Research ArticleCancer

Functional proteomics interrogation of the kinome identifies MRCKA as a therapeutic target in high-grade serous ovarian carcinoma

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Science Signaling  18 Feb 2020:
Vol. 13, Issue 619, eaax8238
DOI: 10.1126/scisignal.aax8238
  • Fig. 1 Characterizing the HGSOC kinome in patient tumors using MIB-MS to identify previously unexplored therapeutic targets.

    (A) Schematic of experimental approach. MIB-MS was used to quantify kinase abundance in HGSOC patient tumors to map the proteomic landscape of the kinome and identify prevalent kinases previously unexplored in HGSOC. m/z, mass/charge ratio. (B) Measurement of kinase abundance in a patient HGSOC tumor sectioned into four pieces, each individually kinome-profiled by MIB-MS, and kinase abundance was determined by s-SILAC quantitation. Pearson’s correlation thereof was determined using Perseus. (C) Proportion of the kinome quantitated by MIB-MS in HGSOC (“HGS”) tumors that represent established (red) and unexplored (gray) targets in HGSOC. Data are from one independent assay per sample in 25 tumor tissues and 10 PDX tumor tissues. The kinome tree was generated in KinMap and was reproduced courtesy of Cell Signaling Technology. (D and E) PCA, including PC1 versus PC2 (D) and PC1 versus PC3 (E), of MIB-MS determined kinome profiles of HGSOC primary and PDX tumors. Data are from one independent assay per sample in 25 tumor tissues and 10 PDX tumor tissues. (F) Volcano plot comparison of HGSOC primary and PDX tumor MIB-MS kinome profiles. Statistical differences in kinase log2 s-SILAC ratios comparing HGSOC primary versus PDX tumors were determined by paired t test Benjamini-Hochberg adjusted P values at an FDR of <0.05. Data are from one independent assay per sample in 25 tumor tissues and 10 PDX tumor tissues. (G) Predominant kinome signature identified by MIB-MS among HGSOC patient and PDX tumors, representing kinases detected at similar abundances among tumors. Statistical differences in kinase log2 s-SILAC ratios comparing HGSOC tumors from the main cluster relative to others were determined by paired t test Benjamini-Hochberg adjusted P values at an FDR of <0.05. Heat map depicts kinase log2 s-SILAC ratios, highlighting established HGSOC drivers, including those with drugs approved or in clinical trials, as well as those previously unexplored in HGSOC. Data are from one independent assay per sample in 25 tumor tissues and 10 PDX tumor tissues. (H) Kinases previously unexplored in HGSOC enriched in the MIB-MS kinome signature from (G). Data are from one independent assay per sample in 25 tumor tissues and 10 PDX tumor tissues. Kinome plot was produced using KinMap. The kinome tree was reproduced courtesy of Cell Signaling Technology. (I) Bar graph depicting kinases that were increased ≥2-fold relative to the s-SILAC reference in HGSOC tumors. The number of HGSOC tumors with kinases exhibiting log2 s-SILAC values (≥1.0) was determined, and the bar graph was generated in Prism software. Related data (additional PCA, correlation, and hierarchical clustering of MIB-MS profiles of primary and PDX HGSOC tumors) can be found in fig. S1.

  • Fig. 2 Knockdown screen targeting kinases from the MIB-MS HGSOC tumor signature identifies MRCKA as a candidate therapeutic target.

