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. 2018 Aug;18(15):e1700388.
doi: 10.1002/pmic.201700388. Epub 2018 Jul 8.

Integrative Analysis of Proteome and Ubiquitylome Reveals Unique Features of Lysosomal and Endocytic Pathways in Gefitinib-Resistant Non-Small Cell Lung Cancer Cells

Wang Li  1 , Heyong Wang  1 , Yan Yang  1 , Tian Zhao  1 , Zhixiong Zhang  1 , Ye Tian  1 , Zhaomie Shi  2 , Xiaojun Peng  2 , Fei Li  3 , Yonghong Feng  1 , Lei Zhang  1 , Gening Jiang  1 , Fan Zhang  1
Affiliations

Integrative Analysis of Proteome and Ubiquitylome Reveals Unique Features of Lysosomal and Endocytic Pathways in Gefitinib-Resistant Non-Small Cell Lung Cancer Cells

Wang Li et al. Proteomics. 2018 Aug.

Abstract

Non-small cell lung cancer (NSCLC) patients carrying EGFR activating mutations treated with gefitinib, a tyrosine kinase inhibitor, will develop drug resistance. Ubiquitylation is one of major posttranslational modifications of proteins affecting the stability or function of proteins. However, the role of protein ubiquitylation in gefitinib resistance is poorly understood. To systematically identify the global change in protein expression and ubiquitylation during gefitinib resistance, a quantitative global proteome and ubiquitylome study in a pair of gefitinib-resistant and sensitive NSCLC cells is carried out. Altogether, changes in expression of 3773 proteins are quantified, and changes in ubiquitylation of 2893 lysine sites in 1415 proteins are measured in both cells. Interestingly, lysosomal and endocytic pathways, which are involved in autophagy regulation, are enriched with upregulated proteins or ubiquitylated proteins in gefitinib-resistant cells. In addition, HMGA2 overexpression or ALOX5 knockdown suppresses gefitinib resistance in NSCLC cells by inhibiting autophagy. Overall, these results reveal the previously unknown global ubiquitylome and proteomic features associated with gefitinib resistance, uncover the opposing roles of HMGA2 or ALOX5 in regulating gefitinib resistance and autophagy, and will help to identify new therapeutic targets in overcoming gefitinib resistance.

Keywords: gefitinib resistance; non-small cell lung cancer; proteome; ubiquitylome.

