Receptor tyrosine kinase inhibitors were purchased from the following sources: AEW541 from Cayman Chemical (Ann Arbor, MI, USA); AG1024 from Enzo Life Sciences (Farmingdale, NY, USA); BMS-754807 and OSI-906 from MedKoo Biosciences (Chapel Hill, NC, USA); ABT-869, AV-951, BAY 73-4506, BMS-536924, BMS-599626, brivaninb, cediranib, CYC116, E-7080, ENMD-2076, GSK1838705A, GSK1904529A, JNJ-38877605, LDN193189, MGCD265, motesanib, MP-470, NVP-TAE684, OSI-930, PF-2341066 (crizotinib), PHA-665752, SB431542, SB525334, SU11274, Tie2 kinase inhibitor, XL184, and XL880 from Selleck Chemicals (Houston, TX, USA); axitinib, dovitinib, gefitinib, GW-2580, lapatinib, lestaurtinib, masitinib, pazopanib, sorafenib, sunitinib, tandutinib, vandetanib, and vatalanib from LC Labs (Woburn, MA, USA). SU11274 alone induced G2 arrest and gefitinib/SU11274 combination sustained the SU11274-induced G2 arrest in these cells. In addition, SU11274/gefitinib combination synergistically reduced the level of ribosomal protein S6 (RPS6) in MSL subtype TNBC cells. In addition, knockdown of RPS6 itself, in Sema3g both HS578T and MDA-MB-231, markedly reduced the proliferation of these cells. Taken Reboxetine mesylate together, our data suggest that dual targeting of EGFR and MET inhibits the proliferation of MSL subtype TNBC cells through down-regulation of RPS6. (25). On the contrary, regardless of high level expression of EGFR, TNBC cells in MSL subtype including HS578T, MDA-MB-231, and MDA-MB-436 are relatively resistant to these combinations (25). Receptor tyrosine kinase crosstalk, providing surrogate or redundant pathways of cell survival against kinase targeted therapy, is one of the mechanisms of drug resistance (26C31). As an attempt to identify potential receptor tyrosine kinase inhibitors (RTKIs) which induce synthetic lethality in the presence of gefitinib, we performed an MTT screening in MDA-MB-231 cells. We further characterized a MET (mesenchymal-epithelial transition factor) inhibitor SU11274 as a synthetic lethal agent with gefitinib in MSL subtype TNBC cells. Materials and methods Cell culture and reagents Reagents for cell culture were purchased from Invitrogen (Carlsbad, CA, USA), Lonza (Basel, Switzerland), or Cellgro (Manassas, VA, USA). HS578T, MDA-MB-231, and MDA-MB-436 were obtained from the Tissue Culture Shared Resource of Georgetown University Medical Center and maintained in the Dulbeccos modified Eagles medium (DMEM) (Lonza) containing Reboxetine mesylate 10% heat inactivated fetal bovine serum (Omega Scientific, Inc., Tarzana, CA, USA) and 100 U/ml penicillin/streptomycin (Lonza). SUM149PT was maintained according to the manufacturers recommendation (Asterand, Detroit, MI, USA). The viability of cultured Reboxetine mesylate cells was monitored by the trypan blue dye exclusion method using the Luna Automated Cell Counter (Logos Biosystems, Gyunggi-Do, Korea). Receptor tyrosine kinase inhibitors were purchased from the following sources: AEW541 from Cayman Chemical (Ann Arbor, MI, USA); AG1024 from Enzo Life Sciences (Farmingdale, NY, USA); BMS-754807 and OSI-906 from MedKoo Biosciences (Chapel Hill, NC, USA); Reboxetine mesylate ABT-869, AV-951, BAY 73-4506, BMS-536924, BMS-599626, brivaninb, cediranib, CYC116, E-7080, ENMD-2076, GSK1838705A, GSK1904529A, JNJ-38877605, LDN193189, MGCD265, motesanib, MP-470, NVP-TAE684, OSI-930, PF-2341066 (crizotinib), PHA-665752, SB431542, SB525334, SU11274, Tie2 kinase inhibitor, XL184, and XL880 from Selleck Chemicals (Houston, TX, USA); axitinib, dovitinib, gefitinib, GW-2580, lapatinib, lestaurtinib, masitinib, pazopanib, sorafenib, sunitinib, tandutinib, vandetanib, and vatalanib from LC Labs (Woburn, MA, USA). Genistein and MG132 was purchase from Sigma (St. Louis, MO, USA). Stock solutions of compounds were made in dimethyl sulfoxide (DMSO) and stored at ?20C in small aliquots. Synthetic lethal screening MDA-MB-231 cells (2,500 cells/ well) in 96-well plates were treated with increasing amount of gefitinib and increasing amount of RTKIs in duplicates in a 65 matrix (Fig. 1A). In an initial screening, the highest concentration of RTKIs was 10 M. The highest concentrations of RTKIs were reduced when significant reduction of cell viability was observed in single agent treatments. The synergism was determined by calculating classification index (CI) with equation of and are the cell viability with individual agent and is the cell viability with the combination (32). We further indexed as follows: strong synergism as index 3 when the CI>1.3 at >5 combination points; medium synergism as index 2 when the CI>1.3 at 3 or 4 4 combination points; weak synergism as index 1 when the CI>1.3 at 1 or 2 2 combination points. Cell viability was determined at ~72 h after treatment of compounds by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described previously except for using 4 mg/ml of MTT solution (25,33). Open in a separate window Figure 1 Synthetic lethal screening of MDA-MB-231. (A) Schematic diagram of synthetic lethal screening. (B) RTK inhibitors (RTKIs) which showed synthetic lethality with gefitinib. Strong, medium, and weak synergisms are defined as described in Materials and.