Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Supplemental Information c-Myc alteration determines the therapeutic response to FGFR inhibitor Hongyan Liu, Jing Ai, Aijun Shen, Yi Chen, Xinyi Wang, Xia Peng, Hui Chen, Yanyan Shen, Min Huang, Jian Ding, Meiyu Geng 1 Supplementary materials and methods Cell Cycle Analysis Cells were seeded in 6-well plates at a density of 2×105 / mL. After 24 hours, the cells were treated with drugs or vehicle (DMSO) for 24 hours. Both adherent and floating cells were harvested and fixed in 70% ethanol over-night at 4°C. Prior to FACS analysis, cells were washed twice with cold D-PBS and re-suspended in D-PBS containing 200 μg/mL RNase, 50 μg/mL propidium iodide,keep away from light and incubated for 30 minutes at room temperature. Quantitation of the cell cycle distribution was performed on a Becton-Dickinson FACS Calibur flow cytometer (Becton-Dickinson). Immunoblotting For immunoblotting, protein extracts were prepared by washing twice in cold D-PBS followed by lysis with SDS-lysis buffer (50 mM Tris-HCl, pH7.4, 2%SDS). Lysates were boiled for 10 minutes and cleared by centrifugation at 12,000 × g for 15 minutes at 4°C. The supernatant was collected and subjected to SDS-PAGE. Immunoblotting analysis were performed according to the antibody manufacturer’s recommendations. Proteins were resolved using the NuPAGER NovexR Midi Gel system on 8% to 15% Bis-Tris Gels. Representative blots are shown from several experiments. Antibodies were obtained from the following resources: Cell Signaling Technology : p-FGFR(Tyr653/654), FGFR1, FGFR3, 2 FGFR4, p-FRS2α(Tyr196), p-FRS2α(Tyr436), PLCγ, STAT3, p-STAT3(Tyr705), p-AKT(Ser473), AKT, p-ERK1/2(Thr202/Tyr204), ERK1/2, E2F1, FOXO1, CDK4, CDK6, CDK2 , RB, P-RB(Ser780), cyclinD1, cyclinD2, cyclinD3, P18, P21, and P27; Santa Cruz: FRS2α, cyclinE; Abcam: FGFR2, c-Myc, P16, GAPDH, and β-actin; MerkMillipore: p-PLCγ(Tyr783); Calbiochem: Rabbit and Mouse IgG-HRP. Cell Viability Assay Cells were seeded in 96-well plates at a density of 3000-30000 cells in triplicates. After 24 hours, the cells were treated with different concentrations of the indicated inhibitors and incubated at 37oC. Then, cells were cultured for 3 days and the number of viable cells was measured by CCK-8 cell viability assay (Dojindo). Gene Silencing by siRNA For siRNA experiments, cells were seeded in 6-well plates at a density of 1.5×105/mL. After 24 hours, cells were transfected with siRNAs with Oligofectamine RNAimax reagent (Invitrogen) according to the manufacturer’s instructions. After 72 hours, cells were harvested for cell cycle analysis or subjected to immunoblotting analysis. Cell viability was determined using Hemocytometer after 0.4% Trypan Blue staining for 10 minutes. The sequence and catalog No. of the siRNAs in this study as follows: c-Myc # 5'-(GGACUAUCCUGCUGCCAAGTT)-3' PLCG1 # 5'-(AAGAAGTCGCAGCGACCCGAG)-3' STAT3 # 5'-(GCAAAAAGUUUCCUACAAA) -3' 3 Dharmacon siRNA: ON-Target plus Control pool Non-Targeting pool Human Cat # D-001810-05 ON-Target plus SMARTpool Human MYC Cat # L-003282-02 ON-Target plus SMARTpool Human PLCG1 Cat # L-003559-00 ON-Target plus SMARTpool Human STAT3 Cat # L-003533-00 Quantitative Real-Time PCR Quantitative real-time PCR was performed using ABIPrism VIIA7 Real-Time PCR System (Life Technologies, Carlsbad, CA). For detection of c-MYC expression, total RNA was extracted with TRIzol reagent (Invitrogen, Grand Island, NY) and subjected to reverse transcription with PrimeScript® RT reagent Kit (Takara, Mountain View, CA). PCR reactions were performed with SYBR® Premix Ex Taq™ kit (Takara). Primer for c- MYC were 5’-TCCCAA AGTACCCAAAGGC-3’(sense), 5’-ACTCCAGCCTTGACCCACTC-3’ (anti-sense) 4 Supplementary Table 1. List of PDX models with FGFR2 amplification in our study (copy number higher than 5) No. Tumor Models Type FGFR2 gene copy number FGFR2 Amplification FGFR2 mRNA (SNP6) (qPCR) (RNAseq) 1 gastric GA0033 14 yes 7.88231 2 gastric GA1224 14 yes 7.74669 3 gastric GA3055 14 yes 7.66415 5 Supplemental Figure and Figure lengends Figure S1. FGFR inhibition induces G1 phase cell cycle arrest in FGFR-addicted cancer cells. A, Dose response curves of BGJ398 and AZD4547 against FGFR over-activated cancer cells were assessed using CCK-8 assay. Bars, means ± SD. B, Cell-cycle distribution was analyzed upon BGJ398 or AZD4547 treatment. Cancer cells were treated with BGJ398 or AZD4547 at indicated concentrations for 24 hours. Cell cycle distribution was analyzed using flow cytometry. Bars, means ± SD. *, p < 0.05, **, p < 0.01, versus vehicle group, using unpaired two-tailed Student’s