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Systemic analysis of tyrosine kinase signaling reveals a common adaptive response program in a HER2-positive breast cancer

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Science Signaling  22 Jan 2019:
Vol. 12, Issue 565, eaau2875
DOI: 10.1126/scisignal.aau2875
  • Fig. 1 Quantitative effects and patterns in the p-Tyr kinome in BT474 cells after anti-HER2 treatments by TMT LC-MS/MS.

    (A) Study design, sample preparation, and dataset analysis. PBS, phosphate-buffered saline; BCA, bicinchoninic acid; DTT, dithiothreitol; IP, immunoprecipitation; IMAC, immobilized metal ion affinity chromatography. (B) Global changes of Tyr peptide phosphorylation in BT474 cells after 6 and 48 hours of continuous treatment with 100 nM TZB, 10 μM ARRY, the combination of ARRY (10 μM) and TZB (100 nM) (A+T), or 100 nM 6L1G, as determined by TMT LC-MS/MS. Increase, decrease, or no change in p-Tyr peptide abundance was based on upper and lower LFC thresholds derived from the 95% CI of DMSO control treatment after 48 hours (fig. S1B). Plots are based on mean values of two biological replicates. (C) Venn diagram of the fractions of p-Tyr peptides identified in (B) of which the abundance was reduced below the threshold (95% CI) after 48 hours of the indicated treatment. Linked tables list the associated kinases and specific Tyr (Y) phosphosites. (D and E) Pearson clustering of 471 unique p-Tyr peptides from the TMT LC-MS/MS dataset (D) and pairwise comparison of LFC values computed by absolute PCC (E). Data are analyzed from the two biological replicates described in (B). PCC, Pearson correlation coefficient.

  • Fig. 2 HER2-dependent off-state network and feedback activation of Tyr kinases in BT474 cells by TMT LC-MS/MS.

    (A and B) Direct protein interaction network derived from a search of the MetaCore database in which p-Tyr peptide phosphorylation was significantly inhibited (below 95% CI) after the 48-hour A+T treatment (Fig. 1B). Node (“hub”) size indicates number of connections (edges) to other nodes (degree) or proteins, as determined by the MetaCore algorithm (n = 2 biological replicates), and edges represent known binding, phosphorylation, or dephosphorylation events between both proteins. (C) Comparison of the fractions of persistent p-Tyr peptides (that is, not decreased) after 48 hours of treatment, arranged by Venn diagram and tables of the associated kinases. A consensus of 54 p-Tyr peptides was identified after 48 hours of treatment with TZB and ARRY (n = 2 biological replicates). (D) Heat map of time-dependent Tyr phosphorylation trends of the 54 consensus peptides from (C). Trends were calculated from the slope of a simple linear regression on LFC values from 6 to 48 hours of treatment based on the mean of two biological replicates. (E) Pairwise comparison of Tyr phosphorylation trends computed by absolute PCC (n = 2 biological replicates).

  • Fig. 3 Peptide chip array, cognate kinase prediction, and combined adaptive kinase response analysis in BT474 cells.

    (A) Heat map of increased and decreased phosphorylation of Tyr (PTK, top) and Ser/Thr (STK, bottom) bait peptides in BT474 cells treated as in Fig. 1B [6 to 48 hours with 100 nM TZB, 10 μM ARRY, the combination thereof (A+T), or 100 nM 6L1G] versus those treated with DMSO, determined by peptide chip array (n = 4 biological replicates). (B) Volcano plots of the bait peptide phosphorylation from PTK (top) and STK (bottom) chip arrays in (A), assessed versus DMSO treatment by ANOVA and post hoc Dunnett’s tests (P ≤ 0.05). (C and D) Sum of scores from group-based prediction system (GPS) based on significantly increased phosphorylation of Tyr (C) and Ser/Thr (D) bait peptides, as determined in (B). (E) Combined adaptive kinase response analysis based on peptide chip array data and on TMT data after 48 hours treatment in a high-confidence interaction network (edges) from STRING DB. Node size and color indicate sum of GPS scores for each kinase that is predicted active after indicated treatment with ARRY (left) or TZB (right) from peptide chip array, as described in (C) and (D). Arrangement of nodes is based on various experimental evidences: Inner core (dark gray) represents proteins from the consensus of the 54 persistent p-Tyr peptides after ARRY and TZB treatment from the TMT data (Fig. 2C), middle layer (light gray) represents persistent p-Tyr peptides from TZB treatment from the TMT data (Fig. 2C) and outer layer (white) represents additional predicted kinases (connected by STRING DB) after TZB treatment from peptide chip array (C and D). Note that the node sample space of TZB was found to include the nodes of ARRY (onion principle), and thus the nodes shown in both plots are the same. (F) Comparison of the up-regulated Tyr and Ser/Thr kinases from (E).

