Requirement for both Shc and phosphatidylinositol 3' kinase signaling pathways in polyomavirus middle T-mediated mammary tumorigenesis

Abstract

Transgenic mice expressing the polyomavirus (PyV) middle T antigen (MT) develop multifocal mammary tumors which frequently metastasize to the lung. The potent transforming activity of PyV MT is correlated with its capacity to activate and associate with a number of signaling molecules, including the Src family tyrosine kinases, the 85-kDa Src homology 2 subunit of the phosphatidylinositol 3' (PI-3') kinase, and the Shc adapter protein. To uncover the role of these signaling proteins in MT-mediated mammary tumorigenesis, we have generated transgenic mice that express mutant PyV MT antigens decoupled from either the Shc or the PI-3' kinase signaling pathway. In contrast to the rapid induction of metastatic mammary tumors observed in the strains expressing wild-type PyV MT, mammary epithelial cell-specific expression of either mutant PyV MT resulted in the induction of extensive mammary epithelial hyperplasias. The mammary epithelial hyperplasias expressing the mutant PyV MT defective in recruiting the PI-3' kinase were highly apoptotic, suggesting that recruitment of PI-3' kinase by MT affects cell survival. Whereas the initial phenotypes observed in both strains were global mammary epithelial hyperplasias, focal mammary tumors eventually arose in all female transgenic mice. Genetic and biochemical analyses of tumorigenesis in the transgenic strains expressing the PyV MT mutant lacking the Shc binding site revealed that a proportion of the metastatic tumors arising in these mice displayed evidence of reversion of the mutant Shc binding site. In contrast, no evidence of reversion of the PI-3' kinase binding site was noted in tumors derived from the strains expressing the PI-3' kinase binding site MT mutant. Tumor progression in both mutant strains was further correlated with upregulation of the epidermal growth factor receptor family members which are known to couple to the PI-3' kinase and Shc signaling pathways. Taken together, these observations suggest that PyV MT-mediated tumorigenesis requires activation of both Shc and PI-3' kinase, which appear to be required for stimulation of cell proliferation and survival signaling pathways, respectively.

FIG. 1

Tissue specificity of transgene expression in MMTV/MT-Y250F transgenic mice. (A and C) Structures of the MMTV/MT-Y315/322F (A) and MMTV/MT-Y250F (C) transgenes. The Bluescript vector backbone is represented by a thin line on either side of the expression cassette, with the cross-hatched region corresponding to the MMTV LTR derived from plasmid pA9, the stippled portion corresponding to the MT-Y250F cDNA with phenylalanine substitutions at amino acids positions 315 and 322 or amino acid position 250, and the solid region corresponding to the transcriptional processing sequences derived from the SV40 early transcription unit. The transcription start site is indicated by the arrow. (B and D) RNA transcripts corresponding to the MMTV/MT-Y315/322F (B) and MMTV/MT-Y250F (D) transgenes in various organs of the MT-Y315/322F transgenic strain as assessed by RNase protection. Tissues were derived from a virgin tumor-bearing female, a virgin female, and a male. The antisense probe used in this RNase protection analysis (MTR) is complementary to a 203-nucleotide fragment corresponding to the amino terminus of PyV MT and is marked by MT-315/322F and an arrow. Also shown is an RNase protection analysis with identical RNA samples and an antisense probe directed against PGK, which protects a 124-nucleotide fragment indicated by PGK and an arrow. M.Gl., mammary gland.

FIG. 2

Histological analyses of mammary glands from FVB/N, MMTV/MT-Y250F, and MMTV/MT-Y315/22F transgenic animals. Photomicrographs comparing the histological (A, C, and E) (magnification, ×100) and whole-mount (B, D, and F) (magnification, ×10) appearances of virgin female FVB (A and B), MTY250F (C and D), and MT-Y315/322F (E and F) mice 12 weeks after birth are shown. Note that the mammary tree from the MT-Y250F mouse (C and D) has extensive formation of side buds along the major ducts. The MT-Y315/322F mammary tree has fewer side buds and a more dilated ductal system (E and F) with multilayered epithelium (hyperplasia) (E) and the formation of solid nests of dysplastic cells (F, arrow).

