Mouse mammary tumor‐like env gene as a molecular marker for breast cancer?

The involvement of the mouse mammary tumor virus (MMTV) in breast cancer has long been postulated but never proven. Two studies have detected the sequence of the MMTV env gene in more than one-third of breast tumor tissues.1, 2 The present study was designed to investigate whether this viral sequence could serve as a molecular marker for the detection of breast cancer. We amplified the viral sequence in 2 cell lines and 18 breast tumor samples, using PCR, PCR Southern hybridization and real-time PCR. Surprisingly, we could not detect the viral sequence in any of these samples despite amplification in positive controls. Our study calls into question the role of MMTV in human breast cancer tumorigenesis. The involvement of retroviruses in human cancer was established long ago, e.g., HPV in cervical and oropharyngeal tumors.3, 4 The participation of a virus similar to MMTV in human breast cancer has long been postulated but never demonstrated. Several lines of evidence suggested the involvement of MMTV-like retroviruses in human breast cancer. Previously, it was shown that MMTV is an agent associated with a high incidence of breast cancer in mice.5 Detectable MMTV env gene–related antigenic reactivity was found in tissue sections of breast cancer,6, 7, 8, 9, 10 breast cancer cell lines,11 serum,12, 13 pleural effusion14 and lymph nodes.15 Moreover, sequence homology to MMTV has been found in human DNA.1, 2, 16, 17, 18, 19, 20, 21 Two studies detected the env gene of MMTV in 37–38.5% of breast tumor tissues but only in 0–2% of normal tissues.1, 2 According to these studies, this viral sequence was highly specific to breast tumor tissues and was not detected in other tumor tissues. The present study was designed to examine whether the env gene of MMTV could be used as a molecular marker for the detection of breast cancer. We planned to evaluate, by real-time PCR, a panel of normal tissues, tumor breast tissues and blood samples from 40 breast cancer patients. MMTV env gene plasmid and MCF-7 and MB-MDA-231 cell lines were used as a positive control, as described in previous studies.1, 2, 11, 17 The viral sequence was first amplified by regular PCR; then, PCR products were hybridized with an internal probe by PCR Southern hybridization; finally, real-time PCR was performed. We were unable to detect the viral sequence in the cell lines or in the breast tumor tissues despite robust amplification from the plasmid control. The MCF-7 and MB-MDA-231 cell lines were purchased from the ATCC (Manassas, VA) and grown in RPMI medium with FBS 10%, penicillin and streptomycin 1%. Eighteen invasive ductal carcinomas of the breast from 17 patients were collected at the Department of Pathology, University Campus BioMedico (Rome, Italy). Tumor specimens were frozen and microdissected on a cryostat so that they contained at least 70% neoplastic cells. DNA from tumor sections and cell lines was digested with 1% SDS/proteinase K, extracted by phenol-chloroform and ethanol-precipitated. MMTV env plasmid was purchased from the ATCC (item number 45006). Seven sets of primers were used to amplify the MMTV sequence and 1 set to amplify the β-globin gene. The first 2 primers were designed as previously described;1 the other primers and probes were designed using Primer Express (Applied Biosystems, Foster City, CA) and Primer 3 (Invitrogen, Carlsbad, CA). Approximately 200 ng of DNA in 1 × PCR buffer [16.6 mM ammonium sulfate, 67 mM TRIS (pH 8.8), 6.7 mM MgCl, 10 mM β-mercaptoethanol, 1% DMSO and 1.25 mM dNTPs] were amplified using 0.5 μl (300 ng/μl) of each primer, 2 units of Taq DNA polymerase (New England Biolabs, Beverly, MA) and 1.5 μl of dNTP in total volume of 25 μl, as previously