Mouse mammary tumor virus and mammary tumorigenesis in wild mice

The current knowledge of the distribution of the mouse mammary tumor virus (MMTV) proviral genomes and the mechanism of mammary tumorigenesis by MMTV in mice, with the main emphasis on Asian feral mice, is reviewed. The relevant earlier discoveries on the mode of MMTV transmission are summarized to provide an outline of the biology of MMTV. Finally, the viral etiology of human breast cancer will be discussed.     

Effect of early thymectomy on growth of transplanted mammary gland tumors arising in C3Hf mice

Mammary gland tumors arising in factorless C3Hf mice, if transplanted into MTV-S⁺ and MTV-S^– recipients, were found to grow at the same rate and early thymectomy likewise had no effect on their growth. The more rapid growth of transplanted tumors in MTV-S⁺ than in MTV-S^– recipients after transplantation of mammary gland tumors from C3H/He mice and the inhibition of tumor growth after early thymectomy are evidently connected with special features of the immunological reaction to virus-induced antigens.Institute of Cytology and Genetics, Academy of Sciences of the USSR, Siberian Branch, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 6, pp. 721–723, June, 1976.     

A Vβ.2-specific superantigen from exogenous mouse mammary tumor virus carried by FM mice

A number of endogenous mouse mammary tumor virus (MMTV) proviruses encode superantigen that have the ability to stimulate T cells with a certain T cell receptor (TCR) β-chain variable region (Vβ) and to mediate the Vβ-specific clonal deletion. The tumorigenic milk-borne MMTV carried by C3H and GR mice also have superantigenic properties in vivo. In the present study we identified and characterized a novel Vβ8.2-specific superantigen of exogenous MMTV carried by FM mice. The open reading frame (ORF) in the 3′ long terminal repeat of the MMTV was cloned by polymerase chain reaction with primers corresponding to conserved regions spanning the ORF coding region. Sequence analysis of the ORF revealed that there is no sequence identical to those in other known MMTV in the carboxy terminus implicated in TCR Vβ recognition. Subcutaneous injection of the virus into adult BALB/c mice induced an approximately three- to fourfold enlargement of draining lymph nodes and a substantial increase of Vβ8.2⁺ CD4⁺ T cells in the lymph nodes within 6 days. The exposure of newborn BALB/c mice to the virus by foster nursing resulted in a marked deletion of Vβ8.2⁺ cells both in CD4⁺ and CD8⁺ T cells. Thus, a novel milk-borne MMTV in FM mice expresses strong superantigenic properties capable of stimulating Vβ8.2⁺ T cells. Vβ8.2⁺ T cells have been demonstrated to be frequently involved in recognition of conventional antigens and responsible for autoimmune diseases such as experimental allergic encephalomyelitis. Therefore, the MMTV (FM) may provide a new mouse model system for inducing immunodeficiency or autoimmune disease by retroviral infection.     

Superantigenic characteristics of mouse mammary tumor viruses play a critical role in susceptibility to infection in mice

Mouse mammary tumor viruses (MMTV) are retroviruses that induce mammary carcinomas. An interesting feature of these viruses is the superantigen (SAg) encoded in an open reading frame within the 3′ long terminal repeat. The mechanism by which ingestion of milk-borne virus results in infection of the host mammary tissue remains incompletely understood. However, a working model has been proposed in which the interaction between viral SAg, T-cell receptor and MHC class II I-E facilitates viral replication and hence infectivity. In this review we summarize current studies demonstrating the role of SAg stimulation in susceptibility to MMTV infection.     

Molecular cloning and primary structure analysis of the mouse mammary tumor virus-related element from dwarf hamster genome

Eleven recombinant bacteriophages carrying mouse mammary tumor virus (MMTV)-related sequences (MRS) were isolated from the genomic library of the dwarf hamster (Phodopus sungorus) using radioactively labeled DNA of MMTV as a hybridization probe. There are approximately 50 copies of MRS-Ps per genome, as estimated by the number of positive signals on the autoradiogram of the primary screening plate. MRS of Phodopus sungorus (MRS-Ps) contain regions homologous to the LTR, pol, and, probably, env, but not gag genes of MMTV. A 0.9 kb MRS-Ps fragment was sequenced and proved to be 63% identical to the MMTV pol gene sequence. However, all absolute frames contain multiple stop codons and do not seem to be expressed.     

