ment resulting in the overexpression of a fusion protein assembled from SND1 and BRAF. SND1 is a multi-functional ribonuclease comprising part of the RNA-induced silencing (RISC) complex [15,16,17]. It plays a role in the function of microRNAs (miRNA) and can regulate transcription through transcriptional co-activa- tion, RNA interference, RNA splicing, and RNA editing [18]. Increased expression of SND1 is associated with colon cancer and prostate cancer [15]. Overexpression of SND1 also promotes angiogenesis in hepatocellular carcinoma xenograft models through induction of angiogenic factors [19]. BRAF is a proto-oncogene that promotes cell growth and proliferation by transducing signals from growth factor receptors as part of the MAP kinase pathway via MEK and ERK. Mutations to this protein such as G469A, E586K, V600E, and K601E can increase BRAF catalytic activity [20]. BRAF V600E has been implicated in papillary thyroid carcinoma [21], colorectal carci- noma [22], and melanoma [23]. Similarly, various fusions of BRAF have been implicated in cancer such as pediatric astrocytomas (KIAA1549-BRAF; exons 9/11) [24], melanocytic nevi (FCHSD1-BRAF; exon 9) [25], papillary thyroid carcinomas (AKAP9-BRAF; exon 9) [26], prostate cancer (SLC45A-BRAF; exon 8) [27], and gastric cancer (AGTRAP-BRAF; exon 8) [27]. In our model, the resultant SND1-BRAF fusion protein contains a constitutively active BRAF kinase which increases phosphorylation of ERK. Functionally, this fusion protein signals downstream of c-Met and bypasses its inhibition by METi. We demonstrate that a MEK inhibitor or the combination of c-Met and RAF inhibitors suppresses phosphorylation of ERK and reduces the proliferation of the resistant clones in vitro. Together, these findings suggest that targeted inhibitors can be bypassed at multiple levels and that inhibiting the nodes where the signal converges might be a more robust strategy for therapy. Materials and Methods Generation of Resistant Clones GTL16 cells were seeded in a 96-well plate at a density of 20,000 cells per well and treated with 0.5 mM PF-0461903 (METi), a selective c-Met kinase inhibitor (Figure S1). The concentration of METi was progressively increased once every two weeks by 0.5 mM increments until a final concentration of 2.5 mM. METi was replenished every 3–5 days as needed. After a total of 4 months, wells with surviving cells at 2.5 mM METi were trypsinized, and subcloned using cloning rings. Three clonal lines, designated GTL16R1, GTL16R3 and GTL16S5 were expanded for further study. The GTL16 gastric cancer cells were a gift from Dr. Paolo Comoglio from the University of Torino Medical School, Candiolo, Italy. Cell Viability Assays Cell viability assays were performed using the GTL16 line and GTL16 resistant clones. Briefly, cells were seeded at a density of 4000 cells/well into 96-well plates and allowed to adhere overnight. The following day, cells were treated with either single agent or combination of METi and a Raf inhibitor, PF-04880594 (RAFi) (Figure S2) or a MEK inhibitor, PD-0325901 (MEKi) [28] as indicated in the figures. For single agent treatment, we administered compound in nine serial concentrations (progres- sively decreasing from 10 mM to 153 pM by a 4-fold ratio) yielding a full sigmoidal curve. For combinations, we added a second compound (RAFi or MEKi) in five serial concentrations ranging from 10 mM to 39 nM by a 4-fold dilution ratio for RAFi, and from 10 nM to 40 pM by a 4-fold dilution ratio for MEKi. After an additional 3-day incubation at 37uC, 30 ml of Cell Titer Glo BRAF Fusion Confers Resistance to c-Met Inhibitor (Promega, Madison, WI) was added to indirectly measure cell viability/proliferation using an Envision multi-reader (Perkin- Elmer, Waltham, MA). The BLISS independence algorithm was used to calculate theoretical combination additivity [29]. The DBLISS score was calculated as the difference between BLISS and experimentally observed inhibition and ranges from 0 (additive) to 1 (synergistic). Graphical surface plots were rendered using the Lattice R library [30]. Data Processing Briefly, readings from the Envision multi-reader were processed using the R package ’drc’ (drug response curves) to generate IC50 values [31]. Cell counts were first adjusted by subtracting the average of the baseline cell counts from untreated cells assessed one day after cell seeding. The Tumor Cell Growth Inhibition (TGI) score for each compound concentration was calculated as: TGI~1-ððwell count-baselineÞ=ðplate control mean-baselineÞÞ . The drc package was then used to fit the TGI as a function of the concentration of the compounds. A four-parameter logistic model was used to fit the dose response curves and infer the IC50, slope, and upper and lower limits. Analysis of Cell Signaling by Inhibitor Treatment GTL16 and resistant clones were grown to approximately 80% confluency and then treated with inhibitor compounds or DMSO vehicle control at the indicated concentrations and time duration. For Western immunoblotting, cells were rinsed with phosphate buffered saline (PBS) and subsequently lysed with cell lysis buffer (Cell Signaling, Danvers, MA) supplemented with 2 mM sodium orthovanadate and 2 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates were harvested, sonicated briefly, and incubated for 1 hr at 4uC. Lysates were then centrifuged at 14,000 rpm for 10 min at 4uC to pellet cell debris, and supernatant was collected. Reverse Phase Protein Array (RPPA) GTL16 and resistant clones were treated with 2.5 mM METi or vehicle control for 1 hr and lysed with CLB lysis buffer according to the vendor’s directions (Zeptosens, Basel, Switzerland). Lysates were sent to Zeptosens for analysis. RPPA data from Zeptosens was processed by the vendor and further processed by median centering. Heatmap plots were generated using a custom R script with scaling to a range of 23 to 3 (arbitrary value). Western Immunoblots The protein concentration of cell lysates was quantified using a DC protein assay kit (Bio-Rad, Hercules, CA). Forty to seventy-six mg of total protein per lane were loaded in a 4–12% gradient BIS- TRIS SDS-PAGE gel (Invitrogen, Carlsbad, CA, NuPAGE) or Criterion XT gel (Bio-Rad), and was transferred onto nitrocellu- lose membrane (Bio-Rad, #162-0233 or Invitrogen, #IB3010- 01). Membranes were blocked with Phosphoblocker (Cell Biolabs, San Diego, CA) and incubated overnight with primary antibody diluted in Phosphoblocker. Primary antibodies were used accord- ing to manufacturer’s recommendation: phospho BRAF S445 (Cell Signaling, #2695); BRAF N-term (Cell Signaling, #9433); BRAF c-TERM C-19 (Santa Cruz Biotechnology, Santa Cruz, CA); phospho c-Met Y1349 (Cell Signaling, #3121); c-Met (Cell Signaling, #3127); phospho ERK T202/Y204 (Cell Signaling, #9106); ERK (Cell Signaling, #9102); phospho AKT S473 (Cell PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39653