Mouse mammary tumor virus (MMTV)-like DNA sequences in the breast tumors of father, mother, and daughter
© Etkind et al; licensee BioMed Central Ltd. 2008
Received: 01 August 2007
Accepted: 28 February 2008
Published: 28 February 2008
The diagnosis of late onset breast cancer in a father, mother, and daughter living in the same house for decades suggested the possibility of an environmental agent as a common etiological factor. Both molecular and epidemiological data have indicated a possible role for the mouse mammary tumor virus (MMTV), the etiological agent of breast cancer in mice, in a certain percentage of human breast tumors. The aim of this study was to determine if MMTV might be involved in the breast cancer of this cluster of three family members.
MMTV-like envelope (env) and long terminal repeat (LTR) sequences containing the MMTV superantigen gene (sag) were detected in the malignant tissues of all three family members. The amplified env gene sequences were 98.0%–99.6% homologous to the MMTV env sequences found in the GR, C3H, and BR6 mouse strains. The amplified LTR sequences containing sag sequences segregated to specific branches of the MMTV phylogenetic tree and did not form a distinct branch of their own.
The presence of MMTV-like DNA sequences in the malignant tissues of all three family members suggests the possibility of MMTV as an etiological agent. Phylogenetic data suggest that the MMTV-like DNA sequences are mouse and not human derived and that the ultimate reservoir of MMTV is most likely the mouse. Although the route by which these family members came to be infected with MMTV is unknown, the possibility exists that such infection may have resulted from a shared exposure to mice.
Slides of tumor tissue
Figure 1A–C represents the hematoxylin and eosin stained slides from the formalin-fixed paraffin-embedded tissue sample blocks obtained from each of the three family members. Samples blocks from mother and daughter contained malignant tissue from their respective breast tumors. Sample blocks from the father were from a lymph node that contained metastatic breast cancer.
Presence of MMTV-like env sequences in breast cancer
Presence of MMTV-like LTR sequences in breast cancer
DNA from the three afflicted family members was amplified by single round PCR with primers specific for a 630-bp region within the MMTV LTR open reading frame (ORF) that codes for the MMTV superantigen (sag) gene [14, 15] and that is not present in HERV-K sequences [6, 11, 12, 16]. Figure 2C represents the ethidium bromide-stained agarose gel electrophoresis of the MMTV LTR primed PCR of the three family members and Figure 2D represents the hybridized Southern blot . Lanes 2, 3, and 4 in both Figure 2C and 2D represent amplified DNA from the mother, daughter, and father respectively. Lane 1 containing no template DNA and lanes 5, 6, and 7 containing normal breast tissue DNA from three separate individuals represent our negative controls for sample contamination. Positive hybridization with the radiolabeled internal 20-bp oligonucleotide probe that contained MMTV-LTR gene sequences indicated that this MMTV-specific sequence was present in the amplified 630-bp fragment and that the bands in the agarose gel were specific for MMTV LTR sequences.
Cloning and sequencing of amplified MMTV-like env from human breast cancer
As shown in Figure 3 the sequenced env clones fell into 7 classes that differed from one another. However no one env clone differed from any other by more than 4 base changes either within the same family member or between family members. One env clone each from the mother (M1), father (F2), and daughter (D2) were identical to each other. Two env clones from the father (F3, F4) were identical to each other, one daughter env clone (D1) and one father env clone (F1) were the same, and two env clones from the mother (M2, M3) were the same. Each family member however, mother (M1, M4), father (F2, F3, F4), and daughter (D2, D3), each contained env clones that included a single base change mutation denoted by the arrow in Figure 3. Each family member also contained env clones (M2, M3, F1, D1, D4) without this single base change. Previously we had identified an identical single base change in the MMTV env sequences that were present in a number of breast tumors and all non-Hodgkin's lymphomas .
Cloning and sequencing of amplified MMTV-like LTR ORF sequences from human breast cancer
The amplified 630 bp MMTV-like LTR ORF sequences present in the primary and metastatic breast tumor tissue were cloned using the Invitrogen TOPO TA Cloning kit for Sequencing. A total of 12 clones (4 for each family member) were sequenced. The U3 region of the MMTV LTR contains the open reading frame (ORF) that encodes the glycoprotein superantigen (sag) that is present in all exogenous and endogenous MMTV viral sequences [14, 15]. Although highly conserved, the MMTV sag sequences are not identical with approximately 35% of the total variation clustered at the hypervariable COOH terminus. This variation present in the COOH terminus of the MMTV sag gene is specific to each MMTV provirus.
