Mammary tumor virus proviral DNA in normal murine tissue and non-virally induced mammary tumors

The Southern DNA filter transfer technique was used to study the involvement of the endogenous mouse mammary tumor virus (MMTV) in the development of mammary tumors of nonviral etiology. The presence of extra MMTV proviruses in the genomes of these non-virally induced mammary tumors would indicate an integration of the provirus of an activated endogenous MMTV. Acquisition of MMTV proviruses did not seem to be an absolute requirement for the development of hormone or carcinogenically induced mammary tumors in strain BALB/c nor for hormone-induced mammary tumors in mouse strains 020, C57BL, and C3Hf. In some hormone-induced mammary tumors we did observe extra MMTV proviruses in submolar quantities, indicating that reintegration may occasionally occur and that only a part of the tumor cells acquired new MMTV DNA information. Hormone-dependent and -independent primary mammary tumors of the mouse strain GR, which are controlled by the Mtv-2 mammary tumor induction gene, all acquired extra MMTV proviruses. Most of these extra MMTV proviral-DNA-containing fragments appeared present in submolar quantities, suggesting that only part of the tumor cells acquired extra MMTV proviral information. These findings indicate that the initially transformed mammary gland cells of non-virally induced mammary tumors do not necessarily acquire extra MMTV proviral DNA information, in contrast to the MMTV-induced mammary tumors, in which all tumor cells contain extra MMTV DNA information.     

A novel mechanism of resistance to mouse mammary tumor virus infection

Exogenous mouse mammary tumor virus (MMTV) is carried from the gut of suckling pups to the mammary glands by lymphocytes and induces mammary gland tumors. MMTV-induced tumor incidence in inbred mice of different strains ranges from 0 to as high as 100%. For example, mice of the C3H/HeN strain are highly susceptible, whereas mice of the I/LnJ strain are highly resistant. Of the different factors that together determine the susceptibility of mice to development of MMTV-induced mammary tumors, genetic elements play a major role, although very few genes that determine a susceptibility-resistance phenotype have been identified so far. Our data indicate that MMTV fails to infect mammary glands in I/LnJ mice foster nursed on viremic C3H/HeN females, even though the I/LnJ mammary tissue is not refractory to MMTV infection. Lymphocytes from fostered I/LnJ mice contained integrated MMTV proviruses and shed virus but failed to establish infection in the mammary glands of susceptible syngeneic (I x C3H.JK)F(1) females. Based on the susceptible-resistant phenotype distribution in N(2) females, both MMTV mammary gland infection and mammary gland tumor development in I/LnJ mice are controlled by a single locus.     

Initial stages of mammary tumor virus infection are superantigen independent

Exogenous mouse mammary tumor virus (MMTV) is transmitted via the milk from infected mothers to newborn pups. Efficient MMTV transmission is dependent on proliferation of T cells with particular TCR beta-chains, which occurs upon recognition of virally encoded superantigen (SAg) bound to MHC class II molecules. It is assumed that infection of these dividing cells favors MMTV amplification. SAg is important for MMTV infection, as mice that lack SAg-cognate T cells due to expression of endogenous Mtv loci or mice that express inappropriate MHC haplotypes unable to present viral SAg efficiently were shown to be resistant to MMTV infection. However, this resistance was not absolute, as these mice developed late onset MMTV-induced mammary tumors. In this study, we show that the success of initial MMTV infection in neonates is independent of SAg function but depends on the developmentally regulated proliferation of target cells. However, SAg was absolutely required for virus spread following completion of this proliferative stage.     

Host genetic background effect on the frequency of mouse mammary tumor virus-induced rearrangements of the int-1 and int-2 loci in mouse mammary tumors.

The frequency with which int-1 and int-2 are rearranged in mouse mammary tumors by mouse mammary tumor virus (MMTV)-induced insertional mutagenesis is a consequence of the host genetic background. In 75% of C3H mammary tumors, int-1 is rearranged by MMTV insertion, whereas only 30% of BALB/cfC3H tumors contain a virus-induced rearrangement of int-1. This difference is significant (P less than 0.005) and could not be accounted for by the potentially additive effect of the genetically transmitted Mtv-1-encoded virus in C3H mice. Similarly, MMTV-induced rearrangement of the int-2 gene in mammary tumors of the R111 mouse strain (59%) occurred at a significantly (P less than 0.025) higher frequency than in BALB/cfR111 (25%) mammary tumors. Moreover, in BALB/cfR111 mammary tumors, there is evidence that rearrangement of int-1 and int-2 does not occur independently (P less than 0.025). These results suggest that the long history of inbreeding for high tumor incidence of C3H and R111 mouse strains has selected for the fixation of host mutations which either complement the action of the particular int gene or affect the sensitivity of specific subpopulations of mammary epithelium to infection by particular strains of MMTV.     

