Abstract
Aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway supports growth of many tumors including those of breast, lung, and prostate. Resistance of breast cancer cells to targeted chemotherapies including tyrosine kinase inhibitors (TKI) has been linked to persistent PI3K activity, which may in part be due to increased membrane expression of epidermal growth factor (EGF) receptors (HER2 and HER3). Recently we found that proteins of the RGS (regulator of G protein signaling) family suppress PI3K activity downstream of the receptor by sequestering its p85alpha subunit from signaling complexes. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. RGS16 overexpression in MCF7 breast cancer cells inhibited EGF-induced proliferation and Akt phosphorylation, whereas shRNA-mediated extinction of RGS16 augmented cell growth and resistance to TKI treatment. Exposure to TKI also reduced RGS16 expression in MCF7 and BT474 cell lines. RGS16 bound the amino-terminal SH2 and inter-SH2 domains of p85alpha and inhibited its interaction with the EGF receptor-associated adapter protein Gab1. These results suggest that the loss of RGS16 in some breast tumors enhances PI3K signaling elicited by growth factors and thereby promotes proliferation and TKI evasion downstream of HER activation.
Highlights
The role of the PI3K3 pathway in cell proliferation and survival, adhesion, metabolism, migration, drug resistance, and cytoskeletal rearrangement is well documented [1,2,3]
Further analysis showed that serum-starved MCF7 cells with reduced expression of RGS16 proliferated significantly more than control cells did over time upon exposure to medium containing either FBS (Fig. 1C) or epidermal growth factor (EGF) (Fig. 1E)
MCF7 cells expressing RGS16-specific shRNA were significantly more resistant to the antiproliferative effect of this compound than cells expressing the control shRNA (Fig. 3B). Because these findings indicated a relationship between quantities of RGS16 and resistance of MCF7 cells to PD168393, we explored the relationship between RGS16 expression and phosphatidylinositol 3-kinase (PI3K) activity in wild type MCF7 cells by examining Akt phosphorylation after tyrosine kinase inhibitors (TKI) treatment
Summary
Cell Culture, Plasmids, Lentiviral Transduction, and Gene Transfection—HEK293T cells were cultured in complete Dulbecco’s modified Eagle’s medium (Invitrogen) (containing 10% (v/v) fetal bovine serum, 1% penicillin and streptomycin at 37 °C and 5% CO2). MCF7 cells expressing either scrambled control or RGS16-specific shRNAs were constructed and maintained in complete RPMI supplemented with neomycin (0.4 mg/ml) as described elsewhere [18]. The interaction of GST-RGS16 to phosphorylated His-p85␣/p110␣ (200 ng, Jena Bioscience) was assessed by incubation with nickel-nitriloacetic acid-agarose beads (Qiagen) in buffer containing 20 mM HEPES, pH 8, 150 mM NaCl, 6 mM MgCl2, 10% glycerol, 0.04% Triton X-100, and 10 mM imidazole. For co-immunoprecipitations, the cells were processed in buffer containing 50 mM Tris, pH 7.5, 250 mM NaCl, 5 mM MgCl2, 5% glycerol, 1% Triton X-100, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor tablets followed by immunoprecipitation with Myc antibody coupled to protein G-Sepharose (Fast Flow 4B; Amersham Biosciences). After incubation for another 24 h, the cells were serum-starved for an additional
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