To our knowledge, this is the first report of breast cancer in father, mother, and daughter. We acknowledge however limitations of our study due to the quality of the DNA of the archival formalin fixed paraffin samples on which this study is based. Limitations include our inability to determine the mutation status of the BRCA1 and BRCA2 genes in these patients . However the late age at which these breast tumors developed argues against their being caused by mutations in the BRCA1 or BRCA2 genes. Quantity and quality of the archival DNA also limited us in that we were able to successfully amplify only parts of two regions, the env and LTR, of the MMTV-like DNA genome and were not able to detect the presence of integrated MMTV-like viral sequences in the cellular genome.
Breast cancer in husband and wife may not be as uncommon as generally thought. At least ten couples have been previously reported since 1975 [29–33]. Russ and Scanlon  reporting on eleven married couples having histologically identical neoplasms, including three couples with breast cancer, noted that the clinical course of the disease tended to be similar in both husband and wife, and that husband and wife were diagnosed usually within approximately five years of each other. Both observations apply to our family. Some studies however have not shown an increased incidence of breast cancer in wives of men with that neoplasm [33–35]. However, Russ and Scanlon noted that the tumors they observed in husband and wife have been suggested either experimentally or indirectly to have viral relationships . Lynch et al  suggested more than twenty years ago that a communicable agent might play a role in the clustering of certain cancers in spouses.
The MMTV-like env sequences that we have detected in the mother, father, and daughter of this family are 98–99.6 percent identical to the GR, C3H and BR6 mouse strains of MMTV [17–19]. Recently the Env protein of MMTV from the mouse has been shown to be capable of transforming mouse and human mammary epithelial cells in vitro . The MMTV Env protein contains an immunoreceptor tyrosine-based activation motif (ITAM) sequence that appears to allow for its transformation ability . The MMTV-like env gene sequences that we have isolated from the primary human breast tumor tissue and the metastatic breast tumor tissue present in a lymph node in the three family members studied in this report and in additional breast tumors previously reported and in non-Hodgkin's lymphomas contain this ITAM sequence [1, 6]. It is not yet known if the MMTV-like Env protein coded for by the MMTV-like DNA sequences which we and others have detected in human breast tumors is capable of transformation. We have sequenced 4 env clones per family member for a total of 12 env clones. Seven of these 12 clones contain an identical single base substitution (Figure 3, ↓) that we have detected in a number of additional breast tumors and in all the non-Hodgkin's lymphomas we have analyzed. This base change of a G to A results in the replacement of an alanine (GCA) with a threonine (ACA) in these samples. Curiously, this single-base change occurs within the ITAM domain .
The 630 bp MMTV-like DNA that we have amplified using primers specific for regions of the ORF of the MMTV LTR codes for the superantigen (sag) gene of MMTV [14, 15]. Although highly conserved, the MMTV sag sequences are not identical with approximately 35 percent of the total variation clustered at the hypervariable COOH terminus. This variation present at the COOH terminus of the MMTV sag gene is specific to each MMTV strain. Each of our cloned sag sequences from each family member was identical or nearly identical to either the MMTV Mtv-8 [20, 21] or Mtv-1 [22, 23] proviral sequence. In a previous publication  we have shown that the cloned sag sequences present in both the breast tumor tissue and non-Hodgkin's lymphoma tissue of eight patients diagnosed with both malignancies also contained sag sequences that were identical or nearly identical to MMTV proviral sequences Mtv-1 and Mtv-8. Similar sequences isolated from human breast tumors by others also contained sag sequences with identity to the Mtv-1 and Mtv-8 proviral sequences [16, 25]. Also, MMTV-like sag sequences isolated from patients diagnosed with primary biliary cirrhosis (PBC) also contained sag sequences with identity to the Mtv-1 and Mtv-8 proviral sequences [26, 27]. Phylogenetic studies from our laboratory and that of others  have shown that such sequences segregate to specific branches of the MMTV phylogenetic tree and do not form a distinct branch of their own thus arguing for their being mouse derived and not human homologues of the mouse sequence [6, 27]. This study, our previously published work , and that of others [26, 27] also indicate that more than one viral strain of MMTV-like DNA sequences may be present in the same individual.
Other works that have shown that MMTV-like DNA sequences are for the most part not found in normal cells [1–7, 25] suggest that the presence of the virus in humans may result from an exogenous infection. Very recently, a virus closely related to the xenotropic murine leukemia viruses (MuLVs) has been detected in the tumor tissue of a certain population of prostate cancer patients. The viral sequence is not found in human genomic DNA thus indicating, as the authors discuss, an exogenous infection [38, 39]. The suggestion that MMTV exogenous infection can occur in humans is a highly controversial topic but is becoming more plausible with two recent publications by Indik et al [40, 41]. In these papers the authors show that MMTV can infect human cells in vitro, make new MMTV, and that this new MMTV can go on to infect other human cells. That the MMTV-like DNA sequences that we and others have found in human breast tumors may be mouse derived is also suggested by the findings that their sag sequences segregate to arms of the MMTV phylogenetic tree and do not form a separate branch of their own [6, 26, 27]. Additional epidemiological findings suggesting an exogenous infection from mice include the findings of Stewart et al  in which it was shown that the incidence of breast cancer was highest in countries in which the mouse strain mus domesticus resides, a mouse strain that carries a large number of endogenous MMTV proviruses. Curiously, a recent study from the Johns Hopkins University Schools of Medicine and Public Health has reported that airborne mouse allergen was found in 84 percent of bedrooms of inner city homes in Baltimore and that the concentration of this mouse allergen may be similar to those found in animal facilities . We do not know the concentration of mouse allergen in the home of the family in this study who lived in a wealthy Baltimore neighborhood.