Hormonal regulation of mouse mammary tumor growth

In view of reports that human breast cancer cells secrete growth factors that can replace estradiol in sustaining tumor growth [1], we have investigated whether hormone independent (HI) GR mouse mammary tumors can sustain growth of estrogen-depleted hormone dependent (HD) tumors. HD GR mammary tumor TSl 106 was grafted subcutaneously in the right flank of estrone plus progesterone treated castrated (020 X GR)F1 mice. After 2 weeks the estrone treatment was stopped and the mice received 50, 100 or 150 mg HI GR mammary tumor TSl 104 in the left flank. However, the regression of the HD tumor due to estrone depletion was not prevented or retarded by the HI grafts. In other experiments we investigated integrations of mouse mammary tumor virus (MMTV) proviral DNA in the DNA of GR mammary tumors. We could demonstrate the presence of two cell populations in tumor TSl 96, both HD but differing in MMTV DNA integration events. Our data indicate that exogenous integrations of MMTV proviruses can take place in mouse mammary tumor DNA without loss of hormone dependency of the tumors. Like in GR/Mtv-2+ mice, mammary tumor transplants differing in MMTV proviral integrations are also observed in 020/Mtv-2+ mice.     

Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co‐expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth‐promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial–mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co‐transfectants exhibited invasive properties in three‐dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf‐1, a specific antagonist of the Wnt/β‐catenin pathway, or short hairpin RNA targeting β‐catenin expression demonstrated that the invasive activity was not mediated by β‐catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context‐dependent. While Rspo2 and Wnt1 act synergistically in the β‐catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures. J. Cell. Physiol. 227: 1960–1971, 2012. © 2011 Wiley Periodicals, Inc.     

Gradual elimination of retroviruses in YBR/Ei mice

Mouse mammary tumor virus (MMTV), a well-characterized retrovirus that causes mammary tumors in susceptible mice, is commonly used to investigate virus-host interactions. We have shown that YBR/Ei mice demonstrate a novel, dominant mechanism of resistance to MMTV infection and MMTV-induced mammary tumors. MMTV can both establish infection in YBR/Ei mice and be transmitted by YBR/Ei mice as an infectious virus. However, virus production is severely attenuated, resulting in gradual clearance of infection in successive generations. Our transfer experiments showed that T cells generated in MMTV-infected resistant mice were required to restrict MMTV replication in susceptible mice. These results emphasize the importance of inducing T-cell responses for effective protection against retroviral infections.     

Mouse mammary tumor virus proviral sequences congenital to C3H/Sm mice are differentially hypomethylated in chemically induced, virus-induced, and spontaneous mammary tumors

C3H/Sm mice have lost the exogenous milk-borne mouse mammary tumor virus (MMTV) characteristic of the C3H strain and have a very low (1.5%) incidence of spontaneous mammary tumors, yet they are highly susceptible to mammary carcinogenesis by either chemical carcinogens or infection with the milk-borne virus. We have analyzed the MMTV proviral DNA content of normal tissues and of spontaneous, virus-induced, and chemically induced mammary tumors by restriction endonuclease digestion and Southern blot analysis. Although the results clearly showed additional MMTV sequences in the virus-induced tumor which are not present in normal liver DNA, none of the spontaneous or chemically induced tumors could be shown to contain either newly acquired exogenous or amplified endogenous MMTV sequences. Interestingly, mammary tumors arising in C3H/Sm mice treated simultaneously with infectious MMTV (C3H) and dimethylbenz[a]anthracene (DMBA) possessed new exogenous MMTV DNA even though no quantitative change in tumor production was observed when these mice were compared with C3H/Sm mice treated with DMBA alone (Smith et al., Int. J. Cancer 26:373-379, 1980). Our data indicate that the endogenous MMTV proviral units are extensively methylated in normal tissues, such as livers and normal nonlactating mammary glands. In the absence of MMTV (C3H), we found that in the rare, spontaneously occurring C3H/Sm mammary tumors, certain endogenous MMTV sequences were specifically hypomethylated. Hypomethylation of endogenous MMTV sequences was also noted in the chemically induced mammary tumors, even though radioimmune competition assays for MMTV gp52 and p28 are negative (Smith et al., Int. J. Cancer 27:81-86, 1981). Our results support the conclusion that amplification of endogenous MMTV sequences is not intrinsic to C3H/Sm mouse mammary tumors arising spontaneously or after induction by chemicals. On the other hand, integration of exogenous MMTV DNA into the genome was a constant feature of mammary tumors developing in MMTV (C3H)-infected C3H/Sm mice, even when DMBA was used as the carcinogen. Hypomethylation of some endogenous MMTV sequences is characteristic of C3H/Sm mammary tumors, whether spontaneous or induced by chemicals, which suggests that these sequences are located in actively transcribing regions of the tumor cell genome.     