FIG. 3

Activation of the PI-3′ kinase by PyV MT is involved in mammary tumor progression. (A) A panel of slide-mounted Mayer’s hematoxylin-stained mammary tissue sections from age-matched mice from nontransgenic FVB/N or transgenic MMTV-Y250F, MMTV/MT-Y315/322F, and wild-type MT strains. Cells were analyzed for apoptotic cell death as described previously (46). Digoxigenin-labeled DNA ends were detected with horseradish peroxidase-conjugated antidigoxigenin antibodies. Note the multiple apoptotic cells in the epithelial hyperplasias derived from the MT-Y315/322F strain and lack of comparable staining in mammary tissues from FVB/N, MT-Y250F, and wild-type MT mice. (B) Structure of Cre-inducible expression cassette carrying the dominant negative p85 inhibitor. The dark shaded box indicates the Mo-MuLV LTR; the arrows flanking the PGK-Neo cassette represent the LOX recombination sites. The unshaded box indicates the cDNA encoding the mutant p85 subunit of the PI-3′ kinase. (C) In situ apoptosis analyses (TUNEL) conducted with PyV MT mammary tumor cells infected with either a control adenovirus expressing a beta-galactosidase reporter (MT-LacZ) or an adenovirus vector expressing the Cre recombinase (MT-Cre). TUNEL analyses with PyV MT tumor cells possessing the inducible dominant negative p85 inhibitor infected with either the LacZ (MTΔ85nl-LacZ) or Cre adenovirus (MTΔ85nl-Cre) are also shown. Both sets of cells were infected at an MOI of 100. Note the presence of numerous cells undergoing apoptotic cell death in the Cre infection panel.

FIG. 4

Kinetics of mammary tumor occurrence and histopathology of mammary tumors derived from MMTV/wild-type MT, MMTV/MT-Y250F, and MMTV/MT-Y315/22F transgenic animals. (A to D) Photomicrographs comparing the histology of invasive malignancies from a wild-type PyV MT (A), an MT-Y250F tumor (B), and an MT-Y315/322F tumor (D) (magnification, ×250) with that of a noninvasive dysplasia (C) (magnification, ×100). The preinvasive MT-Y315/322F dysplastic nodule (C) is found at the end of dialated ducts but is a more extreme form of the hyperplasia observed in Fig. 2E and represents the solid nest in Fig. 2F. (E) Age at which a mammary tumor is first palpable in each transgenic strain. Also shown are the number of animals analyzed for each strain (n) and the median age at which tumors are palpable (T₅₀).

FIG. 5

PyV MT-associated in vitro kinase activities in mammary tumors of the various PyV MT mutants. (A) PyV MT-associated tyrosine kinase activity in tumors induced in the MT-Y315/322F, MT-Y250F, and wild-type MT strains. Mammary tumor lysates derived from MT-Y315/322F, MT-Y250F, wild-type MT, and control MMTV/Neu (N202) (20) strains were subjected to immunoprecipitation with PyV MT-specific antisera and incubated with γ-³²P-labeled ATP in the presence of exogenous enolase substrate. (B) Phosphorimager quantitation of enolase phosphorylation, is normalized to the levels of PyV MT protein. (C) PyV MT-associated PI-3′ kinase activity in tumors induced in the MT-Y315/322F, MT-Y250F, and wild-type MT strains. Mammary tumor lysates derived from MT-Y315/322F, MT-Y250F, wild-type MT, and control MMTV/Neu (N202) strains (21) were subjected immunoprecipitation with PyV MT-specific antisera and incubated with γ-³²P-labeled ATP in the presence of exogenous PI lipid. (D) Phosphorimager quantitation of the phosphorylated PI-3′ lipid, normalized to the levels of PyV MT protein.

