Suppression of Insulin Receptor Substrate 1 (IRS-1) Promotes Mammary Tumor Metastasis

Abstract

The insulin receptor substrate (IRS) proteins are cytoplasmic adaptors that organize signaling complexes downstream of activated cell surface receptors. Here, we show that IRS-1 and IRS-2, despite significant homology, play critical yet distinct functions in breast cancer, and we identify specific signaling pathways that are influenced by IRS-1 using the polyoma virus middle-T (PyV-MT) transgenic mouse model of mammary carcinoma and Irs-1 null (Irs1 ؊/؊ ) mice. The absence of Irs-1 expression enhanced metastatic spread significantly without a significant effect on primary tumor growth. Orthotopic transplant studies revealed that the increased metastatic potential of Irs1-deficient tumor cells is cell autonomous. Mammary tumors that developed in PyV-MT::Irs1 ؊/؊ mice exhibited elevated Irs-2 function and enhanced phosphatidylinositol 3-kinase/ Akt/mTor activity, suggesting that one mechanism by which Irs-1 impedes metastasis is to suppress Irs-2-dependent signaling. In support of this mechanism, reduction of Irs-2 expression in Irs1 ؊/؊ tumor cells restored mTor signaling to wild-type levels. PyV-MT::Irs1 ؊/؊ tumors also exhibited a significant increase in vascular endothelial growth factor expression and microvessel density, which could facilitate their dissemination. The significance of our findings for human breast cancer is heightened by our observation that Irs-1 is inactivated in wild-type, metastatic mammary tumors by serine phosphorylation. Collectively, our findings reveal that inactivation of IRS-1 enhances breast cancer metastasis and support the novel hypothesis that IRS-1 has metastasis suppressor functions for breast cancer.The insulin receptor substrate (IRS) proteins are cytoplasmic docking proteins that function as essential signaling intermediates downstream of activated cell surface receptors, including the insulin, insulin-like growth factor 1 (IGF-1), prolactin, growth hormone (GH), and vascular endothelial growth factor (VEGF) receptors, members of the integrin receptor family, and select cytokine receptors (38,68,70,78,81,85). The IRS proteins are recruited to receptors through pleckstrin homology and phosphotyrosine-binding domains in their N termini and mediate their functions by organizing signaling complexes at sites of receptor activation (81). Upon binding they are phosphorylated on tyrosine residues in their C termini, creating multiple phosphotyrosine binding motifs that recruit downstream effectors, including phosphatidylinositol 3-kinase (PI3K), Grb-2, Fyn, and Shp-2 to initiate intracellular signaling cascades (81). The IRS proteins were originally identified as substrates of the insulin receptor, and they have been predominantly studied for their role in metabolic signaling (81). Although the IRS proteins are highly homologous, there is evidence for unique functions for each of the four IRS family members. Irs-1 null mice are born small and remain runted throughout life, and these mice develop insulin resistance. However, Irs-1 null mice do not develop diabetes, because they mai...

Insulin Receptor Substrate 2-mediated Phosphatidylinositol 3-kinase Signaling Selectively Inhibits Glycogen Synthase Kinase 3β to Regulate Aerobic Glycolysis

“…Phosphorylation of p70-S6 kinase, which we showed previously was activated preferentially by Irs-2, was also reduced (Fig. 4G) (14). Phosphorylation of the Igf-1R was similar in all cells examined, indicating that neither Irs expression nor the ability of Irs-2 to activate PI3K is required for upstream receptor activation (Fig.…”

“…Specifically, in mice expressing PyMT, mammary tumor metastasis is diminished significantly in the absence of Irs-2 expression, and Irs-1 does not compensate for this loss (13). In fact, PyMT:Irs-1 Ϫ/Ϫ tumors have elevated expression and tyrosine phosphorylation of Irs-2, and these tumors are more metastatic when compared with their WT counterparts (14). Cells derived from PyMT:Irs-2 Ϫ/Ϫ tumors are also significantly less invasive and display decreased aerobic glycolysis relative to PyMT:WT or PyMT:Irs-1 Ϫ/Ϫ tumor cells in vitro (13,15).…”

“…The C termini of the IRS proteins are less conserved, and it is these C-terminal differences that likely confer upon the IRS proteins their divergent functions through specific interactions that impact localization and signaling (4). With regard to signaling, activation of the PI3K/mechanistic target of rapamycin (mTor) pathway is enhanced in PyMT:Irs-1 Ϫ/Ϫ tumors that express only Irs-2 and is significantly lower in tumors that lack Irs-2 expression, indicating that Irs-1 does not compensate fully for the activation of this pathway in vivo (14,15). Similarly, in vitro, mammary tumor cells derived from PyMT:Irs-1 Ϫ/Ϫ tumors have enhanced PI3K/mTor signaling, and this increased activity is dependent upon Irs-2 (14).…”

“…With regard to signaling, activation of the PI3K/mechanistic target of rapamycin (mTor) pathway is enhanced in PyMT:Irs-1 Ϫ/Ϫ tumors that express only Irs-2 and is significantly lower in tumors that lack Irs-2 expression, indicating that Irs-1 does not compensate fully for the activation of this pathway in vivo (14,15). Similarly, in vitro, mammary tumor cells derived from PyMT:Irs-1 Ϫ/Ϫ tumors have enhanced PI3K/mTor signaling, and this increased activity is dependent upon Irs-2 (14). The PI3K signaling pathway is one of the most commonly mutated pathways in cancer, including breast cancer (23,24).…”

Modeling Metastatic Breast Cancer in Mice

“…Finally, metastatic potential appears to be independent of hormonal fluctuations with a reproducible progression rate [67]. Several labs have employed the MMTV-PyMT model to define and substantiate a role for genes that have been implicated in tumor progression and metastasis, such as CD44 [68], uPA (69), Irs1 [70] and Plg [71] (Table 3). Also, overexpression of the blood vessel angiogenic factor VEGF-A in MMTV-PyMT mice resulted in accelerated formation of lung metastasis, not only by promoting tumor angiogenesis but also by sustaining tumor proliferation and survival [72].…”