    (A) Cell-Titer Glo assay for cell viability of established HGSOC cell line transfected with siRNAs targeting the indicated kinases or with control siRNA (NT2) and cultured for 120 hours. Data were analyzed as Z scores, presented as means of three independent assays. (B) Cell viability in three additional established HGSOC cell lines to those presented in (A), cultured and assessed after knockdown of select kinases as described in (A). Data were analyzed as % cell viability, presented as means ± SD of three independent assays. *P ≤ 0.05 by Student’s t test. (C) Knockdown efficiency of MRCKA, EIF2AK2, and CHEK1 in HGSOC cell lines presented in (B). Cells were transfected with siRNAs targeting MRCKA, EIF2AK2, and CHEK1/2 or with control siRNAs and cultured for 72 hours. MRCKA and cleaved PARP protein abundance was assessed by immunoblot every 24 hours. Blots are representative of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Apoptosis assessed by immunoblotting for cleaved-PARP abundance in OVCAR4 cells. Cells were transfected with siRNAs targeting MRCKA or with control siRNAs and cultured for 72 hours. MRCKA and cleaved PARP protein abundance was assessed by immunoblot every 24 hours. Blots are representative of three independent experiments. (E) Targeted spectrum in CDC42BPA-coding gene of two distinct siRNAs used in study. CDC42BPA siRNAs 1 and 2 are present in the siGENOME pools. bp, base pair. (F to I) Effect of MRCKA knockdown with two distinct siRNAs on cell viability by Cell-Titer Glo assay and apoptosis by cleaved-PARP immunoblotting in OVCAR4 (F and H) and KURAMOCHI (G and I) cells transfected with CDC42BPA-targeted siRNAs for 48 hours. Data are means ± SD of three independent experiments. *P ≤ 0.05 by Student’s t test.

  • Fig. 3 MRCKA is highly expressed among HGSOC tumors and promotes growth and survival of HGSOC cell lines.

    (A) Analysis of CDC42BPA copy number analysis (CNA), mRNA, and protein expression changes in HGSOC tumors from TCGA and CPTAC studies (1, 18). mRNA levels were determined by U133 microarray, and protein level was determined by MS. Change in MRCKA abundance among tumors was determined at Z score >1 or <−1. Molecular subtype of HGSOC and disease stage are presented in relation to CDC42BPA mRNA levels. (B) Gene set enrichment analysis of HGSOC tumors expressing high or low CDC42BPA mRNA levels. Detailed description of bioinformatics methods is described in Materials and Methods. (C and D) IHC analysis of MRCKA in HGSOC TMAs. Immunoreactivity of MRCKA protein was evaluated by a pathologist, and IHC score was given based on the intensity of protein stain. 0: negative; 1+: mild (low)/weak; 2+: moderate/intermediate; 3+: strong (high)/intensive. MRCKA IHC images were captured at 100 μm. Data are from duplicate analysis of 105 tumor tissues. (E) MRCKA protein abundance among HGSOC cell lines determined by immunoblot. Blots are representative of three independent experiments. (F) Cell-Titer Glo assay for cell viability of HGSOC cells transfected with siRNAs targeting MRCKA or control siRNAs and cultured for 120 hours. Data were analyzed as % cell viability of siRNA control-treated cells, presented as means ± SD of three independent assays. (G) Immunoblot analysis for cell cycle markers in OVCAR4 cells transfected with MRCKA or control siRNAs for 24, 48, or 72 hours. Blots are representative of two independent experiments. (H) FACS analysis of OVCAR4 cells transfected with MRCKA or control siRNAs collected at the indicated time points. The cells were analyzed with a FACScan (BD) flow cytometer, and the data were analyzed using FlowJo (BD). Data were analyzed as % phase of cell cycle, presented as means ± SD of three independent assays. *P ≤ 0.05 by Student’s t test. (I) Apoptosis assessed by immunoblotting for cleaved-PARP abundance in HGSOC cells transfected with siRNAs targeting MRCKA or control siRNA and cultured for 72 hours. PARP cleavage and MRCKA protein levels were determined by Western blot. Blots are representative of three independent experiments. (J) Cell-Titer Glo assay for cell viability of FTSECs transfected with siRNAs targeting MRCKA or control siRNAs and cultured for 120 hours. Data were analyzed as % cell viability of siRNA control-treated cells, presented as means ± SD of three independent assays. (K) Apoptosis assessed by immunoblotting for cleaved-PARP abundance in FTSECs transfected with siRNAs targeting MRCKA or control siRNA and cultured for 72 hours. Blots are representative of three independent experiments. (L) Immunoblot analysis of MLC2 and MYPT1 phosphorylation in COV362 and KURAMOCHI cells transfected with MRCKA or control siRNAs for 72 hours. Blots are representative of three independent experiments. (M) Analysis of cell migration in COV362 cells transfected with MRCKA or control siRNAs and cultured for 72 hours. Migration was monitored over a 24-hour period using the xCELLigence Real-Time Cell Analyzer (RTCA). Data were analyzed as cell index, presented as means ± SD of three independent assays. *P ≤ 0.05 by Student’s t test. Related data (impact of MRCKA expression on overall survival of HGSOC, gene set enrichment analysis, IHC of MRCKA in fallopian tube and ovarian surface epithelium, and immunoblot analysis of MRCKA protein abundance in HGSOC tumors and HGSOC cell lines) can be found in fig. S2.

