Abrogating phosphorylation of eIF4B is required for EGFR and mTOR inhibitor synergy in triple-negative breast cancer

Abstract

Purpose Triple negative breast cancer (TNBC) patients suffer from a highly malignant and aggressive disease. They have a high rate of relapse and often develop resistance to standard chemotherapy. Many TNBCs have elevated epidermal growth factor receptor (EGFR) but are resistant to EGFR inhibitors as monotherapy. In this study we sought to find a combination therapy that could sensitize TNBC to EGFR inhibitors. Methods Phospho-mass spectrometry was performed on the TNBC cell line, BT20, treated with 0.5 μM gefitinib. Immunoblotting measured protein levels and phosphorylation. Colony formation and growth assays analyzed the treatment on cell proliferation while MTT assays determined the synergistic effect of inhibitor combination. A dual luciferase reporter gene plasmid measured translation. All statistical analysis was done on CalucuSyn and GraphPad Prism using ANOVAs. Results Phospho-proteomics identified the mTOR pathway to be of interest in EGFR inhibitor resistance. In our studies, combining gefitinib and temsirolimus decreased cell growth and survival in a synergistic manner. Our data identified eIF4B, as a potentially key fragile point in EGFR and mTOR inhibitor synergy. Decreased eIF4B phosphorylation correlated with drops in growth, viability, clonogenic survival, and cap-dependent translation. Conclusions Taken together these data suggest EGFR and mTOR inhibitors abrogate growth, viability, and survival via disruption of eIF4B phosphorylation leading to decreased translation in TNBC cell lines. Further, including an mTOR inhibitor along with an EGFR inhibitor in TNBC with increased EGFR expression should be further explored. Additionally, translational regulation may play in important role in regulating EGFR and mTOR inhibitor synergy and warrants further investigation.

Inhibitory effects of tetrandrine on epidermal growth factor-induced invasion and migration in HT29 human colorectal adenocarcinoma cells

“…In addition, the expression levels of EGFR, HER2 and HER3 were determined in primary tumors of colorectal cancer cells, and corresponded with lymph node metastases and liver metastases (42,43). The dysregulation of human EGFR pathways by the overexpression or constitutive activation promote s tumor processes, angiogenesis and metastasis in several types of human cancer (4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Previous studies have demonstrated that tetrandrine inhibits cell metastasis in 4T1 breast cancer cells (18) and CT26 colorectal cancer cells in vivo (25).…”

“…Abnormal protein activity and/or expression levels of EGFR have been correlated with the etiology of several types of human cancer, including colorectal cancer (4,5), non-small cell lung cancer (6,7), breast cancer (8,9), head and neck squamous cell carcinoma (10,11), pancreatic cancer (12) and brain cancer (13). EGFR-targeted therapy has been validated in human colorectal cancer (14), and the chemotherapeutics include pharmacological agents, including cetuximab (Erbitux), which is an EGFR inhibitor (15).…”

Enrichment Strategies for Identification and Characterization of Phosphoproteome

“…TiO 2 techniques are usually performed using self-packed or commercial products with microcolumn, 17,56 or beads. 20,25,40 To improve the reproducibility of TiO 2 for phosphopeptide enrichment, Tape and co-worker described that the automatic phosphopeptide enrichment using magnetic TiO 2 beads with IMAC. 40 The automated enrichment system with magnetic TiO 2 beads is achieved by controlling 96-well plates at the eight carousel positions.…”

“…59 In many reports, acidic loading buffers on TiO 2 have been tested and optimized with the modifiers including 2, 5-dihydroxybenzoic acid (DHB), malic acid, lactic acid, and other acidic reagents to enhance the enrichment efficiency of phosphopeptide. [17][18][19][20][21] Capacity of acidic modifier in loading buffer for removing non-specific binding decreased as follows: DHB = salicylic acid = phthalic acid > benzoic acid = cyclohexanecarboxylic acid > phosphoric acid > TFA > acetic acid. 53 In recently, specificity of TiO 2 chromatography was optimized by adding glycerol 60 or citric acid.…”

“…A variety of enrichment and fractionation methods in phosphoproteomics have been reported including the enrichment with immobilized metal affinity chromatography (IMAC) 6,[14][15][16] and metal oxide affinity chromatography (MOAC) as well as titanium dioxide (TiO 2 ), [17][18][19][20][21][22][23][24][25] and fractionation with strong cation exchange (SCX), 26,27 hydrophilic interaction chromatography (HILIC), [28][29][30] and electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). [31][32][33] More recently, the two sequential steps of preparation methods are commonly used in large scale phosphoproteomics.…”