An immunoreceptor tyrosine activation motif in the mouse mammary tumor virus envelope protein plays a role in virus-induced mammary tumors

Abstract

Mouse mammary tumor virus (MMTV) induces breast cancer with almost 100% efficiency in susceptible strains through insertional activation of protooncogenes, such as members of the wnt and fibroblast growth factor (fgf) families. We previously showed that expression of the MMTV envelope protein (Env) in normal immortalized mammary epithelial cells grown in three-dimensional cultures caused their morphological transformation, and that this phenotype depended on an immunoreceptor tyrosine-based activation motif (ITAM) present in Env and signaling through the Syk tyrosine kinase (E. Katz, M. H. Lareef, J. C. Rassa, S. M. Grande, L. B. King, J. Russo, S. R. Ross, and J. G. Monroe, J. Exp. Med. 201:431-439, 2005). Here, we examined the role of the Env protein in virus-induced mammary tumorigenesis in vivo. Similar to the effect seen in vitro, Env expression in the mammary glands of transgenic mice bearing either full-length wild-type provirus or only Env transgenes showed increased lobuloalveolar budding. Introduction of the ITAM mutation into the env of an infectious, replication-competent MMTV or into MMTV/murine leukemia virus pseudotypes had no effect on incorporation of Env into virus particles or on in vitro infectivity. Moreover, replication-competent MMTV bearing the ITAM mutation in Env infected lymphoid and mammary tissue at the same level as wild-type MMTV and was transmitted through milk. However, mammary tumor induction was greatly attenuated, and the pattern of oncogene activation was altered. Taken together, these studies indicate that the MMTV Env protein participates in mammary epithelial cell transformation in vivo and that this requires a functional ITAM in the envelope protein.

FIG. 1.

Whole-mount analysis of mammary glands from 2-month-old virgin C3H/HeN, MMTV-Wnt1, LEL, and HP mice. All pictures were taken at ×4 magnification; the lymph nodes (LN) are included in each picture for comparison.

FIG. 2.

Mutation of the Y residues in the SU ITAM does not alter pseudovirus infectivity or Env incorporation into virions. (A) Pseudovirus titers on TRH3 cells of the particles bearing wild-type and mutant Env proteins. Shown is the average of three independent experiments (infectivity of pseudoviruses bearing the mutant Env normalized to the wild-type Env). (B) Fluorescence-activated cell sorting analysis of HP- (dark line) and HP-Y₁Y₂- (dotted line) transfected NMuMG cells using anti-MMTV Env antisera; unstained cells are shown by the thin line. The mean fluorescense intensity of cells within the M1 window was 183 for HP and 142 for HP-Y₁Y₂. Western blot analysis of virus particles isolated from HP- and HP-Y₁Y₂-transfected NMuMG cell supernatants.

FIG. 3.

Infection of lymphoid tissue. A. Provirus integration was determined by semiquantitative PCR with DNA isolated from the spleens of age-matched mice infected by milk-borne HP or HP-Y₁Y₂ virus. Presented are the relative expression levels in individual mice (see Materials and Methods); shown below are the average values for each group. B. RNA analysis. RNA isolated from the spleens was subjected to RNase protection analysis as previously described (17). Shown are the results from splenic RNA isolated from virgin [HP(v) and Y₁Y₂(v)] and multiparous (HP and Y₁Y₂) females.

FIG. 4.

Mammary gland infection. RNA isolated from the virus fraction of milk or from virgin mammary tissue of age-matched mice infected by milk-borne HP or HP-Y₁Y₂ virus was subjected to RNase protection analysis, as described in the legend to Fig. 3. Shown below each lane is the MMTV signal normalized to β-actin; the boxed values are the average normalized MMTV signal for each group of infected mice. MG, mammary gland.

FIG. 5.

Mammary tumor incidence induced by HP and HP-Y₁Y₂ viruses. Mice infected by milk-borne HP (n = 36) or HP-Y₁Y₂ (n = 21) virus were force bred and monitored for mammary tumors by weekly palpation. Shown are Kaplan-Meier survival curves that were computed for age at tumor onset. The P value for the test in which these two curves are significantly different (null hypothesis of equality) was 0.0066. Shown in the table is the mean and median time to mammary tumor development for the two groups; numbers of mice infected with the mutant virus that had developed tumors at 1 year were insufficient to compute the median.