Abstract
The insulin receptor substrate (IRS) proteins serve as essential signaling intermediates for the activation of PI3K by both the insulin-like growth factor 1 receptor (IGF-1R) and its close family member, the insulin receptor (IR). Although IRS-1 and IRS-2 share significant homology, they regulate distinct cellular responses downstream of these receptors and play divergent roles in breast cancer. To investigate the mechanism by which signaling through IRS-1 and IRS-2 results in differential outcomes, we assessed the involvement of the microtubule cytoskeleton in IRS-dependent signaling. Treatment with drugs that either stabilize or disrupt microtubules reveal that an intact microtubule cytoskeleton contributes to IRS-2- but not IRS-1-mediated activation of AKT by IGF-1. Proximal IGF-1R signaling events, including IRS tyrosine phosphorylation and recruitment of PI3K, are not inhibited by microtubule disruption, indicating that IRS-2 requires the microtubule cytoskeleton at the level of downstream effector activation. IRS-2 colocalization with tubulin is enhanced upon Taxol-mediated microtubule stabilization, which, together with the signaling data, suggests that the microtubule cytoskeleton may facilitate access of IRS-2 to downstream effectors such as AKT. Of clinical relevance is that our data reveal that expression of IRS-2 sensitizes breast carcinoma cells to apoptosis in response to treatment with microtubule-disrupting drugs, identifying IRS-2 as a potential biomarker for the response of breast cancer patients to Vinca alkaloid drug treatment.
Highlights
The insulin receptor substrate (IRS) proteins serve as essential signaling intermediates for the activation of PI3K by both the insulin-like growth factor 1 receptor (IGF-1R) and its close family member, the insulin receptor (IR)
IRS-2 colocalization with tubulin is enhanced upon Taxol-mediated microtubule stabilization, which, together with the signaling data, suggests that the microtubule cytoskeleton may facilitate access of IRS-2 to downstream effectors such as AKT
IRS signaling was measured by assessing the phosphorylation status of AKT, a downstream signaling effector of PI3K, because the IRS proteins are required for the recruitment and activation of PI3K by the IGF-1R [4, 5, 26]
Summary
To investigate the role of the microtubule cytoskeleton in IRS-dependent IGF-1R signaling, MDA-MB-231 cells were stimulated with IGF-1 after short-term (30 min to 1 h) treatment with paclitaxel (Taxol) or nocodazole. The -fold difference in AKT activation observed in shGFP and shIRS2 cells upon microtubule disruption increased with time of stimulation, indicating that microtubules are required to sustain IRS2-dependent signaling (Fig. 2E). Similar to MDA-MB-231 cells, cell death in response to Taxol treatment was not dependent upon Irs expression in PyMT mammary tumor cells (Fig. 5, A and B). PyMT:Irs1Ϫ/Ϫ cells exhibited G2/M arrest, and restoration of IRS-1 expression promoted an increase in G1/G0 cells (Fig. 5, F and G), similar to the profile of the IRS2-deficient cells that signal only through IRS1. These data indicate that breast carcinoma cells that express and signal through IRS-2 are more sensitive to drugs that depolymerize microtubules and respond by undergoing cell cycle arrest and increased cell death, whereas the Taxol response is not determined by IRS-2 expression. The BIM-EL and BIM-L isoforms of BIM were expressed at elevated levels in shGFP cells compared with shIRS2 cells, and expression increased upon co-treatment with MK2206 in shIRS2 cells (Fig. 7B)
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