TY - JOUR
T1 - Antitumor effect of insulin-like growth factor-1 receptor inhibition in head and neck squamous cell carcinoma
AU - Lehman, Christine E.
AU - Khalil, Ashraf A.
AU - Axelrod, Mark J.
AU - Dougherty, Michael I.
AU - Schoeff, Stephen S.
AU - Taniguchi, Linnea E.
AU - Mendez, Rolando E.
AU - David, Abel P.
AU - McGarey, Patrick O.
AU - Hubbard, Matthew A.
AU - Donaldson, Lane
AU - Frierson, Henry F.
AU - Stelow, Edward B.
AU - Bekiranov, Stefan
AU - Wulfkuhle, Julia D.
AU - Petricoin, Emanuel F.
AU - Gioeli, Daniel G.
AU - Jameson, Mark J.
N1 - Funding Information:
Six-well plates were seeded with 250 cells per well. Cells were allowed to adhere overnight and then treated with inhibitor or vehicle and incubated for 14 days, followed by colony fixation in methanol for 15 minutes. Plates were stained with 2% crystal violet, and colonies were counted using ImageJ software (National Institutes of Health, Bethesda, MD). des[1-3]IGF-1 (desIGF1, an N-terminally truncated IGF-1 with very low affinity for IGF-binding proteins) was obtained from Cell Sciences (Canton, MA); alamarBlue, DMEM/F-12 with HEPES, and fetal bovine serum (FBS) from Invitrogen (Carlsbad, CA); OSI-906 and BMS-754807 from Chemietek (Indianapolis, IN); and anti-IGF1Rβ, anti-pIGF1Rβ (Tyr1135/1136), anti-Akt, and anti-pAkt (S473) from Cell Signaling Technology (Beverly, MA). HNSCC cell lines SCC-25 (OC), SCC-9 (OC), Cal27 (OC), and FaDu (hypopharyngeal) were obtained from ATCC (Manassas, VA). SCC-61 (OC), UNC-7 (OC), and UNC-10 (OC) cells were kindly provided by Dr. Wendell Yarbrough (Vanderbilt University, Nashville, TN). OSC-19 (OC) cells were kindly provided by Dr. Jeffrey Myers (The University of Texas MD Anderson Cancer Center, Houston, TX). All cell lines were originally generated from OCSCC except for FaDu cells, which originated from a hypopharyngeal primary. None of the cell lines are human papillomavirus-related. SCC-25GR1 cells were generated by culturing SCC-25 cells with escalating doses of gefitinib up to 5 μM. Cell line identities were confirmed by DNA fingerprinting (University of Arizona). Cells were cultured in DMEM/F-12 with 5% FBS and 400 ng/mL hydrocortisone and maintained in a 37°C humidified 5% CO2 incubator. All cell lines were routinely tested and found to be free of mycoplasma using MycoAlert (Lonza, Allendale, NJ). Two hundred remnant OCSCC samples were obtained with the approval of the University of Virginia Institutional Review Board for Human Subject Research and used to create a tissue microarray (TMA) annotated with clinical outcomes. The TMA was stained with anti-IGF1Rβ. IGF1R expression was independently scored by two senior pathologists with expertise in HNSCC histology. High IGF1R expression was defined as a composite score ≥ 3. Kaplan-Meier survival curves were produced using SPSS Statistics 24 (IBM Corp., Armonk, NY) and correlated with IGF1R expression level. Statistical differences were evaluated using the log-rank test. 96-well plates were seeded with 5 thousand cells per well in DMEM/F-12 with 0.5% FBS. Cells were allowed to adhere overnight and were then treated with inhibitor or vehicle for 72 hours. Cells were incubated with alamarBlue reagent according to the manufacturer's protocol for 2 hours. Fluorescent emission at 540 nm was read using a Synergy2 plate reader (Biotek, Winooski, VT). 