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. 2018 Nov 28:437:1-12.
doi: 10.1016/j.canlet.2018.08.014. Epub 2018 Aug 24.

Integrated proteomic and phosphoproteomic analyses of cisplatin-sensitive and resistant bladder cancer cells reveal CDK2 network as a key therapeutic target

Jae Hun Jung  1 , Sungyong You  2 , Jae Won Oh  1 , Junhee Yoon  3 , Austin Yeon  3 , Muhammad Shahid  3 , Eunho Cho  4 , Vikram Sairam  4 , Taeeun D Park  5 , Kwang Pyo Kim  6 , Jayoung Kim  7
Affiliations

Integrated proteomic and phosphoproteomic analyses of cisplatin-sensitive and resistant bladder cancer cells reveal CDK2 network as a key therapeutic target

Jae Hun Jung et al. Cancer Lett. .

Abstract

Background: Cisplatin-based chemotherapy is currently part of the standard of care for bladder cancer (BC). Unfortunately, some patients respond poorly to chemotherapy and have acquired or developed resistance. The molecular mechanisms underlying this resistance remain unclear. Here, we introduce a multidimensional proteomic analysis of a cisplatin-resistant BC model that provides different levels of protein information, including that of the global proteome and phosphoproteome.

Methods: To characterize the global proteome and phosphoproteome in cisplatin-resistant BC cells, liquid chromatography-mass spectrometry/mass spectrometry experiments combined with comprehensive bioinformatics analysis were performed. Perturbed expression and phosphorylation levels of key kinases associated with cisplatin resistance were further studied using various cell biology assays, including western blot analysis.

Results: Analyses of protein expression and phosphorylation identified significantly altered proteins, which were also EGF-dependent and independent. This suggests that protein phosphorylation plays a significant role in cisplatin-resistant BC. Additional network analysis of significantly altered proteins revealed CDK2, CHEK1, and ERBB2 as central regulators mediating cisplatin resistance. In addition to this, we identified the CDK2 network, which consists of CDK2 and its 5 substrates, as being significantly associated with poor survival after cisplatin chemotherapy.

Conclusions: Collectively, these findings potentially provide a novel way of classifying higher-risk patients and may guide future research in developing therapeutic targets.

Keywords: Biological network; Bladder cancer; Cisplatin resistance; Competing interests; Global proteome; Phosphoproteomics.

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Conflict of interest statement

Competing interests

The authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of this review.