  • Fig. 4 FAK1 activation in response to AKT inhibition and effects of combination treatments.

    (A) XTT cell proliferation assays of HER2-positive breast cancer cell lines with or without PIK3CA point mutations [BT474 (K111 N), SKBR3 (WT), HCC1419 (WT), MDA-MB361 (E345K), MDAMB453 (H1047R), and UACCC893 (H1047R)] treated for 4 days with the indicated concentration of HER2-targeted therapy (TZB, 6L1G, ARRY, and A+T), AKT inhibitor (MK2206), or FAK1 inhibitor (PF562271). Data are means ± SD of n = 3 experiments. WT, wild type; O.D., optical density. (B) Western blot analysis of a HER2-dependent signaling cascade after 6 (top) and 48 hours (bottom) of treatment with the indicated drug (DMSO control, 100 nM TZB, 100 nM 6L1G, 10 μM ARRY, 10 μM PF562271, or 5 μM MK2206) or combination (A+T, 10 μM ARRY and 100 nM TZB). Blots are representative of two to four independent experiments. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. (C) XTT cell proliferation assays of BT474 and MDAMB361 cells after 4 days of continuous treatment with titration of PF562271, and subsequent addition at day 0 (less than 5 min) of the indicated anti-HER2 agents TZB (100 nM) and 6L1G (100 nM), or the AKT inhibitor MK2206 (5 μM). Data are means ± SD of n = 3 experiments. (D) High-throughput microscopy analysis of breast cancer cells continuously treated for 3 days with 10 μM ARRY or 10 μM PF562271 in combination with 100 nM TZB or 6L1G and then stained with propidium iodide (PI) and Hoechst-33342 to detect the number of membrane-permeable (PI positive, inferred as dead) cells in the population. Data are means ± SD of n = 5 experiments. ***P ≤ 0.005 by one-sided pairwise t test. (E) Western blot analysis for the indicated proteins in BT474 and MDA-MB361 cells treated for 2 days with 10 μM PF562271 or 5 μM MK2206, either alone or in combination with either 100 nM TZB or 6L1G. Blots are representative of two independent experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/565/eaau2875/DC1

    Fig. S1. HER2/HER3 signaling after anti-HER2 treatments and the distribution of phosphopeptides in the TMT LC-MS/MS dataset and peptide signature.

    Fig. S2. Peptide chip array and kinase predictions.

    Fig. S3. Counter-activation of FAK1-AKT1 signaling and the effect of combination treatments.

    Table S1. LFC of peptide abundance from TMT LC-MS/MS dataset.

    Table S2. LFC and P values for Tyr peptide phosphorylation from kinase activity profiling.

    Table S3. LFC and P values for Ser/Thr peptide phosphorylation from kinase activity profiling.

  • The PDF file includes:

    • Fig. S1. HER2/HER3 signaling after anti-HER2 treatments and the distribution of phosphopeptides in the TMT LC-MS/MS dataset and peptide signature.
    • Fig. S2. Peptide chip array and kinase predictions.
    • Fig. S3. Counter-activation of FAK1-AKT1 signaling and the effect of combination treatments.
    • Legends for tables S1 to S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). LFC of peptide abundance from TMT LC-MS/MS dataset.
    • Table S2 (Microsoft Excel format). LFC and P values for Tyr peptide phosphorylation from kinase activity profiling.
    • Table S3 (Microsoft Excel format). LFC and P values for Ser/Thr peptide phosphorylation from kinase activity profiling.

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