Mouse mammary tumor viruses expressed by RIII/Sa mice with a high incidence of mammary tumors interact with the V beta-2- and V beta-8-specific T cells during viral infection

The mouse mammary tumor viruses (MMTVs) that induce mammary adenocarcinomas in mice are transmitted from mother to offspring through milk. MMTV infection results in the deletion of specific T cells as a consequence of interaction between the MMTV-encoded superantigen (Sag) and specific V beta chains of the T cell receptor. The specificity and kinetics of T cell deletion for a number of highly oncogenic MMTVs, such as C3H- and GR-MMTVs, have been studied in great detail. Some work has also been done with the MMTVs expressed in two substrains of RIII mice, BR6 and RIIIS/J, but the nature of the interaction between T cells and the virus(es) that the parental RIII-strain of mice express has not been investigated. Since RIII mice (designated henceforth as RIII/Sa) have a very high incidence (90-98%) of mammary tumors, and they have been extensively used in studies of the biology of mammary tumor development, we have presently determined the pattern of V beta-T cell deletion caused by RIII/Sa-MMTV-Sag(s) during viral infection. T cells were isolated from lymph nodes and thymus of young RIII/Sa mice, as well as from BALB/c (BALB/cfRIII/Sa), C57BL (C57BLfRIII/Sa), and RIIIS/J (RIIIS/JfRIII/Sa) mice after they were infected with RIII/Sa-MMTV(s) by foster nursing. The composition of the T cells was analyzed by FACS using a panel of monoclonal antibodies specific to a variety of V betas. Our results show that milk-borne RIII/Sa-MMTV(s) infection leads to the deletion of CD4(+) V beta-2, and to a lesser extent V beta-8 bearing peripheral and central T cells in RIII/Sa, RIIIS/J, BALB/c, and C57BL mice. Our results are in contrast to the findings that C3H-, GR-, and BR6-MMTVs delete V beta-14- and/or V beta-15-specific T cells.     

T cell receptor Vβ repertoire in mice lacking endogenous mouse mammary tumor provirus

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) Vβ segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, ?7, ?8 and ?9) and 38CH (Mtv^?) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, ?7, ?8 or ?9. The TcR Vβ chain (TcR Vβ) usage in these mice was analyzed using monoclonal antibodies specific for TcR Vβ2,Vβ3, Vβ4,β5,Vβ6,Vβ7,Vβ8,Vβ11,Vβ12 and Vβ14 segments. Both Mtv-8⁺ mice and Mtv-9⁺ mice deleted TcR Vβ5⁺ and Vβ11⁺ T cells. Moreover, we also observed the deletion of TcR Vβ12⁺ cells by Mtv-8 and Mtv-9 products. Mtv-6⁺ and Mtv-7⁺ animals deleted TcR Vβ3⁺ and Vβ35⁺ cells, and TcR Vβ6⁺,Vβ7⁺ and Vβ8.1⁺ cells, respectively. Unexpectedly, TcR Vβ8.2⁺ cells were also deleted in some backcross mice expressing Mtv-7. TcR Vβ8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn → Asp) in position 19 on the TcR Vβ8.2 fragment. Reactivities of BALB.D2 TcR Vβ8.2 and 38CH TcR Vβ8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR Vβ2⁺, Vβ4⁺ and Vβ8⁺ CD4⁺ T cell subsets in Mtv-8⁺ and Mtv-9⁺ mice, and TcR Vβ4⁺ CD4⁺ T cells in Mtv-6⁺ and Mtv-7⁺ mice, when compared with the T cell repertoire of Mtv^? mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.     

Deletion of CD4+ T cells and thymocytes by apoptosis in mouse mammary tumor virus (C4)-infected Vβ2 transgenic mice

