Mouse mammary tumor virus (MMTV)-like DNA sequences in the breast tumors of father, mother, and daughter

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Hematoxylin and eosin stained slides of formalin-fixed, paraffin-embedded tissue sample blocks of breast tumor tissue and metastatic breast tumor tissue in lymph node. Panel A (father): metastatic ductal carcinoma of breast in axillary lymph node. The tumor is almost completely replacing the normal tissue in this 2-cm node. Note the rim of residual subcapsular lymphoid tissue. Panel B (mother): invasive moderately differentiated ductal carcinoma of breast. Note the prominent lymphocytic response. Panel C (daughter): invasive and in situ lobular carcinoma of breast. Only a portion of the round edge of a lobule containing lobular carcinoma in situ is seen here. Magnification is 200X.

Figure 2

Amplification of 250 bp of MMTV-like env gene and 630 bp of MMTV-like LTR gene by PCR. A, Ethidium bromide stained 1% agarose gel electrophoresis of amplified MMTV-like env sequences; B, Southern blot [13] hybridization of A using 5' P³² end-labeled 23-mer probe. Lanes 2, 3 and 4 represent the amplified DNA from the daughter, mother and father respectively. C, Ethidium bromide stained 1.8 % agarose gel electrophoresis of amplified MMTV-like LTR sequences; D, Southern blot [13] hybridization of C using 5' P³² end-labeled 20 mer probe. Lanes 2, 3, and 4 represent amplified DNA from mother, daughter and father respectively. In Panels A-D, Lane 1 containing no template DNA and lanes 5, 6, and 7 containing normal breast tissue DNA from three separate individuals represent negative controls for sample contamination. M is the molecular weight marker ΦX174 RF DNA cut with the restriction enzyme HaeIII.

Figure 3

Sequences of the 250 bp PCR MMTV-like env gene product amplified from the DNA of primary breast cancer tissue of mother and daughter and metastatic breast tumor tissue in lymph node of father compared with the sequences in this region of the env gene of GR, C3H and BR6 strains of MMTV. The numbers 1, 2 and 3 indicate the three locations where the BR6 strain differs from the GR and C3H strains in this region of the MMTV env gene [17,18,19]. The A and ↓ indicate the location at which the identical single base change described in the text occurs. The numbers 7656 and 7905 indicate the location of the MMTV 250 bp env gene sequence within the MMTV genome [17]. Clones are designated as M (mother), D (daughter), and F (father) followed by a number (1–4) denoting the order in which they were cloned. - denotes the same nucleotide.

Figure 4

Sequence homology of the highly variable COOH-terminal region of the MMTV sag gene of MMTV proviruses Mtv-1 [22,23] and Mtv-8 [20,21] to the amplified highly variable MMTV sag sequences cloned from the primary breast tumor tissue of the mother and daughter and metastatic breast tumor tissue in lymph node of the father. Clones are named as M (mother), D (daughter), and F (father) followed by a dash and the letter s for sag and numbers (1–4) identifying the order in which they were cloned. - denotes the same nucleotide.

Figure 5

Phylogenetic analysis of human breast tumor MMTV-like LTR sequences showing that the human and mouse sequences do not cluster as two distinct species. The 12 human MMTV-like LTR sequences from the three family members as well as the human sequences previously isolated from human breast tumors, non-Hodgkin's lymphomas, and primary biliary cirrhosis tissue, clustered with their murine counterparts. Boxes denote LTR sequences from mother (M-S1-4), daughter (D-S1-4), and father (F-S1-4). Previously published human isolates AF346815, AY325271, AF243039, AY652977, AY652968, AY652964, AY652975, AY652974, AY652967, AY652969, AY652973, from human breast tumors [6,16,27], AY652970, AY652976, AY652978, AY652965, AY652971, AY652966, AY652972 from human non-Hodgkin's lymphomas [6], and AF513913, AF513923, from human primary biliary cirrhosis patients [26,27]. The mouse sequences, JYG, FM, and SW21 from Asian mice that were used to root the tree, the endogenous MMTV proviral sequences Mtv-8, Mtv-1, and Mtv-6, and the exogenous MMTV sequences BR6, HEJ, and C3H are bolded. Numbers on branches indicate percent frequencies of assortment in an individual branch after the bootstrap procedure (45) and indicate the robustness of branch assignments. Branch lengths are indicative of the number of nucleotide changes to individual branch points (see scale bar).