12-well plates were seeded with 50 thousand cells per well, allowed to adhere overnight, and were then treated with inhibitor or vehicle for 72 hours. The media from each well was collected; adherent cells were then trypsinized and collected with the corresponding conditioned media. Cell suspensions were centrifuged at 3 thousand g for 3 minutes and then resuspended in 1 mL PBS. A 100 μL sample of the cell suspension was stained with 100 μL trypan blue (Invitrogen) to assess live and dead cell numbers using the TC20 Automated Cell Counter (Bio-Rad, Hercules, CA). Six-well plates were seeded with 250 cells per well. Cells were allowed to adhere overnight and then treated with inhibitor or vehicle and incubated for 14 days, followed by colony fixation in methanol for 15 minutes. Plates were stained with 2% crystal violet, and colonies were counted using ImageJ software (National Institutes of Health, Bethesda, MD). After treatment, cells grown in 6-cm dishes were washed with ice-cold PBS containing 2 mmol/L sodium orthovanadate, collected, resuspended in lysis buffer, and processed as described previously. Proteins were resolved on 12% SDS polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). After blocking and treatment with primary and secondary antibody, proteins were detected using the Odyssey imaging system (LI-COR Biosciences, Lincoln, NE). Cells were cultured in 6-cm dishes and grown to 70% confluence, starved with 0.5% FBS for 24 hours, and treated with inhibitor or vehicle for 72 hours. Conditioned medium from each well was collected, and then adherent cells were trypsinized and collected with the corresponding media. Samples were centrifuged at 4°C, washed with cold PBS, and resuspended in fluorescein isothiocyanate-conjugated anti-cleaved poly(ADP)ribose polymerase (PARP) or anti-cleaved caspase-3 antibody and phycoerythrin according to the manufacturer's recommendations (Millipore). Flow cytometry was conducted in the Flow Cytometry Core Facility, University of Virginia. Cal27, SCC61, SCC9, UNC10, FaDu, OSC19, and SCC25 were treated with 100 nM BMS-754807 or 1 μM OSI-906 for 2 hours, followed by a 15-minute stimulation with 1 nM des-IGF1. Pathway activation mapping was performed by reverse phase protein array (RPPA). Cells were lysed with 2.5% 2-mercaptoethanol (Sigma, St. Louis, MO) in 1:1 Tissue Protein Extraction Reagent (T-PER, Thermo Scientific, Waltham, MA) and 2X Tris-Glycine SDS Sample Buffer (Invitrogen). The lysates were printed in triplicate on glass-backed nitrocellulose array slides (Grace Bio-Labs, Bend, OR) using an Aushon 2470 arrayer (Aushon BioSystems, Burlington, MA) equipped with 185 μm pins. Arrays were blocked (I-Block, Applied BioSystems, Foster City, CA) for 1 hour and subsequently probed with primary antibodies. Detection was performed using a fluorescence-based tyramide signal amplification strategy using Streptavidin-conjugated IRDye680 (LI-COR) as detection reagent. All antibodies were previously validated for single band specificity and ligand-induction by immunoblotting. Each array was scanned using a Tecan LS laser scanner (Tecan, Durham, NC). Spot intensity was analyzed; data were normalized to total protein; and a standardized value was generated for each sample on the array by MicroVigene software V2.999 (VigeneTech, North Billerica, MA). Normalized log2 RPPA data were subjected to a moderated paired t test using the Bioconductor in R. The limma package was used to perform paired t tests to determine differentially expressed genes and to derive false discovery rate-adjusted P values. Figure was generated using the ggplot2 package. Statistical significance was calculated using Student 2-tailed t tests, unless indicated.
Publisher Copyright:
© 2019 The American Laryngological, Rhinological and Otological Society, Inc.
