Mechanisms of acquired resistance to insulin-like growth factor 1 receptor inhibitor in MCF-7 breast cancer cell line

No studies have yet clarified the mechanism of acquired resistance to insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase inhibitor (TKI). Our previous study of 16 breast cancer cell lines found that only MCF-7 expressed high levels of insulin receptor substrate (IRS)-1 and was sensitive to the IGF-1R-TKI NVP-AEW541. We developed a model of acquired resistance to NVP-AEW541 by continuously exposing MCF-7 cells to NVP-AEW541, naming the model MCF-7-NR. Examination of the effects of NVP-AEW541 on cell growth and IGF-1R signaling in MCF-7 and MCF-7-NR cells showed much lower levels of IRS-1 in the latter than the former. While phosphorylation of Akt was completely inhibited by administration of NVP-AEW541 (3 µM) in both cell lines, phosphorylation of S6K remained high only in MCF-7-NR. The notion of Akt-independent S6K phosphorylation in MCF-7-NR was further supported by the fact that cell growth and phosphorylation of S6K was affected by administration of the Akt inhibitor perifosine to a lesser degree in MCF-7-NR than in MCF-7. Further, the mTOR inhibitor everolimus inhibited phosphorylation of S6K and cell growth equally in both lines. Screening of MCF-7 and MCF-7-NR lines for phosphorylation of 42 receptor tyrosine kinases with and without 3 µM NVP-AEW541 showed that Tyro3 phosphorylation remained high only in MCF-7-NR cells. Gene silencing of Tyro3 using siRNA resulted in reduced cell growth, decreased phosphorylation of phosphoinositide-dependent kinase-1 (PDK-1) and protein kinase C α/βII, reduced expression of cyclin D1 in the MCF-7-NR line, with minimal effects evident in the MCF-7 line. In summary, Akt-independent activation of mTOR/S6K appears to induce acquired resistance to NVP-AEW541, and an mTOR inhibitor may have therapeutic value in overcoming it. The Tyro3 upregulation and migration of control of cell growth and cyclin D1 expression from the IGF-1R- to Tyro3-dependent signal may also cause resistance to NVP-AEW541

Direct interaction, instrumental for signaling processes, between LacCer and Lyn in the lipid rafts of neutrophil-like cells

Lactosylceramide [LacCer; \u3b2-Gal-(1-4)-\u3b2-Glc-(1-1)-Cer] has been shown to contain very long fatty acids that specifically modulate neutrophil properties. The interactions between LacCer and proteins and their role in cell signaling processes were assessed by synthesizing two molecular species of azide-photoactivable tritium-labeled LacCer having acyl chains of different lengths. The lengths of the two acyl chains corresponded to those of a short/medium and very long fatty acid, comparable to the lengths of stearic and lignoceric acids, respectively. These derivatives, designated C18-[3H]LacCer-(N3) and C24-[3H]LacCer-(N3), were incorporated into the lipid rafts of plasma membranes of neutrophilic differentiated HL-60 (D-HL-60) cells. C24-[3H] LacCer-(N3), but not C18-[3H]LacCer-(N3), induced the phosphorylation of Lyn and promoted phagocytosis. Incorporation of C24-[3H]LacCer-(N3) into plasma membranes, followed by illumination, resulted in the formation of several tritium-labeled LacCer-protein complexes, including the LacCer-Lyn complex, into plasma membrane lipid rafts. Administration of C18-[3H]LacCer-(N3) to cells, however, did not result in the formation of the LacCer-Lyn complex. These results suggest that LacCer derivatives mimic the biological properties of natural LacCer species and can be utilized as tools to study LacCer-protein interactions, and confirm a specific direct interaction between LacCer species containing very long fatty acids, and Lyn protein, associated with the cytoplasmic layer via myristic/palmitic chains