Genetics of mouse mammary tumor virus-induced mammary tumors: linkage of tumor induction to the gag gene

Retroviruses are believed to induce tumors by acting as insertional mutagens that activate expression of cellular protooncogenes. Indeed, almost 90% of mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/He mice show upregulation of Int protooncogenes. We have analyzed three different MMTV variants [MMTV(C3H), MMTV(HeJ), and a genetically engineered MMTV hybrid provirus (HP)] for tumorigenicity in mice from two distinct genetic backgrounds. All three viruses were tumor causing in BALB/cJ mice. However, only MMTV(C3H), but not MMTV(HeJ) or HP, induced mammary tumors in C3H/He mice. All of the viruses were infectious on either background and up-regulated expression of Int genes in tumors they induced. Like HP, MMTV(HeJ) was found to be a genetic recombinant between endogenous Mtv1 provirus and exogenous MMTV(C3H). Sequence comparison of MMTV variants linked the tumorigenicity of MMTV(C3H) to the gag region of the retrovirus.     

A retrovirus vector expressing the putative mammary oncogene int-1 causes partial transformation of a mammary epithelial cell line

In mammary tumors induced by the mouse mammary tumor virus (MMTV), the int-1 gene is frequently activated by adjacent proviral insertions and is thereby strongly implicated in tumorigenesis. To seek a direct biological effect of int-1 that would validate its proposed role as an oncogene, we constructed a retrovirus vector containing the gene and examined its effects on tissue culture cells. Expression of int-1 in a mammary epithelial cell line caused striking morphological changes, unrestricted growth at high cell density, and focus formation on a monolayer, although the cells were not tumorigenic in vivo. This partial transformation induced by int-1 was not observed in cells infected by an otherwise identical virus bearing a frameshift mutation in the gene. These findings strongly support the hypothesis that int-1 plays a functional role in MMTV-induced mammary tumorigenesis.     

Endogenous MMTV in the normal mammary gland and in dimethylanthracene-induced mammary cancer in BALB/c mice

Immunization of DMBA-treated mice by the glycoprotein fraction from mammary glands of mice BALB/c did not decrease the frequencies of induction of mammary tumors. This is in contrast to the results obtained during immunization by the formaldehyde treated preparation of Mouse Mammary Tumor Virus (MMTV). EcoRI and HindIII cleaved DNA from the DMBA-induced mammary tumors did not contain the additional virus specific fragments. In mammary tumors the expression of p27 MMTV was registered in contrast to normal mammary glands and mammary epithelium cultures in which the proteins of MMTV are not expressed even after induction.     

Transfer, by selective breeding, of the pathogenic Mtv-2 endogenous provirus from the GR strain to a wild mouse line free of endogenous and exogenous mouse mammary tumor virus.

The GR laboratory mouse strain has five endogenous proviral copies of the mouse mammary tumor virus (MMTV). One of these, Mtv-2, is unique because it causes mammary carcinomas in virtually 100% of breeding GR females prior to 1 year of age. To facilitate studies of this locus in particular, and mammary tumorigenesis in general, we genetically tailored a new mouse line, WXG-2, which bears Mtv-2 as its only endogenous MMTV provirus. The WXG-2 line was constructed by making hybrids between the GR strain and a wild mouse line free of both endogenous and exogenous MMTV, backcrossing to the MMTV-free line, and fixing the Mtv-2 locus in a population with the desired genotype. Mammary tumors were observed in 5 of the 20 hybrid females carrying the endogenous Mtv-2 provirus. The WXG-2 line represents a new model system for studying MMTV-induced mammary tumorigenesis in the absence of multiple endogenous proviruses.     

ErbB-beta-catenin complexes are associated with human infiltrating ductal breast and murine mammary tumor virus (MMTV)-Wnt-1 and MMTV-c-Neu transgenic carcinomas

Simultaneous deregulation of both Wnt and ErbB growth factors has previously been shown to result in the cooperative induction of mammary gland tumors. Using the murine mammary tumor virus (MMTV)-Wnt-1 transgenic model of mammary carcinoma, we have identified an unvarying association between beta-catenin and epidermal growth factor receptor/c-Neu (ErbB1/ErbB2) heterodimers in mammary gland tumors, indicating a requirement for ErbB signaling in Wnt-mediated tumorigenesis. Expansion of these observations to a second transgenic model, MMTV-c-Neu, demonstrated similar tumor-specific interactions, including an ErbB1 ligand-inducible phosphorylation of both beta-catenin and c-Neu. Direct relevance of these findings to human breast cancer was established upon examination of a set of human infiltrating ductal breast adenocarcinoma and lymph node metastasis tissues taken at surgery. These data revealed increased levels of beta-catenin in tumors and metastases versus normal breast as well as an association between beta-catenin and c-Neu that measurably occurs only in neoplasia, most strongly in metastatic lesions. These studies have identified a seemingly indispensable interaction between beta-catenin and epidermal growth factor receptor/c-Neu heterodimers in Wnt-1-mediated breast tumorigenesis that may indicate a fundamental signaling event in human metastatic progression.