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1 Supplementary Materials for Dynamic Reprogramming of Signaling Upon Met Inhibition Reveals a Mechanism of Drug Resistance in Gastric Cancer Andrea Z. Lai, Sean Cory, Hong Zhao, Mathieu Gigoux, Anie Monast, Marie-Christine Guiot, Sidong Huang, Ali Tofigh, Crista Thompson, Monica Naujokas, Victoria A. Marcus, Nicholas Bertos, Bita Sehat, Rushika M. Perera, Emily S. Bell, Brent D. G. Page, Patrick T. Gunning, Lorenzo E. Ferri, Michael Hallett, Morag Park* The PDF file includes: *Corresponding author. morag.park@mcgill.ca Published 22 April 2014, Sci. Signal. 7, ra38 (2014) DOI: /scisignal Fig. S1. Met IHC staining ranges in intensity in gastroesophageal primary tumors and lymph node metastases. Fig. S2. Met inhibitor decreases the number of soft agar colonies. Fig. S3. HGF-induced IEGs decrease upon Met inhibition. Fig. S4. DUSP4 and DUSP6 are induced upon growth factor stimulation. Fig. S5. Late expression of genes after HGF stimulation and inhibition indicates proteins involved in the abrogation of cell proliferation. Fig. S6. FOXO3A expression and downstream genes are differentially regulated upon Met inhibition. Fig. S7. STAT3-targeted genes are differentially expressed in gastric cancer cell lines upon Met inhibition. Fig. S8. Not all MAPK pathways are dependent on Met kinase activity. Fig. S9. E2F1-targeted genes are differentially expressed in gastric cancer cell lines upon Met inhibition. Fig. S10. DUSP loss upon Met inhibition is MEK-dependent, and subsequent ERK reactivation promotes phosphorylation of downstream effectors. Fig. S11. Met inhibitors crizotinib and EMD recapitulate effects on signaling observed with. Fig. S12. Inhibition of STAT3 hinders cell proliferation. Fig. S13. STAT3 phosphorylation is dependent on Met kinase activity. Fig. S14. Met inhibition promotes variable cytotoxicity in gastric cancer cell lines. Table S1. MET amplification in primary gastric tumors.
2 SUPPLEMENTARY MATERIALS Figure S1 - Met IHC staining ranges in intensity in gastroesophageal primary tumors and lymph node metastases. (A) Representative images illustrating strong, medium, and weak Met IHC staining (40x magnification). (B) Quantification of Met positivity in tumors (n=31) illustrating the range of Met staining in each of the three categories. Horizontal bars represent means; error bars show distribution. (C) An example of a Met-negative sample in a signet ring cell carcinoma. (D) Met staining in a normal stomach tissue (10x magnification). (E) Percentage of Ki67 positive cells in tumors scored with high (3), medium (2) or low (1) Met IHC staining as determined by a pathologist. Horizontal bars denote the mean percentage; error bars show distribution. a.u., arbitrary units.
3 A # microscopic colonies * 0 untreated B Okajima MKN45 H 2 O Crizotinib Figure S2 - Met inhibitor decreases the number of soft agar colonies. (A) Quantification of soft agar colonies from 3 independent experiments. Means ± SEM are shown. * P < (B) Representative images of soft agar colonies formed by cells cultured in H 2 O or 0.1 µm Crizotinib.
4 Figure S3 - HGF-induced IEGs decrease upon Met inhibition. EGR1, FOS, FOSL1, IER3 expression upon 0.5 nm HGF stimulation (first column), 0.1µM treatment (second column), and EGF stimulation (third column) are plotted. Dashed lines represent the vehicle control. Log 2 expression values from 132 arrays are plotted. EGF-dependent expression data is re-plotted from (41).
5 Figure S4 - DUSP4 and DUSP6 are induced upon growth factor stimulation. (A) Expression of DUSP4 and DUSP6 upon 0.5 nm HGF (left column) and EGF (right column are plotted as log 2 expression values. EGFdependent expression data is re-plotted from (41). (B) Western blot of T47D 2A cells were stimulated with 0.5 nm HGF. (C) DUSP4 and DUSP6 protein upon HGF stimulation were quantified from 3 independent experiments. Data are means ± SEM from 3 independent experiments.
6 Figure S5 - Late expression of genes after HGF stimulation and inhibition indicates proteins involved in the abrogation of cell proliferation. (A) Heat map of genes that significantly change in expression upon HGF (0.5 nm) stimulation for 3 hours in T47D 2A cells is depicted on the left. The corresponding changes in expression of these genes upon (0.1 µm) treatment in the gastric cancer cell lines are shown in the heatmap on the right. h, hours; t, time. (B) Genes that are differentially expressed at 24 hours of HGF stimulation are shown in the left heatmap. As in (A), the corresponding changes in expression of these genes upon Met inhibition are shown in the right heatmap. (C) Of the genes whose expression is positively correlated between HGF and treatment at 24 hours [from (B)], the top 20 pathways enriched with the most genes as determined through Gene Ontology pathway analysis are plotted. (D) Schematic model illustrating the induction of immediate early genes (IEGs, < 1 hour), delayed early genes (DEGs, < 3 hours), and secondary response genes (SRGs, > 3 hours) upon HGF stimulation, and the loss of these in the absence of an activating Met signal.
