Involvement of Insulin Receptor Substrate 2 in Mammary Tumor Metastasis

Analysis of IRS-2 involvement in PyV-MT mammary tumor development. Female IRS-2^+/− mice were bred with male IRS-2^+/− mice that were also transgenic (+/−) for the PyV-MT antigen. (A) Female WT and IRS-2^−/−, PyV-MT^+/− mice were monitored for the age at which mammary tumors were first palpable. (B) Female WT and IRS-2^−/− PyV-MT^+/− mice were analyzed for their total tumor burden at 100 or 110 days of age. Shown is the mean tumor volume (± standard errors of the means) from each of the time points. The number of mice analyzed for each time point is indicated. WT at 100 days versus IRS-2 at 100 days, P = 0.69; WT at 110 days versus IRS-2 at 110 days, P = 0.68.

Whole-mount analysis and histological characteristics of WT (A, C, G) and IRS-2 null (B, D-F, H) PyV-MT mammary tumors. (A and B) The number 4 inguinal mammary glands from WT and IRS-2^−/−, PyV-MT^+/− mice were fixed and stained with carmine alum stain. Bar, 1 mm. (C and D) Solid nodular high-grade tumors that lack glandular structure and contain minimal stroma. (E) Glandular, low-grade tumor region from an IRS-2^−/− mouse. Myoepithelial cells are still present surrounding the dilated glands, as demonstrated by the positive smooth-muscle actin staining (F). (C-F) Bar, 100 μm. (G and H) Mammary tumors from WT (G) and IRS-2^−/− (H) mice stained with CD31 to detect tumor angiogenesis. Bar, 50 μm.

Biochemical analysis of representative WT and IRS-2^−/−, PyV-MT mammary tumors. Aliquots of mammary tumor extracts that contained equivalent amounts of total protein were immunoblotted with IRS-1-, IRS-2-, β4-, p85-, cyclin D1-, ERα-, and PR-specific antibodies. Aliquots of mammary tumor extracts that contained equivalent amounts of total protein were immunoprecipitated and then immunoblotted with antisera specific for the IGF-1R β subunit. WT, mice that are homozygous positive for the IRS-2 gene; IRS-2^−/−, mice that are homozygous null for the IRS-2 gene.

Analysis of IRS-2 involvement in PyV-MT mammary tumor metastasis. (A) Lungs from female WT and IRS-2^−/−, PyV-MT^+/− mice were sectioned and screened microscopically for the presence of metastatic lesions. Five representative sections from each lung were analyzed. Shown is the percentage of the mice that scored positively for metastasis at each time point. (B) PyV-MT mRNA was amplified and quantitated from the lungs of 110-day-old WT (1 to 9) and IRS-2^−/− (10 to 16) PyV-MT-expressing mice by RQ-PCR. Number 17, control RNA from the lungs of a mouse lacking PyV-MT expression. WT, mice that are homozygous positive for the IRS-2 gene; IRS-2^−/−, mice that are homozygous null for the IRS-2 gene.

Characterization of WT and IRS-2-null mammary tumor cell lines. (A) Aliquots of cell extracts from WT, IRS-2^−/−, and IRS-1^−/− mammary tumor cells that contained equivalent amounts of total protein were immunoblotted with antibodies specific for IRS-1, IRS-2, the β4-integrin subunit, and the p85 regulatory subunit of PI3K as a loading control. PyV-MT mammary tumor cells were stimulated for 10 min with IGF-1 (100 ng/ml), and aliquots of cell extracts that contained equivalent amounts of total protein were immunoprecipitated with antisera specific for the IGF-1R β subunit. The immunoprecipitates were immunoblotted with phosphotyrosine-specific antibodies (pIGF-1Rβ). The immunoblots were subsequently stripped and reprobed with IGF-1R β-subunit-specific antisera (IGF-1R). Two independent subclones for each tumor genotype are shown. (B) H&E-stained section from an IRS-2^−/− tumor grown orthotopically in the mammary fat pad of a WT mouse. The tumors are solid, with little glandular structure, and are high grade. (C) CD31 staining of an IRS-2^−/− tumor grown orthotopically in the mammary fat pad of a WT mouse. WT, mice that are homozygous positive for the IRS-2 gene; IRS-2^−/−, mice that are homozygous null for the IRS-2 gene; IRS-1^−/−, mice that are homozygous null for the IRS-1 gene.

Analysis of WT and IRS-2-null mammary tumor cell function. (A) Mammary tumor cells were assayed for their ability to invade Matrigel. The data shown are from two individual subclones of each cell type and are the mean values (± standard deviations) of three (IRS-1^−/−) or four (WT and IRS-2^−/−) independent assays done in triplicate. (B) Mammary tumor cells were maintained in culture medium containing either 10% FCS or 0.1% BSA for 24 h. Adherent and nonadherent cells were collected and stained with propidium iodide. The DNA content of the cells was analyzed by flow cytometry. The data shown represent the percentage of sub-G₁ cells in the presence of 0.1% BSA and 10% FCS and are the mean (± standard deviations) of three (WT) or four (IRS-1^−/− and IRS-2^−/−) independent experiments. (C) WT and IRS-2^−/− mammary tumor cells were maintained in culture medium containing either 10% FCS or 0.1% BSA for 24 h. Adherent cells were incubated in the presence of FITC-VAD-FMK to detect apoptotic cells. Representative images of the cells maintained in 0.1% BSA are shown. The Hoechst nuclear stain verifies the apoptotic morphology of the fluorescein isothiocyanate-labeled cells. WT, mice that are homozygous positive for the IRS-2 gene; IRS-2^−/−, mice that are homozygous null for the IRS-2 gene; IRS-1^−/−, mice that are homozygous null for the IRS-1 gene.