Mouse mammary tumor virus MMTV(C4) encodes a Vβ2-specific superantigen. In Vβ2 transgenic (TG2) mice more than 98 % of peripheral T cells express Vβ2. Infection of Tg2 mice with MMTV(C4) at birth through their mothers' milk or at 6–8 weeks of age by intravenous injection resulted in massive deletion of peripheral CD4⁺ T cells and suppressed thymopoiesis. The number of peripheral CD8⁺ T cells was not affected in neonatally infected mice. In older mice injected with MMTV(C4), splenic CD8⁺ T cells were significantly elevated. Suppressed thymopoiesis was observed in both neonatally infected and older mice injected with MMTV(C4). Thymocytes which expressed high level CD3 or Vβ2 were deleted. To determine if T cells or thymocytes were deleted through apoptosis, DNA fragmentation was examined by flow cytometry and diphenylamine (DPA) binding assay. Approximately 31 % of CD4⁺ T cells from MMTV(C4)-infected Tg2 mice as compared to 6% from normal Tg2 mice contained fragmented nuclear DNA by flow-cytometric analysis. The DPA binding assay showed significantly increased total soluble DNA in lymph node cells and thymocytes from MMTV(C4)-infected mice. The kinetics of T cell and thymocyte apoptosis correspond to their deletion, supporting apoptosis as the mechanism of T cell and thymocyte deletion. CD4⁺ T cell and thymocyte deletion by MMTV(C4) in Tg2 mice provides a sensitive system for the analysis of retrovirus superantigen-induced apoptosis.     

Distribution of mouse mammary tumor virus-related sequences does not correlate with the taxonomic position of their hosts

Sequences (MRS) distantly related to mouse mammary tumor virus (MMTV) were found in genomes of a wide range of mammalian species using blot hybridization. The number of MRS copies and the degree of their homology with the hybridization probe varied and did not correlate with the taxonomic position of the species. Nevertheless, within a genus the set of MRS was species specific and reflected the taxonomic relation between the species. MRS were also found in avian genomes and the degree of their homology did not correlate with the taxonomic position of the species either. The origin and distribution of MRS is discussed on the basis of the authors' and published data.     

Massive mammary gland infection and pregnancy-dependent mammary tumor development in mice infected neonatally with mouse mammary tumor virus (SW) but not in mice infected as adults,despite a dramatic local response

Mouse mammary tumor virus (MMTV) (SW) caused a high incidence (65%) of pregnancy-dependent adenocarcinomas in BALB/c(SW) mice infected as new-borns by suckling their mothers' milk. These tumors were type B adenocarcinomas which developed early, at about 1 year of age. Uninfected breeding females and those infected at an age of 8 weeks by injection of virus had the same low incidence of malignant tumors (13%), and the tumors developed later (at approx. 23–24 months). The low incidence of tumors in adult-infected mice was correlated with partial infection of the mammary glands, and delayed transmission of MMTV(SW) to the offspring. Although the virus was rapidly disseminated in both types of infection, the responses of neonatally infected and adult-infected mice to MMTV(SW) infection and viral superantigen (vSAG) presentation were different. Activation by and presentation of the vSAG was impaired in mice infected neonatally, and tolerance induction by clonal deletion was delayed. Local activation was dramatic in mice infected as adults and clonal deletion followed rapidly. Although interaction between B and T cells is needed for completion of the virus life cycle and viral amplification, the strong local immune response to MMTV(SW) in adult-infected mice limits mammary gland infection, and protects them against mammary tumor development.     

Conditional deletion of the Lkb1 gene in the mouse mammary gland induces tumour formation

Heterozygous germline mutations in the LKB1 (STK11) gene cause Peutz‐Jeghers syndrome (PJS), an autosomal dominant disorder characterized by hamartomatous polyposis of the gastrointestinal tract and an increased risk of colorectal, breast, and other cancers. To model the role of LKB1 mutation in mammary tumourigenesis, we have used a conditional gene targeting strategy to generate a mouse in which exons encoding the kinase domain of Lkb1 were deleted specifically in the mammary gland. Mammary gland tumours developed in these mice with a latency of 46–85 weeks and occurred in the thoracic or inguinal glands. These tumours were grade 2 invasive ductal carcinomas or solid papillary carcinomas with histological features similar to those described in breast cancers arising in patients with PJS. This mouse model of Lkb1 deficiency provides a potentially useful tool to investigate the role of Lkb1 in tumourigenesis and to guide the development of therapeutic approaches. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Expression and demethylation of germinally-transmitted BALB/c mouse mammary tumor virus DNA in Abelson MuLV B-lymphoid cell lines