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Figures

Figure 1
Figure 1
Characterization of protein ubiquitylome in gefitinib‐resistant NSCLC cells. A) Proteome and ubiquitylome project workflow. PC9/GR cells were labeled with light (L) isotopic 12C lysine, and PC9 cells were labeled with heavy (H) isotopic 13C lysine. B) Quantiles‐based clustering of quantified proteins in the proteome study (upper panel) or quantified ubiquitylated lysine sites in the ubiquitylome study (lower panel), in PC9/GR versus PC9 cells. The quantified proteins or ubiquitylated lysine sites were divided into four quantiles according to the log2 ratio (L: H): Q1 (0–25%; dark green), Q2 (25–50%; light green), Q3 (50–75%; light red), and Q4 (75–100%; dark red). C) Comparison of protein expression in PC9/GR versus PC9 cells using two different methods. Log2 ratio (L:H) of indicated proteins from the proteome study (left panel). N/A stands for the data not available in the proteome study. Western blotting (WB) validation of differentially expressed proteins in PC9/GR and PC9 cells (right panel). GAPDH serves as a loading control in WB. D) Venn diagram showing the overlapping groups of proteins among up‐ or downregulated proteins and proteins with up‐ or downregulated ubiquitylated lysine sites in PC9/GR cells versus PC9 cells. The bar graph shows the number of proteins in each overlapping group of proteins. E) Bar graph showing 12 proteins containing both up‐ and downregulated ubiquitylation sites within the same protein. F) Distribution of the number of ubiquitylated lysine sites per protein in PC9/GR versus PC9 cells. The black or red bar indicates proteins either identified or quantified in the ubiquitylome study.
Figure 2
Figure 2
Analysis of the characters of the ubiquitylated peptides in PC9/GR and PC9 cells. A) Ubiquitylation motifs were constructed with the Motif‐X software. The central K (at position 0) indicates the ubiquitylated lysine. All the surrounding amino acid residues are indicated with the letters in different heights which is consistent with their frequency in respective position. B) Heat map showing the frequency of the amino acid residues around the ubiquitylated lysine site based on analysis of all the ubiquitylated peptides in each motif. C) Number of the ubiquitylated peptides in each motif. D) Heat map showing the enriched motifs in the representative KEGG pathways. z score = −log10 (Fisher's exact test p value). Green represents the negative z score, and red represents the positive z score.
Figure 3
Figure 3
Classification of molecular functions and pathway enrichment analysis for differentially expressed proteins or proteins with lysine sites undergoing differential regulation in ubiquitylation in PC9/GR versus PC9 cells. Molecular functions for (A) upregulated or (B) downregulated proteins in PC9/GR versus PC9 cells. Molecular functions for proteins containing lysine sites undergoing (C) upregulated or (D) downregulated ubiquitylation in PC9/GR versus PC9 cells. Pathway enrichment analysis for (E) differentially expressed proteins or (F) proteins with differentially regulated ubiquitylation in PC9/GR versus PC9 cells. The top x axis represents the number of proteins enriched in the pathway, the bottom x axis represents – log (p value), and the y axis represents the name of KEGG pathway.
Figure 4
Figure 4
Lysosomal and endocytic pathways are enriched with upregulated proteins or protein ubiquitylation in PC9/GR cells. A) The lysosomal pathway was significantly overpresented with upregulated proteins. B) Lysosomal and endocytosis pathways were significantly overpresented with upregulated protein ubiquitylation in PC9/GR cells. Proteins with upregulated (red), downregulated (dark green), or similar (light green) expression or ubiquitylation (red) in PC9/GR cells are shown.
Figure 5
Figure 5
Ubiquitylated sites in proteins revealed by diGly proteomics in PC9/GR cells. Ubiquitylated proteins are listed in the following pathways: for SNARE interactions in vesicular transport pathway (red), the ubiquitylated targets are STX3, STX19, BET1, USE1, SNAP23, VAMP8, VAMP4, VAMP5, VAMP7, and VAMP3; for the endocytosis pathway (blue), the target proteins are: PLD1, HSPA8, EGFR, CLTA, CLTC, TGFBR1, ITCH, ITCH, CAV1, TFRC, HLA‐C, RAB5C, RAB7A,RAB11B, PML, AP2M1,TSG101, VPS37A, MVB12B, CHMP2A,LDLR, RUFY1, ARFGAP1, CAV2, and NEDD4L; for the lysosome pathway (blue), they are: ATP6V0A4, ATP6AP1, CLTA, CLTC, ABCA2, SLC17A5, MFSD8, CTNS, LAPTM4A, CLN3, and AP1G1; for the ABC transporter pathway (grey), they are: ABCA2, ABCA12, TAP2, ABCB6, ABCC1, ABCC2, and ABCC4; for the phagosome pathway (pink), the targets are: ATP6V1C1, ATP6V1E1, ATP6V0A4, ATP6AP1, RAC1, TAP2, ACTB, ITGB1, ITGA2, TFRC, HLA‐C, TUBA1B, TUBB4B, RAB5C, RAB7A, OLR1, DYNC1H1, and VAMP3; for the ubiquitin mediate proteolysis pathway (brown), the target proteins are: CDC27, FBXW11, CUL3, WWP2, ITCH, ITCH, PML, UBE2G1, UBE2N, HUWE1, PRPF19, TRIM32, DDB1, SAE1, UBA3, UBA6, NEDD4L, and BIRC2; for the endocrine and other factor‐regulated calcium reabsorption pathway (red), the targets are: ATP1A1, PRKCB, PRKACB, GNAS, GNAQ, CLTA, CLTC, ATP2B1, and AP2M1. Dotted lines indicated cell organelle where the pathways function. ↑and↓ indicate protein level decreases or increases in gefitinib‐resistant PC9/GR cells.
Figure 6
Figure 6
Overexpression of HMAG2 or knocking down of ALOX5 suppresses gefitinib resistance by inhibiting autophagy. A) WB validation of HMAG2 overexpression in PC9/GR cells stably expressing the control (Control OE, #1 and #2) or HMGA2 overexpression (HMGA2 OE, #1 and #2) plasmids. The ratio of band intensity of HMGA2 versus GAPDH (HMGA2/GAPDH) was shown underneath each condition. B) Comparison of gefitinib‐resistant colony formation between PC9/GR cells stably expressing the control or HMGA2 overexpression plasmid. PC9/GR cells were cultured in 5 or 10 μm gefitinib. The number of colonies is quantified in the bar graph on the right side. Each condition was repeated in triplicates. This is true for all the rest of the experiments. C) Quantification of HMAG2 knockdown level in PC9/GR cells stably expressing the control (sh‐Control) or shRNA targeting HMGA2 (sh‐HMGA2, #1 and #2) by RT‐qPCR. D) Comparison of gefitinib‐resistant colony formation between PC9/GR cells stably expressing sh‐Control or sh‐HMGA2 #1, cultured in 5 or 10 μm gefitinib. E) WB validation of flag tagged ALOX5 overexpression in PC9/GR cells stably expressing the control (Control OE) or ALOX5 overexpression (Flag‐ALOX5 OE) plasmid. F) Comparison of gefitinib‐resistant colony formation between PC9/GR cells stably expressing the control or ALOX5 overexpression plasmid. G) Quantification of ALOX5 knockdown level in PC9/GR cells stably expressing the control (sh‐Control) or shRNA targeting ALOX5 (sh‐ ALOX5, #1 and #2) by RT‐qPCR. H) Comparison of gefitinib‐resistant colony formation between PC9/GR cells stably expressing sh‐Control or sh‐ ALOX5 #1. I) Comparison of gefitinib‐resistant colony formation between PC9/GR cells treated with the control (DMSO) or 200 um Zileuton. J) WB detection of LC3B II (autophagy marker) expression in PC9/GR cells overexpressing control (Control OE) or HMGA2 (HMGA2 OE) plasmid at 1 or 4 days. K) WB detection of LC3B and ALOX5 expression in PC9/GR cells overexpressing the control shRNA (sh‐Control) or shRNA targeting ALOX5 (sh‐ALOX5, #1 and #2).
Figure 7
Figure 7
Proteins or genes found up‐ or downregulated in drug‐resistant cancer cells from other studies. A) Proteins or genes found upregulated (left panel) or downregulated (right panel) in PC9/GR cells (from our proteomic study) and erlotinib‐resistant head and neck SCC cells (from the GSE62061 study) by Venn diagram analysis. B) Proteins or genes found upregulated (left panel) or downregulated (right panel) in PC9/GR cells (from our proteomic study) and paclitaxel‐resistant MDA‐MB‐231 cells (from the GSE12791 study) by Venn diagram analysis. The proteins or genes shared by both studies were listed besides the Venn diagram. HMGA2 and ALOX5 are highlighted in red.
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