described.22 PCR conditions were as follows: 1 cycle at 95°C for 1 min; 35 cycles at 95°C for 0.5 min, 60°C for 1 min and 72°C for 1 min; followed by a final 5 min extension at 72°C. Seven sets of primers were designed to recognize the mmtv env gene sequence (primer sets 1–7). Primer set 1 (5L, CCA GAT CGC CTT TAA GAA GG, and 3L, TAC AGG TAG CAG CAC GTA TG) amplified a 660 bp sequence; primer set 2 (2N, CCT ACA TCT GCC TGT GTT AC, and 3, ATC TGT GGC ATA CCT) amplified a 250 bp sequence; primer set 3 (env F1, AGA CGA GTC TGC TCC TCC AC and env R TGG CAG ATT CAT CAA ACC AT) amplified a 596 bp sequence; primer set 4 (env F2, AGG CCA GGA TTT CAA GAA CA, and env R, TGG CAG ATT CAT CAA ACC AT) amplified a 259 bp sequence; primer set 5 (TAQ1 F, GCC TAC GAC TAT GCA GCG ATC, and TAQ1 R, CCA TGG TTC ATC ACC AAT ATC TACA) amplified a 78 bp sequence; primer set 6 (TAQ2 F, CTG CCA GAT CGC CTT TAA GAA, and TAQ2 R, GGT CAG GGA AGG CGC AA) amplified an 86 bp sequence; primer set 7 (TAQ3 F, TTC ACT GCA CTA GTC CCC CAT AC, and TAQ3 R, GAT ATG CCC AGG ATA ATG GCT G) amplified a 401 bp sequence. One primer set was designed to amplify the β-globin sequence, primer set 8 (β-globin F, 5′-TAAGGTGAAGGCTCATGGCAA-3′, and β-globin R, 5′-GCAGCTCACTCAGTGTGGCA-3′). Eighteen breast cancer samples were amplified by fluorescence-based real-time PCR using a 384-well reaction plate format in an ABI 7900 Sequence Detector (Applied Biosystems). Multiple primers for MMTV env were used as in the conventional PCR. In addition, primers were designed for the β-globin gene as an internal reference for input DNA (Invitrogen). Dual-labeled fluorogenic probes were designed for the different primer sets (Applied Biosystems). Fluorogenic PCRs were set up in a reaction volume of 20 μl. The final PCR reaction mixture consisted of 600 nM of each primer; 200 nM of probe; 200 μM each of dATP, dCTP, dGTP and dTTP; 16.6 mM ammonium sulfate; 67 mM Trizma; 6.7 mM MgCl; 10 mM mercaptoethanol; 0.1% DMSO; and 3 μl of template DNA. PCRs were performed using the following conditions: 95°C for 2 min, followed by 50 cycles at 95°C for 15 sec and 60°C for 1 min. Three dual-labeled fluorogenic probes were designed for the different primer sets: (i) β-globin probe 5′-FAM-TTTAGTGATGGCCTGGCTCACCTGGAC-TAMRA-3′, (ii) MMTV probe 1 5′-FAM-ACGAGTCTGCTCCTCCACGGTGGTT-TAMRA-3′ for primer sets 1 and 6 and (iii) MMTV probe 2 5′-FAM-CAAGAGGCCGCCATATGTGCTGCTA-TAMRA-3′ for primer sets 4, 5, and 7. All samples were run in duplicate, and each PCR plate included serial dilutions of the plasmid and MCF-7 cell line DNA as positive controls, for construction of standard curves as well as multiple water-blanks. Further duplicates were run for any equivocal sample results. To verify the specificity of the bands, we separated PCR products on 0.9 % agarose gel. The gel was washed with 0.25N HCl for 10 min, then with denaturing buffer (0.5N NaOH, 1.5N NaCl) for 1 hr and transferred to a Zeta-Probe GT Genomic blotting membrane (Bio-Rad, Hercules, CA) for 24 hr. Using T4 kinase (Life Technologies, Gaithersburg, MD), 100 ng of the internal probe were end-labeled with [32P]-ATP (20 mCi; Amersham, Piscataway, NJ) in a total volume of 20 μl. The labeled probe was then hybridized with the membrane in Rapid Hybridization Buffer (Stratagene, La Jolla, CA) at 50°C for 2 hr, then washed in room temperature and at 58°C and exposed to film (Eastman-Kodak, Rochester, NY). Seven sets of primers and 2 probes were designed to recognize different parts of the env gene (2 of the primers were used in previous studies and considered optimized for viral sequence amplification). First, the 2 positive control cell lines were subjected to PCR for the env gene. Identification of the viral sequence in these cell lines was compared to that in the