Figures

Figure 1.
Figure 1.. Quantitative measuring of the proteome and phosphoproteome using TMT labeling.
A. Experimental design to study the effects of EGF stimulation across 3 time points. A total of 3 and 2 biological replicates from T24S and T24R cells were analyzed for their global proteome and phosphoproteome, respectively. B. Overall experimental workflow for multiplex TMT labeling and comprehensive profiling of the global proteome and phosphoproteome. Both T24S and T24R cells were treated with EGF (0, 10, or 30 mins). Proteins were extracted using RIPA buffer and peptides were FASP digested with trypsin. Peptide samples were derived at 6 different time points, labeled using TMT reagents, mixed, and separated using mid-pH reverse phase chromatography. Fractions were combined in a non-contiguous way into 15 fractions for proteome analysis (5% of total proteins) and 10 fractions for phosphoproteome analysis (95% of total proteins). All peptides and phosphopeptides were analyzed on a Q Exactive Mass Spectrometer. Protein identification and. quantification was achieved using the MSGF+ search engine and MASIC (reporter ion intensity extractor). C and D. Numbers of identified (C) proteins or (D) phosphopeptides from T24S and T24R cells. Venn diagrams depict the number of common and uniquely identified proteins or phosphopeptides in T24S and T24R cells after 0, 10 and 30 mins of EGF stimulation
Figure 2.
Figure 2.. Altered proteins in the global proteome and phosphoproteome between T24R and T24S cells.
A and B. Volcano plots display differentially expressed (A) proteins or (B) differentially phosphorylated peptides. Red and blue dots represent upregulated and downregulated proteins or peptides, respectively. C. Protein expression and phosphorylation levels were compared and represented in the scatterplot. Proteins and phosphorylation sites were considered to be perturbed by cisplatin resistance based on the integrative hypothesis testing method (FDR < 0.05). Comparison between biological replicates of MS-based quantitative proteomic and phosphoproteomic experiments of T24R and T24S cells. Each dot represents one protein. D. Enrichment analysis using DAVID software showed that differentially expressed proteins (cyan) and differentially phosphorylated peptides (purple) were enriched for various cellular functions.
Figure 3.
Figure 3.. Distinct protein alteration patterns in T24R and T24S cells after EGF stimulation.
A and B. Density plots are shown for the log2 fold changes of the (A) global proteome and (B) phosphoproteome for T24R and T24S cells after 10 mins and 30 mins of EGF treatment. Only phosphopeptides or proteins quantified in at least two samples were plotted. Log2 fold changes in T24S cells at 10 mins (S10) vs. 0 min (S0) of EGF treatment, T24S cells at 30 mins (S30) vs. 0 min (S0) of EGF treatment, T24R cells at 10 mins (R10) vs. 0 min (R0) of EGF treatment, T24R cells at 30 mins (R30) vs. 0 min (R0) of EGF treatment, and R0 vs. S0 are displayed. C. Volcano plots display differentially expressed phosphopeptides in T24R and T24S cells after 10 mins and 30 mins of EGF treatment. Red and blue dots indicate up- and downregulated phosphopeptides, respectively. Only those quantified in at least two samples were plotted. D and E. Number of phosphoproteins associated with cisplatin resistance mechanisms that are involved in EGFR signaling in (D) T24S and (E) T24R cells. Venn diagram depicts the overlapping altered phosphoproteins in (D) T24S or (E) T24R cells with or without EGF treatment. F and G. Enriched cellular functions of the overlapping proteins after 10 mins and 30 mins of EGF treatment in (F) T24S and (G) T24R cells.
Figure 4.
Figure 4.. Identification of key kinases linked to cisplatin resistance through the activation of EGFR signaling.
A. Schematic diagram describing the process of reconstructing the networks of proteins significantly altered by EGF stimulation in T24R and T24S cells. Venn diagram depicts number of altered proteins involved in cisplatin resistance in downstream EGFR signaling. Using kinase-substrate interaction information of the significantly altered proteins and their upstream kinases, 27 kinases were initially selected. Of those, 3 kinases were found to be significant altered in either protein expression or phosphorylation after EGF treatment. B. List of the 3 key kinases with the number of significantly altered substrates and number of interactions. C. Network model describing the interactions of the 3 kinases and their substrates. Node and border color represent proteins phosphorylation ratios in T24R vs. T24S cells after 10 mins or 30 mins of EGF treatment, respectively. Red and green indicate up- and downregulated phosphorylation following EGF treatment, respectively.
Figure 5.
Figure 5.. Functional role of CDK2 associated with cisplatin resistance.
A. Western blot analysis of the key protein kinases. After stimulation with 10 ng/ml of EGF treatment for 0, 10, or 30 mins, cells were harvested for protein extraction and western blot analysis. Representative western blot images were selected after experiments were repeated at least 3 times. B. Gene silencing of CDK2 enhanced cisplatin sensitivity in T24R cells. T24R cells transiently transfected with siRNAs targeting CDK2, CHEK1, or ERBB2 were incubated in culture medium with 10 μM cisplatin. T24R cells transfected with control siRNAs (siCtrl) were used as controls. The cell viability rate was measured at various time points (0, 8, 24, 32, 40, 48, and 72 hrs). Experiments were done in triplicate. *p<0.05 (Student’s t-test). Representative western blot analysis data demonstrated that the levels expression of CDK2, CHEK1, or ERBB2 in these experiments were downregulated by targeting siRNAs. C. Cisplatin resistant BC cell lines (R24R, J82R, or RT4R) were treated with cisplatin alone, CDK2 inhibitor (CDKi), or a combination of both for 72 hr. Cell survival rates were quantified as describe in Methods. C. Increased expression levels of CDK2 in BC. IHC analysis was performed to measure the protein expression of CDK2 in bladder tissues from BC patients. Violin plots showing expression of CDK2 in NAT and tumor tissue. Representative images of NAT and tumor cores are shown.
Figure 6.
Figure 6.. CDK2 network may have clinical implications in BC.
A. Correlation of 72 network proteins in levels of gene expression vs. protein expression (left) or phosphorylation (right). Dots on the scatter plot represent proteins and lines represent regression lines. Correlation was measured using the Spearman’s rank correlation method. B. Selection criteria used to identify the 6 proteins from the 72 network proteins. C and D. Box plots display differential gene expression of the selected 6 proteins from the network model using the gene expression datasets from (C) Als et al. and (D)Lee et al. Box plots at the far right display differential representation of CDK2 network scores computed by the Z-score method. Significance levels of differential gene expression between BC patients who were alive and deceased after cisplatin treatment were computed via the Wilcoxon rank-sum test. E. Regression coefficient and significance of the 6 genes based on multivariate Cox regression analysis using Lee at al. dataset. F. Kaplan-Meier curves for overall survival based on the expression of the CDK2 network genes are shown for the high (upper 50th percentile; n=97) and low (lower 50th percentile; n=91) z-score groups from the Lee et al. patient dataset. P-values and hazard ratios (HRs) were taken from a Cox regression analysis. G. Box plots display differential expression of the 6 genes in the TCGA BC cohort. Wilcoxon rank-sum test was performed to compute significance of differential expression between high and low grade samples.
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