7 Figure S6 - FOXO3A expression and downstream genes are differentially regulated upon Met inhibition. (A) FOXO3A gene expression increases in cell lines upon Met inhibition. Solid lines, -treated cells; dashed lines, controls. (B) Heat map illustrating differential expression of FOXO3A-regulated genes upon Met inhibition.
8 Figure S7 - STAT3-targeted genes are differentially expressed in gastric cancer cell lines upon Met inhibition. Heatmaps of differentially expressed STAT3 target genes upon Met inhibition in gastric cancer cell lines. Genes are from STAT3-targeted gene expression signatures obtained from (A) GM12878 and (B) HeLa cells in (54).
9 A C Okajima MKN45 pjnk / Jnk (ratio of t=0) Time(h) pmek Time (h) D p-p38 / p38 (ratio of t=0) Mek pmek Mek pmek Mek pmek Mek Time (h) Time(h) pjnk Figure S8 - Not all MAPK pathways are dependent on Met kinase activity. (A) Western blots of phosphorylated MEK (pmek) abundance upon inhibition of Met with 0.1 µm. (B) Western blots of components of other MAPK pathways upon treatment with 0.1µM. (C and D) Amount of (C) phosphorylated JNK (pjnk) at Thr 183 /Tyr 185 or (D) phosphorylated p38 (p-p38) at Thr 180 /Tyr 182 after addition of 0.1 µm. Data are means ± SEM from all 4 cell lines quantified from 3 independent experiments. B MKN45 Okajima Jnk p-p38mapk p38mapk pjnk Jnk p-p38mapk p38mapk pjnk Jnk p-p38mapk p38mapk pjnk Jnk p-p38mapk p38mapk
10 Figure S9 - E2F1-targeted genes are differentially expressed in gastric cancer cell lines upon Met inhibition. Heatmaps of differentially expressed E2F1-target genes upon Met inhibition in gastric cancer cell lines. Genes are from E2F1-targeted gene expression signatures obtained from (A) MCF7 and (B) HeLa cells in (54).
11 A D U0126 U h perk Erk Actin Crizotinib (24h) Selumetinib(2h) pmet Met pmek Mek perk Erk DUSP4 DUSP6 Actin B Fluorescence Intensity C Fluorescence Intensity EGFR HER2 EGFR HER2 E perk / Erk (ratio of H 2 O value) Met H 2 O Selumetinib HER3 FGFR1 FGFR3 FGFR4 InsR IGF-1R TrkA TrkB Ron Ret Met Crizotinib Selu / Criz Met HER3 FGFR1 FGFR3 FGFR4 InsR IGF-1R TrkA TrkB Ron Ret F DUSP/Actin (ratio of H 2 O value) ALK PDGFR c-kit FLT3 M-CSFR EphA1 EphA2 EphA3 Met ALK PDGFR c-kit FLT3 M-CSFR EphA1 EphA2 EphA3 H 2 O EphB1 EphB3 EphB4 Tyro-3 DUSP4 DUSP6 DUSP4 DUSP6 Crizotinib Axl Tie2 VEGFR2 EphB1 EphB3 EphB4 Tyro-3 Axl Tie2 VEGFR2 G 24h U0126 Sorafenib Tpl2i S + T U0126 Sorafenib Tpl2i S + T U0126 Sorafenib Tpl2i S + T U0126 Sorafenib Tpl2i S + T 2h p-p90rsk RSK1/2/3 ps6 (short exp) ps6 (long exp) S6 Figure S10 - DUSP loss upon Met inhibition is MEK-dependent, and subsequent ERK reactivation promotes phosphorylation of downstream effectors. (A) Western blot of phosphorylated ERK (perk; Thr 202 /Tyr 204 ) abundance in and cells after short-term treatment with, 20 µm U0126, or 0.1 µm. (B) Abundance of the specified phosphorylated RTKs in cells in the presence of or 0.1 µm -treated for 24 hours as determined with a phospho-rtk array. Images of the arrays are shown in the inset. (C) As described in (B) but with cells. (D) Western blot of components of the Met-ERK signaling pathway upon treatment with 0.1µM Crizotinib for 24h (where specified) followed by or 5 µm Selumetinib (2 hours). (E) Quantification of perk/erk in and cells (normalized to control H 2 O-treated samples) treated as described in (D). (F) Quantification of DUSP4 or DUSP6 abundance in cells treated with 0.1 µm Crizotinib normalized to matched H 2 O control values. Data in (E) and (F) are means ± SEM from 3 independent experiments. (G) Western blot of and cells treated with or 0.1 µm for 24 hours, followed by addition of, 20 µm U0126, 10 µm Sorafenib, 10 µm Tpl2 inhibitor, or a combination of Sorafenib and Tpl2 inhibitor (S + T) for 2 hours. h, hours.