plasmid. The only bands detected were from plasmid DNA in which 6 of the 7 primer sets produced bands of the expected size (Fig. 1). One of the primer sets produced a detectable band in the cell line DNA (Fig. 1a, lane 5) but was not the exact size expected (about 300 bp instead of 259 bp). We therefore performed PCR followed by Southern hybridization using TAQ1 as an internal probe. The probe clearly hybridized to the plasmid PCR product but not to the PCR product from the MCF-7 cell line (Fig. 2). PCR amplification of the MCF-7 cell line and the mmtv env plasmid. PCR was performed using 7 different sets of primers. (a) PCR products were separated on a 1.5% polyacrylamide gel. Lanes 1–3, (primer set 2), 3. Lanes 4–6, (primer set 4). Lanes 7–9 (primer set 5). Lanes 10–12, (primer set 6). (b) PCR products were separated on a 1% polyacrylamide gel. Lanes 1–3, (primer set 1). Lanes 4–6, (primer set 3). Lanes 7–9, (primer set 7). M, m.w. marker; W, water; D, cell line DNA; P, plasmid DNA. Expected sizes of PCR products are listed in the Material & Methods section. PCR Southern hybridization of the MCF-7 cell line and the mmtv env plasmid. PCR products from the MCF-7 cell line and the mmtv env plasmid were separated on a 0.9% agarose gel, transferred to a Zeta-Probe GT Genomic blotting membrane and then hybridized to the [32P]-ATP end-labeled TAQ1 F primer, which was used as an internal probe. Lanes 1–3, 660 bp PCR product (5L, 3L). Lanes 4–6, 410 bp PCR product (TAQ3 F, R). Lanes 7–9, 259 bp PCR products (MMTV env F2, R). W, water; D, DNA; P, plasmid. We than amplified 18 breast tumor tissues and the 2 cell lines by real-time PCR. Plasmid DNA was again used as a positive control. As for conventional PCR, plasmid DNA produced a robust amplification with all primer sets, while the tumor samples and cell line DNA yielded no amplification with any of the MMTV primer sets (Fig. 3). β-Globin was robustly amplified from the latter samples (except plasmid DNA), indicating the presence of sufficient DNA for amplification. Real-time PCR analysis by Taqman assay. (a) Serial 10-fold dilutions of plasmid containing the MMTV env gene. From 107 copies to 0.01 copy. (b) Duplicate β-globin curves for 18 breast tumor samples. All breast samples and the MCF-7 cell line were negative for the MMTV env gene (not shown). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] We thus used several different tools, PCR, PCR hybridization and real-time PCR, to detect the env sequence from MMTV in breast cancer cell lines and primary breast cancer. Despite the sensitivity of these tools (especially real-time PCR), we did not detect viral sequences in the cell lines or breast tumor samples. The difference between the present data and previous results1, 2, 20 might be explained by recognition of nonspecific sequences after conventional and nested PCR. In the human genome, there are many incorporated endogenous retroviral sequences, e.g., HERV-K, which are related to MMTV. Under low-temperature conditions, such closely related sequences might be recognized and amplified nonspecifically by PCR. We thus failed to detect MMTV viral sequences for use as a molecular marker for breast cancer. HPV DNA in oral cancer and EBV DNA in nasopharyngeal tumors can be used to detect and monitor patients with cancer.23, 24 The identification of new viral sequences in breast cancers could provide the basis for a similar approach in breast cancer patients. Yours sincerely, Rachel ZANGEN, Susan HARDEN, Daniel COHEN, Paola PARRELLA and David SIDRANSKY Rachel Zangen*, Susan Harden*, Daniel Cohen*, Paola Parrella*, David Sidransky*, * Department of Otolaryngology–Head and Neck Surgery, Head and Neck Cancer Research Division, Johns Hopkins University School of Medicine, Baltimore, MD, USA.