PY - 2020/6/1
Y1 - 2020/6/1
N2 - Objectives: The insulin-like growth factor-1 receptor (IGF1R) has been implicated in therapeutic resistance in head and neck squamous cell carcinoma (HNSCC), and small molecule tyrosine kinase inhibitors (TKIs) of IGF1R activity may have anticancer activity. Therefore, the relationship between survival and IGF1R expression was assessed for oral cavity (OC) cancer, and the antitumor effects of two IGF1R-TKIs, OSI-906 and BMS-754807, were evaluated in HNSCC cell lines in vitro. Methods: Clinical outcome data and tissue microarray immunohistochemistry were used to generate IGF1R expression-specific survival curves. Immunoblot, alamarBlue proliferation assay, trypan blue exclusion viability test, clonogenic assay, flow cytometry, and reverse phase protein array (RPPA) were used to evaluate in vitro responses to IGF1R-TKIs. Results: For patients with stage III/IV OCSCC, higher IGF1R expression was associated with poorer overall 5-year survival (P = 0.029). Both BMS-754807 and OSI-906 caused dose-dependent inhibition of IGF1R and Akt phosphorylation and inhibited proliferation; BMS-754807 was more potent than OSI-906. Both drugs reduced HNSCC cell viability; only OSI-906 was able to eliminate all viable cells at 10 μM. The two drugs similarly inhibited clonogenic cell survival. At 1 μM, only BMS-754807 caused a fourfold increase in the basal apoptotic rate. RPPA demonstrated broad effects of both drugs on canonical IGF1R signaling pathways and also inhibition of human epidermal growth factor receptor-3 (HER3), Src, paxillin, and ezrin phosphorylation. Conclusion: OSI-906 and BMS-754807 inhibit IGF1R activity in HNSCC cell lines with reduction in prosurvival and proliferative signaling and with concomitant antiproliferative and proapoptotic effects. Such antagonists may have utility as adjuvants to existing therapies for HNSCC. Level of Evidence: NA Laryngoscope, 130:1470–1478, 2020.
AB - Objectives: The insulin-like growth factor-1 receptor (IGF1R) has been implicated in therapeutic resistance in head and neck squamous cell carcinoma (HNSCC), and small molecule tyrosine kinase inhibitors (TKIs) of IGF1R activity may have anticancer activity. Therefore, the relationship between survival and IGF1R expression was assessed for oral cavity (OC) cancer, and the antitumor effects of two IGF1R-TKIs, OSI-906 and BMS-754807, were evaluated in HNSCC cell lines in vitro. Methods: Clinical outcome data and tissue microarray immunohistochemistry were used to generate IGF1R expression-specific survival curves. Immunoblot, alamarBlue proliferation assay, trypan blue exclusion viability test, clonogenic assay, flow cytometry, and reverse phase protein array (RPPA) were used to evaluate in vitro responses to IGF1R-TKIs. Results: For patients with stage III/IV OCSCC, higher IGF1R expression was associated with poorer overall 5-year survival (P = 0.029). Both BMS-754807 and OSI-906 caused dose-dependent inhibition of IGF1R and Akt phosphorylation and inhibited proliferation; BMS-754807 was more potent than OSI-906. Both drugs reduced HNSCC cell viability; only OSI-906 was able to eliminate all viable cells at 10 μM. The two drugs similarly inhibited clonogenic cell survival. At 1 μM, only BMS-754807 caused a fourfold increase in the basal apoptotic rate. RPPA demonstrated broad effects of both drugs on canonical IGF1R signaling pathways and also inhibition of human epidermal growth factor receptor-3 (HER3), Src, paxillin, and ezrin phosphorylation. Conclusion: OSI-906 and BMS-754807 inhibit IGF1R activity in HNSCC cell lines with reduction in prosurvival and proliferative signaling and with concomitant antiproliferative and proapoptotic effects. Such antagonists may have utility as adjuvants to existing therapies for HNSCC. Level of Evidence: NA Laryngoscope, 130:1470–1478, 2020.
KW - Head and neck squamous cell carcinoma
KW - insulin-like growth factor-1 receptor
KW - targeted molecular therapy
KW - tyrosine kinase inhibitor
UR - http://www.scopus.com/inward/record.url?scp=85071230230&partnerID=8YFLogxK
U2 - 10.1002/lary.28236
DO - 10.1002/lary.28236
M3 - Article
C2 - 31433065
AN - SCOPUS:85071230230
SN - 0023-852X
VL - 130
SP - 1470
EP - 1478
JO - Laryngoscope
JF - Laryngoscope
IS - 6
ER -