The presence of MMTV-like DNA sequences in the malignant tissues of all three family members who lived in the same house for decades argues for the possibility of MMTV as a common environmental etiological agent. Phylogenetic data suggest that the MMTV-like sequences are mouse and not human derived and that the ultimate reservoir of MMTV is most likely the mouse. Although the route by which these three family members came to be infected with MMTV is unknown, the possibility exists that such infection may have resulted from an exposure to mice.
Formalin-fixed, paraffin-embedded tissue sample blocks of breast tumor tissue (mother and daughter) and metastatic breast tumor in lymph node (father) were obtained from the Johns Hopkins Medical Center, Baltimore, Maryland, courtesy of Drs. Elizabeth Montgomery and Arlene Forastiere, under a protocol approved by the Institutional Review Board of Our Lady of Mercy Medical Center (OLMMC). Hematoxylin and eosin stained slides of each paraffin block were made and read to determine the location of malignancy in each block. Blocks were shaved into 5 μm thick serial sections and two sections from each specimen were used for DNA extraction. DNA was isolated from the blocks using a microwave technique . Normal breast tissue samples were obtained from the Pathology Department at OLMMC under a protocol approved by the Institutional Review Board of OLMMC. DNA was extracted from normal breast tissue using the QIAgen DNA Mini Kit (Qiagen Inc, Germantown, MD). To determine the quality of the isolated DNA from both paraffin blocks of tumor tissue and fresh normal breast tissue, globin primers were used in PCR and the resulting amplified products were electrophoresed in 1.8 % agarose gels.
Conditions and primer sequences for MMTV env and LTR gene amplification were those described by Wang et al  and Liu et al  respectively. A reaction without template DNA was routinely tested to detect possible contamination of master mix components. Reactions with normal breast DNA were done to rule out contamination of tumor samples. The PCR product was analyzed by electrophoresis in 1.8 % agarose gels. ΦX174 RF DNA cut with the restriction enzyme Hae III was used as a marker to identify the size of the PCR products.
PCR products were hybridized on Southern blots  under stringent hybridization conditions to either a 23-base pair (bp) probe specific for DNA sequences present in exogenous MMTV env sequences but not present in human endogenous retroviral sequence (HERV-K) DNA [1, 4, 11, 12] or to a 20-bp probe specific for the MMTV LTR [6, 16] and also not present in HERV-K DNA [11, 12]. The 23-bp env probe, which extended from position 7822–7845 in the MMTV genome, and the 20-bp LTR probe, which extended from position 972–991 or 9545–9564 in the MMTV genome , were end-labeled with [32P]ATP using the T4 kinase forward reaction (Invitrogen Life Technologies, Inc., Carlsbad, CA). Stringent hybridization conditions were as described previously [1, 11].
Cloning and sequencing of PCR products
Amplified DNA products were cloned directly from the PCR tube using the TOPO TA Cloning kit for Sequencing (Invitrogen). DNA sequencing was performed by Genemed Synthesis (San Antonio, TX). The resulting sequences were compared to known published sequences and to sequences in the Genbank.
The LTR sequences obtained from cloned fragments after PCR amplification were compared to known exogenous and endogenous MMTV sequences in the Genbank database. LTR sequences were aligned using DNAssist 2.0 software and analyzed using the phylogenetic Analysis Using Parsimony (PAUP 4.0b 10) program  as previously described .
We thank Dr. Stanley Oiseth of the Department of Pathology at Our Lady of Mercy Medical Center (OLMMC) for supplying normal breast tissue and for his expertise in evaluating our paraffin blocks for study and for photos of our tissue samples. We thank Drs. Elizabeth Montgomery and Arlene Forastiere of the Johns Hopkins Medical Center for their contribution of paraffin blocks for this study. We also thank Marie Alice Lilazois and Julio Aviles in OLMMC Pathology for their excellent skill in preparing shavings of paraffin blocks for DNA isolation. This work was funded in part by the A.L. Levine Family Foundation, Inc. of Wayne, NJ.
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