Monolayers were washed with PBS then incubated in medium containing inhibitor or vehicle for 2 h followed by treatment with growth factor for 48 h. cells secrete IGFs (5, 6). In certain settings, the IGF1R is required for transformation by other agents (including the EGFR) (7), and the IGF1R encourages and supports properties of the transformed phenotype. In addition, IGF-1 is also involved in other aspects of cancer progression, Ibrutinib-biotin such as angiogenesis and inflammation (8). Recent studies have demonstrated that elevated serum IGF-1 levels are associated with increased risk of a variety of epithelial cancers (9C12), and that reduced IGF-1 levels may be protective (13). Thus, it has been proposed that reduction of IGF signaling in some cancer types may have therapeutic benefit (4, 14). Augmenting this concept is the Ibrutinib-biotin recent demonstration that the IGF1R can promote therapeutic resistance to multiple treatment approaches including radiation, cytotoxic chemotherapy, and molecular targeted therapy (15C18). The significance of these findings in HNSCC is, as yet, unknown. In the present study, we demonstrate that activation of the IGF1R in HNSCC cells can overcome growth inhibition caused by EGFR-TKIs via a primarily anti-apoptotic mechanism. This validates the concept that, in the context of EGFR blockade, an alternate growth factor can continue to sustain tumor cell growth, and suggests that IGF1R signaling may be a mechanism of resistance to targeted anti-EGFR therapy Thus, coinhibition of the EGFR and the IGF1R may lead to increased clinical response rates. MATERIALS AND METHODS Reagents Recombinant human IGF-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), des[1C3]IGF-1 from GroPep (Adelaide, Australia), EGF from Sigma (St. Louis, MO), and fetal bovine serum (FBS) from Invitrogen (Carlsbad, CA). U0126, PD158780 and LY294002 were obtained from EMD Biosciences (San Diego, CA), gefitinib and erlotinib from LC Laboratories (Woburn, MA), and PQ401 from Tocris Bioscience (Ellisville, MO). Anti-IGF1R was acquired from Santa Cruz Biotechnology, anti-p-Erk from Sigma, anti-p-Tyr and anti-PARP from BD Biosciences (San Jose, CA), anti-Akt, anti-p-Akt (S473), anti-Erk, anti-p-IGF1R, and anti-p-EGFR from Cell Signaling Technology (Beverly, MA). Tissue Culture & Human Tissue Specimens HNSCC cell lines included SCC-25, SCC-9, Cal27, and FaDu cells obtained from ATCC (Manassas, VA), and SCC-61 and UNC-7 cells kindly provided by Dr. Wendell Yarbrough (Vanderbilt University, Nashville, TN). These were selected to evaluate a variety of anatomic sites in the upper aerodigestive tract and because they exhibit a wide range of IGF1R expression. None of these cell lines had detectable basal IGF1R activation. They were grown in Rabbit polyclonal to PLEKHA9 D-MEM/F12 supplemented with 400 ng/mL hydrocortisone and 5% FBS at 37C and 5% CO2. for 15 min at 4C and sample buffer containing 0.1 M DTT was added. Proteins were resolved on an 8% SDS-polyacrylamide gel then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). The PVDF was quenched, incubated with primary antibody, washed and incubated with secondary antibody according to the manufacturers instructions. Proteins were visualized by reaction with Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Millipore). Flow Cytometry Monolayers were grown to 70% confluence and then incubated in 0.5% serum for 24 h. Monolayers were washed with PBS then incubated in medium containing inhibitor or vehicle for 2 h followed by treatment with growth factor for 48 h. The medium from each well was collected; cell monolayers were trypsinized, resuspended in the corresponding medium, centrifuged at 1,000 for 7 min at 4C, Ibrutinib-biotin washed once with cold PBS, and resuspended in Annexin V-FITC and propidium iodide according to the manufacturers recommendations (Millipore). Flow cytometry was performed in the UVA Flow Cytometry Core Facility within 24 h of completion of the stimulation. CellTiter 96? Aqueous Cell Proliferation Assay Cells were plated at 5,000 cells/well in a Ibrutinib-biotin 96-well plate, grown for 24 h and serum-starved for 24 h. Cells were washed with PBS, incubated for 2 h with inhibitor, and stimulated for 48 h with growth factor. MTS and PMS were added to each well according to the manufacturers protocol (Promega,.