the PR delta6/2 ASV was detected more frequently in breast cancer tissues than in noncancerous tissues. The detection of PR delta6/2 might be useful for distinguishing non-risk from at-risk cells. This is, to the best of our knowledge, the first report of a possible relationship between the expression of PR delta6/2 and breast cancer.
Balleine et al.5 and Nagao et al.6 described alternatively spliced PR transcripts, including PR delta2, delta3, delta4, delta6, delta4/2, delta2+3, delta5+6 and PR delta4+6 that were unrelated to the pathogenesis, but they missed the detection of the PR delta6/2 ASV, probably due to having used Northern blot analysis for the identification of the ASVs. We identified the novel ASV using a sensitive PCR analysis. We also identified PR delta3+6/2 lacking both exon 3 and 52 bp in exon 6, and PR delta4+6/2 lacking both exon 4 and 52 bp in exon 6. Both ASVs were detected at a low frequency (2–7%) in cancerous tissues, but were not related to the pathogenesis or development of breast cancer.
In summary, the relationship between the PR delta6/2 ASV mRNA and breast cancer could provide a novel strategy for the genetic diagnosis of breast cancer. Further studies, however, with a larger number of samples are required to confirm these results.
We are grateful to Mariko Arimura (SRL Inc., Kobe, Japan) for her kind assistance in this study.