  • Fig. 4 MRCKA knockdown remodels the kinome of HGSOC cells.

    (A) Application of MIB-MS and global phosphoproteomics to explore the consequence of MRCKA knockdown on HGSOC signaling. (B) Volcano plot depicts kinases with induced or repressed MIB binding after MRCKA knockdown. OVCAR4 cells were transfected with siRNAs targeting MRCKA or control siRNAs for 48 hours and subjected to MIB-MS analysis. Statistical differences in kinase log2 s-SILAC ratios comparing MRCKA relative to control siRNA were determined by analysis of variance (ANOVA) Benjamini-Hochberg (BH) adjusted P values at an FDR of <0.05. Kinase log2 s-SILAC ratios were determined by comparing ratio of ratios (siMRCKA/s-SILAC relative siControl/s-SILAC). MIB-MS profiling was performed in biological duplicate. (C) Volcano plot depicts kinases predicted to be activated or inhibited in response to MRCKA knockdown using Kinase Substrate Enrichment Analysis (KSEA). Phosphoproteomics datasets were queried using PhosphoSitePlus at a P value cutoff of 0.05, a NetworkKIN score cutoff of 2, and a set substrate count cutoff of 10. Statistical differences in log2 s-SILAC ratios of phosphosites comparing siMRCKA/s-SILAC relative to siControl/s-SILAC were determined by paired t test, Benjamini-Hochberg adjusted P values at an FDR of <0.05. Phosphoproteomics profiling was performed in biological duplicate. (D) Immunoblot analysis of kinase remodeling in OVCAR4 cells transfected with MRCKA or control siRNAs and cultured for 72 hours. Protein abundance and phosphorylation levels were assessed 24, 48, or 72 hours after MRCKA knockdown. Blots are representative of three independent experiments. (E) Densitometric analysis of immunoblots presented in (D). Values indicate the optical density of total protein levels from an immunoblot normalized to total protein content (loading control, GAPDH). Quantitation of immunoblot bands was performed in ImageJ using three independent biological replicates. *P ≤ 0.05 by Student’s t test. Related data (hierarchical clustering of MIB-MS and phosphoproteomics profiles, a KSEA bar graph, and additional immunoblot and densitometry analysis) can be found in fig. S3.

  • Fig. 5 MRCKA knockdown blocks focal adhesion signaling impairing spheroid formation and sensitizes HGSOC cells to carboplatin or PAK inhibitors.