t test. C, DMS114 cell were treated with BGJ398 or AZD4547 at 0.5 μM for 2, 6, 12, 24, 48 hours and subjected to immunoblotting. Figure S2. c-Myc downregulation is essential for FGFR inhibition caused growth arrest in FGFR-addicted cells. A, Immunoblotting of analysis. NCI-H1581 and DMS114 cells were treated with BGJ398 at 0.5 μM. NCI-H520 cells and NCI-H2444 cells were treated with AZD4547 at 1 μM or 2 μM for indicated time (2, 6, 12, 24 hours). B, KG1 cell treated with BGJ398 and AZD4547 at 0.5 μM for indicated time (12, 24 hours), followed by immunoblotting analysis. C, NCI-H1581 and KATOIII cells were treated with scramble or c-Myc siRNAs for 24 hours, followed by AZD4547 treatment for 72 hours (0.5 μM for NCI-H1581, 0.1 μM for KATOIII) before immunobloting was analyzed (parallel tests for Figure 2B and 2C, left panel). D, NCI-H1581 and KATOIII cells were treated with scramble or c-Myc siRNAs for 72 hours, 6 followed by immunobloting analysis (parallel tests for Figure 2B and 2C, Right panel). E, c-Myc was disrupted using (+)-JQ1 at 1 μM in DMS114 cells before subject to immunoblotting. Cell viability was analyzed. Bars, means ± SD. Figure S3. c-Myc alteration determines the response to FGFR2 inhibition in FGFR2-addicted cancer cells. A and B, KATOIII (A) and MFM-223 (B) cells were treated with BGJ398 and AZD4547 (0.1 μM for KATOIII, 0.5 μM for MFM-223) for indicated time (2, 6, 12, 24 hours), followed by immunoblotting analysis. C and D, SUM52PE (C), and NCI-H716 (D) cells treated with BGJ398 and AZD4547 at 0.5 μM for indicated time (2, 6, 12 hours), followed by immunoblotting analysis. Figure S4. c-Myc downregulation determines the response to FGFR3 inhibition in FGFR3-addicted cancer cells. A and B, UMUC14 (A) and RT112 (B) were treated with BGJ398 and AZD4547 at 0.5 μM for indicated time (2, 6, 12 hours), followed by immunoblotting analysis. C, c-Myc was disrupted using c-Myc siRNAs for 72 hours in UMUC14 cell, followed by cell viability and immunoblotting analysis. Bars, means ± SD. *, p < 0.05, versus scramble, using unpaired two-tailed Student’s t test Figure S5. c-Myc downregulation determines the response to FGFR inhibition of FGFR constitutively activated BaF3/TEL-FGFR cells. A, IC50s values of BGJ398 and AZD4547 against BaF3/TEL-FGFR1, BaF3/TEL-FGFR3 and BaF3/TEL-FGFR4 cells were assessed using CCK-8 assay. Bars, means ± SD. B, BaF3/TEL-FGFR constitutive activated cells 7 were treated with BGJ398, or AZD4547 (0.5 μM for BaF3/TEL-FGFR1 and BaF3/TEL-FGFR3, 1 μM for BaF3/TEL-FGFR4) for 24 hours, then subjected to immunoblotting with indicated antibodies. Figure S6. FGFR inhibition facilitates c-Myc protein degradation in FGFR-addicted cancer cells. A, NCI-H1581, KATOIII, MFM-223 and UMUC14 cells were treated with BGJ398 or AZD4547 at 0.5 μM for indicated time (2, 6, 12, 24 hours). c-Myc mRNA level was examined by RT-PCR and normalized by that of vehicle control group. Bars, means ± SD. B, sensitivity of SUM52PE cells stably transfected with MSCV-c-Myc or MSCV-c-MycT58A. SUM52PE cells stably expressing wild type c-Myc or T58A mutant were treated with AZD4547 at 0.5 μM for the indicated time, and then subjected to immunoblotting. C, Dose response curve of BGJ398 and AZD4547 in NCI-H1581 cells stably expressing c-Myc wild type or c-Myc T58A. Cell viability was assessed using CCK-8 assay. D, NCI-H1581, KATOIII, NCI-H716, and UMUC14 cells were treated with scramble or PLCγ siRNAs for 72 hours, followed by immunoblotting analysis. E, KATOIII and SUM52PE cells were treated with scramble or STAT3 siRNAs for 72 hours, followed by immunoblotting analysis. Figure S7. c-Myc indicates the therapeutic response to FGFR inhibition in vivo. A, NCI-H1581 xenograft-bearing nude mice were treated with AZD4547 at the indicated doses or vehicle for 3 or 10 consecutive days. 8 Tumor tissues resected after last dose were subjected to immunoblotting with indicated antibodies. B and C, NCI-H716 xenograft-bearing nude mice were received AZD4547 at the indicated doses or vehicle for 21 consecutive days (n = 6 for treated group, n = 12 for vehicle group). Tumor volumes were measured twice a week. The relative tumor volume was shown as mean ± SEM.*, p < 0.05, **, p < 0.01, versus vehicle group, using unpaired two-tailed Student’s t test (B). Tumor tissues resected after last dose were subjected to immunoblotting with indicated antibodies. (C). D and E, GA3055 and GA1224 xenograft-bearing nude mice received BGJ398 at the indicated doses or vehicle for 3 days. Tumor tissues resected after last dose were subjected to immunoblotting with indicated antibodies. 9