FIG. 6

Binding properties of Shc and the p85 subunit of the PI-3′ kinase with the various mutant PyV MTs expressed in mammary tumors. (A) Shc immunoblot analysis of MT-specific immunoprecipitates (IP) isolated from MT-Y250F (lanes 2 to 4), reverted MT-Y250F (dl MT) (lanes 5 and 6), MT-Y315/22F (lanes 7 and 8), and wild-type MT (lanes 9 and 10) mammary tumors. As a nonspecific control, a protein lysate derived from an MMTV/Neu mammary tumor (lane 1) was also included. The MT-associated 52-kDa Shc protein is indicated by the arrow. (B) The same PyV MT immunoprecipitates were subjected to p85 immunoblot analyses. As a nonspecific control, a protein lysate from an MMTV/Neu tumor (lane 1) was also included (18). The p85 kDa subunit of the PI-3′ kinase is indicated by the arrow. (C) PyV MT immunoblot analysis of MT-specific immunoprecipitates isolated from the same sets of mammary tumor samples. As a nonspecific control, mammary tumor protein lysate derived from an MMTV/Neu tumor was included. Indicated by the arrows are the wild-type 56-kDa PyV MT and deleted MT (dlMT) mutant proteins.

FIG. 7

Restoration of Shc binding in the MT-Y250F mutant can occur through somatic mutations in the transgene during tumor progression. (A) Stringent RNase protection analyses on RNA samples derived from either primary mammary tumors (lanes 1 to 7) or lung metastases (lanes 8 to 13) arising in the MT-Y250F strains. Thirty micrograms of total RNA was hybridized with an antisense riboprobe (MTsn301) spanning the wild-type MT Shc binding site. The arrows indicate the expected protected bands for a wild-type MT (MT) or MT-Y250F transcript. Also shown is expected cleavage occurring at tyrosine residue 250. Note that the 1760 RNA sample (lane 5) displays an RNase protection profile that is deleted relative to the expected MT-Y250F pattern. (B) DNA sequence analyses of the transcripts detected in the MT-Y250F samples. Sequence analysis of cloned RT-PCR products from these tumors confirmed the presence of a phenylalanine residue substitution at site 250 for RNA tumor samples exhibiting the expected MT-Y250F RNase protection profile (lanes 1 to 4, 6, 9, 12, and 13 in panel A). DNA sequence analyses of the tumor samples derived from tumor samples displaying wild-type MT RNase protection profile (lanes 7, 10, and 11 in panel A) revealed the presence of a T-to-A mutation leading to conversion of the phenylalanine to tyrosine. Sequence analyses of the RT-PCR product derived from deleted transcript revealed the presence of an 18-nucleotide in-frame deletion spanning the binding site tyrosine residue (nucleotides 730 to 783). Also indicated are critical amino acid residues implicated in Shc and 14-3-3 binding. Note that the 18-nucleotide deletion restores the Shc binding consensus NPXY. The relative incidence of either the point mutation (WT MT) or the deletion (dlMT) is indicated in both primary tumors (BT) and lung metstases (LUNG METS).

FIG. 8

Elevation of ErbB-2 and ErbB-3 expression during tumor progression in the mutant PyV MT strains. (A) Immunoblot analyses of mammary epithelial hyperplasias (HP) or breast tumors (BT) from the MT-Y315/322F strain. One hundred micrograms of total protein lysate for four different hyperplasias (2480, 7184, 8786, and 8789) and tumors (7450, 7887, 8673, and 8064) was subjected to immunoblot analyses with either ErbB-2- or ErbB-3-specific antibodies. (B) Immunoblot analyses of mammary epithelial hyperplasias or breast tumors from the MT-Y250F strain. Sixty micrograms of total protein lysate for four different hyperplasias (8137, 8814, 8458, and 9086) or breast tumors (8809, 8566, 6879, and 9687) was subjected to immunoblot analyses with either ErbB-2 or ErbB-3.