    (A) Immunoblot analysis of FAK1 activating phosphorylation and total FAK1 protein abundance in HGSOC cells transfected with control or MRCKA siRNAs for 72 hours. Blots are representative of three independent experiments. (B) Densitometric analysis of immunoblots presented in (A). Values indicate the optical density of total protein levels from an immunoblot normalized to total protein content (loading control, GADPH) expressed as a percent change (si-MRCKA/si-control). Quantitation of immunoblot bands was performed in ImageJ using three independent biological replicates. (C) Immunoblot analysis of focal adhesion signaling markers in COV362 cells transfected with MRCKA or control siRNAs for 24, 48, or 72 hours. Blots are representative of two independent experiments. (D) Confocal fluorescence microscopy of focal adhesion and actin cytoskeleton in COV362 cells transfected with MRCKA or control siRNAs for 72 hours. DAPI: nuclear staining by DAPI; vinculin: focal contacts revealed by anti-vinculin antibody; actin: F-actin detected by TRITC-conjugated phalloidin; merge: merged stain of DAPI, phalloidin, and vinculin. Images are representative of three independent experiments. Scale bars, 50 μm. (E) Quantitation of focal adhesion contacts in COV362 and KURAMOCHI cells transfected with MRCKA or control siRNAs for 72 hours. Data were analyzed as number of focal contacts, presented as means of three independent assays. *P ≤ 0.05 by Student’s t test. (F and G) Assessment of spheroid formation in COV362 and KURAMOCHI cells transfected with MRCKA or control siRNAs for 72 hours. Representative microscopy images of KURAMOCHI or COV362 spheroids from three independent biological replicates. Scale bars, 1000 μm. (G) Quantitation of spheroid density determined by ImageJ. *P ≤ 0.05 by Student’s t test. (H and I) Cell-Titer Glo assay for cell viability of KURAMOCHI (H) or COV362 (I) cells transfected with siRNAs targeting MRCKA or control siRNAs, cultured for 72 hours (KURAMOCHI) or 120 hours (COV362) and treated with increasing doses of carboplatin or DMSO. Data were analyzed as percentage of DMSO control, presented as means of three independent assays. GI50 were determined using PRISM. (J) Apoptosis assessed by immunoblotting for cleaved-PARP abundance in HGSOC cells transfected with siRNAs targeting MRCKA or with control siRNAs, cultured for 72 hours, and treated with carboplatin (25 μM) or DMSO. Blots are representative of two independent experiments. (K) Immunoblot analysis of phosphorylated and total levels of PAK1 in HGSOC cells transfected with MRCKA or control siRNAs for 72 hours. Blots are representative of three independent experiments. *P ≤ 0.05 by Student’s t test. (L) Cell-Titer Glo assay for cell viability of OVCAR4 or COV362 cells transfected with siRNAs targeting MRCKA or control siRNAs, cultured for 120 hours, and treated with PF-03758309 (3.5 nM OVCAR4 or 50 nM COV362) or DMSO. Data were analyzed as percentage of DMSO control, presented as means of three independent assays. Related data (immunoblot analysis of FAK1 protein levels using two distinct siRNAs, FAK1 mRNA levels after MRCKA knockdown, confocal fluorescence microscopy of focal adhesion and actin cytoskeleton in KURAMOCHI cells, and Bliss synergy analysis of carboplatin and MRCKA siRNA drug synergy) can be found in fig. S4.

  • Fig. 6 Characterizing MRCKA small-molecule inhibitors in HGSOC cells.

    (A and B) In vitro radioisotope-based 33P assays testing the effect of BDP9066 on MRCKA and MRCKB activity. BDP9066 was tested in a 10-dose IC50 mode with threefold serial dilution starting at 10 μM. Reactions were carried out at 1 μM ATP. Curve fits were performed where MRCKA or MRCKB activities at the highest concentration of BDP9066 were less than 65%. Hill slope and EC50 values were determined in molar (M). (C) MIB-MS kinome profile of OVSAHO cells treated with BDP9066 for 4 hours. SILAC-labeled OVSAHO cells were treated with 2 μM BDP9066 or DMSO for 4 hours, and lysates were incubated with CTx-0249885 beads. Line graph depicts average SILAC-determined log2 fold changes in kinase MIB binding as a ratio of BDP9066/DMSO. Biological duplicates of SILAC heavy-BDP9066/light-DMSO or light-BDP9066/heavy-DMSO were performed. (D) Immunoblot analysis of MLC2 phosphorylation in HGSOC cells treated with increasing concentrations of BDP9066 (0, 0.5, 1, and 3 μM) for 4 hours. Blots are representative of three independent experiments. (E) Confocal fluorescence microscopy of actin cytoskeleton in COV362 treated with DMSO or 1 μM BDP9066 for 48 hours. F-actin was detected by TRITC-conjugated phalloidin, and nuclear staining was detected by DAPI. Images are representative of three independent experiments. Scale bars, 50 μm. (F) Analysis of cell migration in COV362 cells treated with DMSO or 1 μM BDP9066. Migration was monitored over a 24-hour period using the xCELLigence RTCA. Data were analyzed as cell index, presented as means ± SD of three independent assays. *P ≤ 0.05 by Student’s t test. Related data (in vitro kinase assays testing BDP5290 selectivity, a comparison of BDP9066 and A-674563 MIB-MS profiles, and the consequence of BDP9066 treatment on actin cytoskeleton and migration of KURAMOCHI cells) can be found in fig. S5.