12 Figure S11 Met inhibitors crizotinib and EMD recapitulate effects on signaling observed with. (A) Western blot of cells treated with H 2 O or 0.1µM Crizotinib for 2 or 24 hours (2h or 24h) and immunoblotted for signaling components downstream from Met. (B to D) Quantification of (B) phosphorylated Met (pmet; Tyr 1234/1235 ), (C) pakt (Ser 473 ), and (D) pstat3 (Tyr 705 ) against the corresponding whole protein abundance from Western blots of all 4 cell lines treated with Crizotinib or H 2 O as indicated, each from 3 independent experiments. (E) Ratio of perk (Thr 202 /Tyr 204 ) to ERK abundance in crizotinib (Criz)-treated cells normalized to that in H 2 O-treated cells. Data are means ± SEM from Western blots of all 4 cell lines each from 3 independent experiments. (F) Western blots of cells treated with or 0.1 µm EMD and immunoblotted for signaling proteins downstream from Met. (G to I) Quantification of (G) pmet, (H) pakt, and (I) pstat3 against the corresponding whole protein abundance from Western blots from 3 independent experiments in which cells were treated with or EMD (J) Average perk/erk ratios, normalized to the corresponding value, in cells treated with either or EMD (EMD). Data are means ± SEM from 3 independent experiments. a.u., arbitrary units.
13 A Absorbance / SH454 Cell Proliferation BP S Okajima MKN45 Stattic B Okajima MKN45 BP1-102 C D 1.5 plko shrna1 shrna2 shrna3 shrna4 shrna5 E F G plko shrna1 shrna2 shrna3 shrna4 shrna5 pstat3 STAT3 Actin pstat3 STAT3 Actin Normalized to parental (a.u.) Normalized to parental (a.u.) STAT3/Actin STAT3/Actin *** **** pstat3/actin Parental plko shrna1 shrna2 shrna3 shrna4 shrna5 pstat3/actin Parental plko shrna1 shrna2 shrna3 shrna4 shrna5 % Confluence plko shrna1 shrna2 shrna3 shrna4 shrna Time (h) ** *** **** H I J plko shrna1 shrna2 shrna3 shrna4 shrna5 MKN45 pstat3 STAT3 Actin Normalized to parental (a.u.) 1.5 MKN STAT3/Actin pstat3/actin 4 MKN Figure S12 - Inhibition of STAT3 hinders cell proliferation. (A) Proliferation of cells cultured for 5 days in, 20 µm SH454, 30 µm BP-1-102, 200 µm S31-201, 5 µm Stattic, or 0.1 µm. Proliferation was determined with an MTS assay and normalized to the -treated control. (B) Representative images of soft agar colonies formed by cells cultured in or 30 µm BP (C) Western blot of cells stably expressing STAT3 shrna or plko control vector. (D) Quantification of STAT3 and pstat3 amounts from samples from (C). (E) Western blot of cells stably expressing STAT3 shrna or plko control vector. (F) Quantification of whole and phosphorylated (p) STAT3 protein abundance from blots in (E). (G) Cell proliferation of II cells stably expressing one of five different shrna to STAT3 or a control vector (plko). (H to J) Western blots, quantification, and growth curves as described for (E-G) but with MKN45 cells. All plotted data (in A, D, F, G, I, and J) are means ± SEM from 3 biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < a.u., arbitrary units. Parental plko shrna1 shrna2 shrna3 shrna4 shrna5 % Confluence plko shrna1 shrna2 shrna3 shrna4 shrna5 Time (h) * ****
14 Figure S13 STAT3 phosphorylation is dependent on Met kinase activity. (A) Western blot of cells treated with 0.1 µm Ruxolitinib (Rux), 0.1 µm or vehicle control for 2 hours. (B) Western blot analysis of cells were treated with 10 µm PP2, 0.1 µm Dasatinib, 4 µm Imatinib, or 0.1 µm for 2 hours. Representative western blots from three independent experiments are shown.
15 Figure S14 - Met inhibition promotes variable cytotoxicity in gastric cancer cell lines (A) Cell viability in gastric cancer cell lines cultured for up to 7 days in vehicle control or 0.1 M, ascertained using the Trypan Blue dye exclusion assay. Data are mean cell viability ± SEM from 3 independent biological replicates. a.u., arbitrary units. (B) Western blot showing the amount of cleaved PARP in gastric cancer cells after treatment with or 0.1 M. (C) Volume of established tumors after treatment with control solvent or Crizotinib (30 mg/kg) for up to 15 days. Data are means ± SEM from 20 tumors for each condition. * P < 0.05, *** P < 0.001, **** P <
16 Table S1. MET amplification in primary gastric tumors Tumors are from patients diagnosed with gastric cancer and treated with chemotherapy prior to surgical resection. Met IHC and FISH scores were determined by pathologists. Shaded area indicates tumors that were not assessed for MET copy number with FISH. Sample Tissue Type Met IHC score (TMA) MET qpcr (fold) MET FISH (copy number) 27 Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor Tumor
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