Murine mammary tumor virus (MMTV) RNA expression, DNA organization and DNA demethylation were examined in BALB/c B-lymphoid cell lines produced by transformation with the Abelson murine leukemia virus (AbMuLV). The MMTV DNA sequences in AbMuLV B cell lines, based on restriction mapping with EcoRI, PstI, BglII, BamHI and SacI and molecular hybridization with cloned probes of the MMTV LTR, gag-pol or env gene regions, were identical to the germinally-transmitted MMTV DNA complement of BALB/c mice. Several AbMuLV B cell lines expressed MMTV poly(A+)-RNA at detectable levels. MMTV poly(A+)-RNA for the env gene, 3.8 kb, and the long terminally redundant (LTR) region, 1.7 kb, were detected in some AbMuLV B cell lines. MMTV DNA sequences in the AbMuLV B cell lines were at least partially sensitive to digestion by the methylation-sensitive restriction endonucleases HhaI and HpaII. HhaI-sensitive sites were present in Units I, II and III of the germinally-transmitted MMTV DNA and were localized specifically near the 5' end of the 5' and 3' LTRs of both Units II and III. HpaII-sensitive sites were localized near the 3' end of the 3' LTRs of Units II and III, and at a cellular site 2.1 kbp 5' to the 5' LTR. These observations demonstrate that the germ line MMTV DNA sequences of BALB/c mice are expressed in cells of B lymphocyte origin, and suggest a correlation between MMTV RNA expression and selective demethylation in the LTR regions of germinally-transmitted MMTV DNA sequences.     

Differential reactivity of TCR Vβ10 alleles to a mouse mammary tumor virus superantigen

Mouse mammary tumor virus (MMTV) expresses a superantigen (SAg) which plays a critical role in the viral life cycle. We have recently described the new infectious MMTV (SIM) encoding a Vβ4-specific SAg in mice with a TCR-Vβ^b haplotype. We have now compared the SAg activity of this virus in BALB / c mice harboring the TCR-Vβ^a, TCR-Vβ^b or TCR-Vβ^c haplotypes which differ by a central deletion in the TCR-Vβ^a and TCR-Vβ^c locus and by mutations in some of the remaining Vβ elements. Injection of MMTV (SIM) led to a strong stimulation of Vβ4 + CD4 + T cells in TCR-Vβ^b mice, but only to a weak stimulation of these cells in TCR-Vβ^a or TCR-Vβ^c mice. A large increase in the percentage of Vβ10 + cells was observed among CD4 + T cells in mice with the Vβ ^a or Vβ ^c, but not the Vβ ^b TCR-Vβ haplotype. Vβ 10+ cells dominated the response when Vβ10^a/c and Vβ 4 subsets were present together. This is the first report of a viral SAg interacting with murine Vβ10 + cells. Six amino acid differences between Vβ10^{a / c} and Vβ10^b could account for the gain of reactivity of Vβ10^{a / c} to the MMTV(SIM) SAg. No mutations were found in the hypervariable region 4 (HV4) of the TCR. Mutations at positions 22 and 28 introduce into Vβ10^{a / c} the same amino acids which are found at these positions in the MMTV(SIM)-reactive Vβ4. Tridimensional models indicated that these amino acids lie close to HV4 and are likely to be important for the interaction of the SAg with the TCR.     

Mammary preneoplasia and tumorigenesis in the BALB/c mouse: structure and modification of mouse mammary tumor virus DNA sequences

Mouse mammary tumor virus (MMTV) DNA sequences were examined in mammary tissues from BALB/c mice, including both preneoplastic hyperplastic alveolar nodule (HAN) outgrowth lines, tumors derived from those preneoplastic tissues, and DMBA-induced mammary tumors. Over 60 different HAN and tumors samples were analyzed. The D2 HAN line contained one additional provirus, whereas Cv-2 and Cv-4 HAN lines that are infected with exogenous virus exhibited multiple virus integration events. D2 tumors showed the same provirus pattern as D2 HANs, whereas Cv-2 and Cv-4 tumors exhibited a subset of the acquired proviruses found in the parental HAN populations. Differential methylation patterns of virus-specific sequences were observed that resembled those described for other tissues in which viral DNA replication and integration has occurred, i.e., acquired proviruses were hypomethylated and endogenous proviruses were methylated. In tumors that arose from HAN lines and exhibited only endogenous proviruses, demethylation of the subgenomic Mtv-6 locus was observed. Demethylation of Mtv-6 was not detected in any of the preneoplastic tissues. Altered methylation of Mtv-8 and -9 was observed in both Cv-2 and Cv-4 tumors. Finally, mammary tumors induced by DMBA carried no acquired proviruses and demethylation of endogenous MMTV proviruses was demonstrated.     