  • Fig. 7 Treatment of HGSOC cells with BDP9066 blocks cell growth, induces apoptosis, and inhibits focal adhesion signaling, impairing spheroid formation of HGSOC cells.

    (A) Cell-Titer Glo assay for cell viability of HGSOC cell lines treated with increasing concentrations of BDP9066 or DMSO and cultured for 120 hours. Data were analyzed as % cell viability of DMSO control, presented as means of three independent assays. GI50 values for BDP9066 were generated in Prism. (B) Long-term 14-day colony formation assay of HGSOC cells treated with BDP9066 or DMSO. Colony formation was assessed by crystal violet staining. Representative images of three biological replicates are shown. (C) Apoptosis assessed by immunoblotting for cleaved-PARP abundance in HGSOC cells treated with DMSO or 1 μM BDP9066 for 72 hours. Blots are representative of three independent experiments. (D) Immunoblot analysis of FAK1 phosphorylation and total FAK1 protein abundance in HGSOC cells treated with escalating doses of BDP9066 (0, 0.5, 1, and 3 μM) for 48 hours. Blots are representative of three independent experiments. (E) Immunoblot analysis of phosphorylation of FAK1 and paxillin in HGSOC cells treated with 2 μM BDP9066 for 48 hours. Blots are representative of three independent experiments. (F) Densitometric analysis of immunoblots presented in (E). Values indicate the optical density of phosphorylated or total protein levels from an immunoblot normalized to total protein content expressed as a percent change (BDP9066/DMSO). Phosphorylated proteins were normalized to loading control and then normalized to total abundance of the respective protein. Quantitation of immunoblot bands was performed in ImageJ using three independent biological replicates. *P ≤ 0.05 by Student’s t test. (G) Confocal fluorescence microscopy of focal adhesion and actin cytoskeleton in KURAMOCHI cells treated with DMSO or 1 μM BDP9066 for 24 hours. DAPI: nuclear staining by DAPI; vinculin: focal contacts revealed by anti-vinculin antibody; actin: F-actin detected by TRITC-conjugated phalloidin; merge: merged stain of DAPI, phalloidin, and vinculin. Images are representative of three independent experiments. Scale bars, 50 μm. (H and I) Assessment of spheroid formation in HGSOC cells treated with DMSO or 1 μM BDP9066 for 72 hours. Representative images of HGSOC cell spheroids (H) from three independent biological replicates are shown. Scale bars, 1000 μm. (I) Quantitation of spheroid density determined by ImageJ. *P ≤ 0.05 by Student’s t test. (J) Model of the mechanism, depicting the integral role of MRCKA in promoting proliferation, migration, and survival of HGSOC cells, thus highlighting MRCKA as a new kinase inhibitor target for the treatment of HGSOC. Related data (knockdown of MRCKB’s impact on HGSOC cell viability, drug synergy studies involving BDP9066 and carboplatin or PAK1 inhibitors, as well as quantitation of number of focal contacts in KURAMOCHI cells in response to BDP9066) can be found in fig. S6.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/619/eaax8238/DC1

    Fig. S1. Quantitation of kinase abundance in HGSOC tumors using MIB-MS and s-SILAC.

