Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels

Abstract

Protein abundance and phosphorylation convey important information about pathway activity and molecular pathophysiology in diseases including cancer, providing biological insight, informing drug and diagnostic development, and guiding therapeutic intervention. Analyzed tissues are usually collected without tight regulation or documentation of ischemic time. To evaluate the impact of ischemia, we collected human ovarian tumor and breast cancer xenograft tissue without vascular interruption and performed quantitative proteomics and phosphoproteomics after defined ischemic intervals. Although the global expressed proteome and most of the >25,000 quantified phosphosites were unchanged after 60 min, rapid phosphorylation changes were observed in up to 24% of the phosphoproteome, representing activation of critical cancer pathways related to stress response, transcriptional regulation, and cell death. Both pan-tumor and tissue-specific changes were observed. The demonstrated impact of pre-analytical tissue ischemia on tumor biology mandates caution in interpreting stress-pathway activation in such samples and motivates reexamination of collection protocols for phosphoprotein analysis.

Fig. 1.

Quantitative proteome and phosphoproteome analysis of human ovarian tumors and xenograft breast tumors subjected to controlled ischemia. A, experimental design to study effects of post-excision delay time before freezing across four time points. After excision, tumor samples were cut into four equal pieces and incubated for the indicated times at room temperature before freezing. A total of four different ovarian tumors and three pooled breast cancer xenograft samples for the basal and for the luminal subtype were analyzed. All of these samples were biologically distinct and can be considered as biological replicates. B, quantitative proteomics and phosphoproteomics workflow using 4-plex iTRAQ labeling. Tumor samples were cryofractured and proteins were extracted with urea lysis buffer prior to digestion into peptides using trypsin. Peptide samples derived at four different ischemic time points were labeled using iTRAQ reagents, mixed equably, and separated using high-pH reversed-phase chromatography. Fractions were combined in a noncontiguous way into 24 fractions for proteome analysis (5% of the total material) and 12 fractions for phosphoproteome analysis (95% of the total material). Ovarian cancer samples were analyzed on an LTQ-Orbitrap Velos, and xenograft breast cancer samples were analyzed on a Q Exactive mass spectrometer. Phosphosite and protein identification and quantification were achieved using Spectrum Mill.

Fig. 2.

Global proteome analysis revealed no changes in protein abundance level, but specific alterations in the phosphoproteome induced by ischemia were observed. Density plots are shown for averaged phosphosite iTRAQ ratios (labeled with “pSTY”) and protein iTRAQ ratios (small insets) for the ovarian cancer (OC) samples and the basal-like and luminal breast cancer (BC) samples. Only phosphosites/proteins were plotted that were quantified in at least three ovarian tumors and at least two basal-like or luminal tumor samples.

Fig. 3.

Temporal dynamics of phosphorylation changes resulting from cold ischemia. A, fuzzy c-means clustering of temporal profiles for all regulated phosphosites observed in the ovarian and breast cancer samples. We detected six up-regulated (U) clusters, which were further grouped pairwise into early (U1), middle (U2), and late clusters (U3), and three down-regulated (D1, D2, D3) clusters using a fuzzyfication parameter m = 1.6. Phosphosites were assigned to each cluster with a membership value α > 0.7. T1/2 indicates the median over all half-maximum time points for all phosphosites within a cluster. Half-maximum time points were determined via first-order kinetic modeling analysis. B, number of regulated phosphosites assigned to each cluster for the ovarian and breast cancer tumors. C, gene enrichment analysis of regulated human phosphoproteins across early, middle, and late clusters. We used DAVID Bioinformatics Resources 6.7 (39) to test for enrichment of GO BPs in each cluster relative to a list of all proteins containing nonregulated sites using a modified Fisher's exact test (EASE score). GO BP categories with p < 0.01 and a minimum occurrence of ≥10 genes/proteins were called significant. p values were −Log10 transformed, and the transformed values for each annotation were plotted as a heat map in Gene-E.

Fig. 4.

Ischemia induced common phosphorylation events in ovarian and basal-like/luminal breast cancer tumor samples. A, scatter plots of averaged phosphosite ratios (5:0, 30:0, and 60:0) over at least three ovarian tumors (OC) and at least two basal-like (BA) or luminal (LU) breast cancer samples. X and Y indicate which tumor type is plotted on the respective axis. Intertumor Pearson correlation coefficients are indicated for each tumor type comparison. B, C, Venn diagrams of the overlap of up-regulated (B) and down-regulated (C) phosphosites between ovarian and basal-like/luminal breast cancer samples.

Fig. 5.

Stress-response kinases were affected by cold ischemia. A, kinases detected with and without regulated phosphosites were mapped in a dendrogram of the human kinome (40) using KinomeCluster. Ischemia-regulated kinases were observed across all major kinase subfamilies. B, protein–protein interaction and kinase-substrate network of phosphoproteins commonly regulated in all analyzed ovarian and breast cancer samples. High confidence, experimentally validated protein–protein interaction information was obtained from the STRING protein interaction database (26). Kinase–substrate relationships were derived from the PhosphoSite database (27). Protein–protein interaction and site-specific kinase–phosphosite relationships were illustrated using Cytoscape (41), with blue edges indicating protein–protein interactions and red dashed arrows indicating kinase–substrate relationships. Up-regulated phosphosites are shown in green, and down-regulated sites are in red. Kinases are depicted as squares, whereas all other proteins appear as circles.