Increased erbB3 promotes erbB2/neu-driven mammary tumor proliferation and co-targeting of erbB2/erbB3 receptors exhibits potent inhibitory effects on breast cancer cells

The kinase deficient erbB3 receptor frequently co-expresses and interacts with erbB2 in human breast cancer to activate the oncogenic signaling pathways, and thus promote breast cancer cell survival/proliferation. In the current study, we discovered that the expression of endogenous mouse erbB3 was increased in the mammary tumors-derived from wild type (wt) rat erbB2/neu-transgenic mice, and the co-expression of erbB2 and erbB3 significantly promoted mammary tumor proliferation in vivo. Co-immunoprecipitation assays detected a heterodimeric complex consisting of the transgene encoded protein rat erbB2 and the endogenous mouse erbB3 in the mammary tumors. Specific knockdown of mouse erbB3 dramatically inhibited proliferation of the mammary tumor cell lines-derived from the transgenic mice. Elevated expression of erbB3 protein, but not mRNA, was abserved in human breast cancer cells upon ectopic expression of erbB2. Additional studies revealed that overexpression of erbB2 downregulated three erbB3-targeting miRNAs, miR-125a, miR-125b, and miR-205, whereas the erbB2 kinase inhibitor (lapatinib) significantly enhanced expression of the three miRNAs in breast cancer cells, suggesting that erbB2 might regulate erbB3 expression through a miRNA-dependent mechanism. Furthermore, an anti-erbB3 monoclonal IgG1 antibody (Ab) in combination with Herceptin mainly inactivated Akt and significantly inhibited proliferation of erbB2-overexpressing breast cancer cells. Collectively, our data indicate that increased expression of erbB3 plays a pivotal role in activating downstream PI-3K/Akt pathway and promoting erbB2-driven mammary/breast tumorigenesis. Simultaneous targeting of erbB2 and erbB3 with two IgG1 Abs may be an effective strategy to treat breast cancer patients whose tumors overexpress both erbB2 and erbB3.     

Maternal transfer of infectious mouse mammary tumor retroviruses does not depend on clonal deletion of superantigen-reactive Vβ14+ T cells

Female C3H/HeJ mice maternally transmit through their milk an infectious mouse mammary tumor retrovirus (MMTV) which causes clonal deletion of T cell receptor (TcR)Vβ14⁺ T cells reactive to the retroviral superantigen (SAG). To test whether CD4⁺ or CD8⁺ T cells are crucial for intestinal infection and maternal transfer of exogenous retroviruses, newborn mice lacking CD4 or CD8 molecules after gene targetting were raised by surrogate C3H/HeJ mothers. In CD8^?/? mice, clonal deletion of TcRVβ14⁺ cells reactive to the SAG from this exogenous MMTV occured with delayed kinetics. Deletion of TcRVβ14⁺ cells was not observed in CD4^?/? mice up to 12 months after exposure to the retrovirus. In both CD4^?/? and CD8^?/? mice TcRVβ5⁺ and TcRVβ11⁺ T cells were deleted in the presence of genomically integrated endogenous MMTV (Mtv), indicating that the lack of SAG-induced clonal deletion was not due to a general defect in these mutant mouse strains. Although TcRVβ14⁺ T cells were not deleted in CD4^?/? mice, female CD4^?/? mice nursed on C3H/HeJ milk maternally transmitted the retrovirus to their offspring, albeit with delayed kinetics. These data demonstrate that CD4⁺ and CD8⁺ lymphocytes influence clonal deletion events and that the mechanisms responsible for clonal deletion of SAG-reactive TcRVβ14⁺ T cells may be different from mechanisms which allow the mammary tumor virus to enter the mammary gland and complete its infectious cycle.     

A new infectious mammary tumor virus in the milk of mice implanted with C4 hyperplastic alveolar nodules

We have previously characterized an infectious mouse mammary tumor virus [(MMTV(SW)] which induces a strong superantigen response in vivo. Here we describe the isolation and characterization of MMTV(C4) which was derived from milk of mice implanted with hyperplastic alveolar nodules. MMTV(C4) stimulates Vβ2 expressing T cells after local injection in vivo. Comparison with known open reading frame (orf) sequences revealed high homology to Mtv-6, an endogenous virus interacting with Vβ3-expressing T cells. The carboxyl-terminal amino acids were, however, altered. High homology including the carboxyl-terminal orf amino acids were found with MMTV(C3H-K). We show here that MMTV(C3H-K) has lost its superantigen function. Sequence comparisons permitted the characterization of few key amino acids which could be important for T cell receptor interaction and superantigen processing.