    Fig. S2. Characterization of MRCKA in HGSOC tumors and HGSOC cell lines.

    Fig. S3. Exploring MRCKA knockdown in HGSOC cells using MIB-MS and phosphoproteomics.

    Fig. S4. MRCKA promotes focal adhesion signaling in HGSOC cells.

    Fig. S5. Evaluation of MRCKA kinase inhibitors in HGSOC cells.

    Fig. S6. Characterization of proliferation, migration, apoptosis, and spheroid formation in BDP9066-treated HGSOC cells.

    Data file S1. Raw and processed s-SILAC ratios from MIB-MS profiling of sections from a single HGSOC tumor tissue.

    Data file S2. Characteristics of HGSOC primary and PDX tumors used in MIB-MS studies.

    Data file S3. Raw and processed s-SILAC ratios from MIB-MS profiling of HGSOC primary and PDX tumors.

    Data file S4. Prevalent MIB-MS kinome signature among HGSOC primary and PDX tumors.

    Data file S5. IHC analysis of MRCKA protein levels in HGSOC tumor sections, normal fallopian tube, and ovarian surface epithelial tissues.

    Data file S6. Raw and processed s-SILAC ratios from MIB-MS profiling of MRCKA knockdown in HGSOC cells.

    Data file S7. Raw and processed s-SILAC ratios from phosphoproteomics analysis of MRCKA knockdown in HGSOC cells.

    Data file S8. In vitro kinase inhibitor profiling report for BDP5290 and BDP9066.

    Data file S9. Raw and processed SILAC ratios from MIB-MS profiling of OVSAHO cells treated with BDP9066 or A-674563.

    Data file S10. Reagents used in the study, including small molecules, siRNAs, and antibodies.

  • The PDF file includes:

    • Fig. S1. Quantitation of kinase abundance in HGSOC tumors using MIB-MS and s-SILAC.
    • Fig. S2. Characterization of MRCKA in HGSOC tumors and HGSOC cell lines.
    • Fig. S3. Exploring MRCKA knockdown in HGSOC cells using MIB-MS and phosphoproteomics.
    • Fig. S4. MRCKA promotes focal adhesion signaling in HGSOC cells.
    • Fig. S5. Evaluation of MRCKA kinase inhibitors in HGSOC cells.
    • Fig. S6. Characterization of proliferation, migration, apoptosis, and spheroid formation in BDP9066-treated HGSOC cells.
    • Legends for data files S1 to S10

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Raw and processed s-SILAC ratios from MIB-MS profiling of sections from a single HGSOC tumor tissue.
    • Data file S2 (Microsoft Excel format). Characteristics of HGSOC primary and PDX tumors used in MIB-MS studies.
    • Data file S3 (Microsoft Excel format). Raw and processed s-SILAC ratios from MIB-MS profiling of HGSOC primary and PDX tumors.
    • Data file S4 (Microsoft Excel format). Prevalent MIB-MS kinome signature among HGSOC primary and PDX tumors.
    • Data file S5 (Microsoft Excel format). IHC analysis of MRCKA protein levels in HGSOC tumor sections, normal fallopian tube, and ovarian surface epithelial tissues.
    • Data file S6 (Microsoft Excel format). Raw and processed s-SILAC ratios from MIB-MS profiling of MRCKA knockdown in HGSOC cells.
    • Data file S7 (Microsoft Excel format). Raw and processed s-SILAC ratios from phosphoproteomics analysis of MRCKA knockdown in HGSOC cells.
    • Data file S8 (Microsoft Excel format). In vitro kinase inhibitor profiling report for BDP5290 and BDP9066.
    • Data file S9 (Microsoft Excel format). Raw and processed SILAC ratios from MIB-MS profiling of OVSAHO cells treated with BDP9066 or A-674563.
    • Data file S10 (Microsoft Excel format). Reagents used in the study, including small molecules, siRNAs, and antibodies.

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