Heterogeneity of pancreatic cancer metastases in a single patient revealed by quantitative proteomics

Abstract

Many patients with pancreatic cancer have metastases to distant organs at the time of initial presentation. Recent studies examining the evolution of pancreatic cancer at the genetic level have shown that clonal complexity of metastatic pancreatic cancer is already initiated within primary tumors, and organ-specific metastases are derived from different subclones. However, we do not yet understand to what extent the evolution of pancreatic cancer contributes to proteomic and signaling alterations. We hypothesized that genetic heterogeneity of metastatic pancreatic cancer results in heterogeneity at the proteome level. To address this, we employed a model system in which cells isolated from three sites of metastasis (liver, lung, and peritoneum) from a single patient were compared. We used a SILAC-based accurate quantitative proteomic strategy combined with high-resolution mass spectrometry to analyze the total proteome and tyrosine phosphoproteome of each of the distal metastases. Our data revealed distinct patterns of both overall proteome expression and tyrosine kinase activities across the three different metastatic lesions. This heterogeneity was significant because it led to differential sensitivity of the neoplastic cells to small molecule inhibitors targeting various kinases and other pathways. For example, R428, a tyrosine kinase inhibitor that targets Axl receptor tyrosine kinase, was able to inhibit cells derived from lung and liver metastases much more effectively than cells from the peritoneal metastasis. Finally, we confirmed that administration of R428 in mice bearing xenografts of cells derived from the three different metastatic sites significantly diminished tumors formed from liver- and lung-metastasis-derived cell lines as compared with tumors derived from the peritoneal metastasis cell line. Overall, our data provide proof-of-principle support that personalized therapy of multiple organ metastases in a single patient should involve the administration of a combination of agents, with each agent targeted to the features of different subclones.

Fig. 1.

A SILAC strategy combined with high-resolution mass spectrometry for differential proteome analysis. A, schematic of a SILAC-based quantitative proteomics for differential proteome analysis of cells isolated from metastatic pancreatic cancer in distal organs. B, highly accurate mass measurements were carried out with sub-ppm error on the LTQ-Orbitrap Elite mass spectrometer. C, distribution of Mascot scores of all peptide-spectrum matches at 1% false discovery rates shows a median Mascot score of ∼59 derived from high-quality tandem mass spectrometry data acquired in this study. D, over 4,200 proteins were observed in this experiment. When two metastatic pancreatic cancer proteomes (i.e. peritoneal metastasis versus liver metastasis) were compared, ∼28% of proteins were found to vary by >2-fold among metastases, although most of the proteins remained unchanged.

Fig. 2.

Receptor tyrosine kinases as a highly regulated class of proteins in metastatic pancreatic cancer revealed by quantitative proteomics analysis. A, proteomes of liver metastasis and lung metastasis were found to be more similar to each other than to that of peritoneum metastasis. This result is consistent with genetic data from in our previous study showing that lung pancreatic metastasis is evolutionarily closer to liver pancreatic metastasis. This indicates that a pattern of proteome expression among genetically close clones is closely related. In other words, a genetically different clone may have a different proteome expression. B, the pie chart shows that ∼42% of proteomes in cells derived from distal metastatic pancreatic cancers were categorized in proteins that were highly variable when pairwise comparison of three metastases was carried out. C, bioinformatics analysis showed that receptor/signal transducer activities were enriched in altered proteome.

Fig. 3.

Unique activation of different signaling pathways in metastatic pancreatic cancer revealed by quantitative tyrosine phosphoproteomics analysis. A, a pattern of tyrosine phosphorylation levels among pancreatic cancer metastases was briefly screened via Western blot experiment using anti-phosphotyrosine antibody. B, schematics of SILAC-based quantitative tyrosine phosphoproteomics of multiple pancreatic cancer metastases. Tyrosine phosphorylated peptides were enriched via immunoaffinity purification using anti-phosphotyrosine antibodies. C, the pie chart shows that a greater portion (∼89%) of identified tyrosine phosphorylation sites was found to be altered.

Fig. 4.

Axl receptor tyrosine kinase as a potential therapeutic target revealed by inhibitor screening assay and in vivo mouse study. A, the heat map shows the differential sensitivity of metastatic pancreatic cancer cells derived from peritoneum, lung, and liver to small molecule inhibitors. B, mice xenografted with metastatic pancreatic cancer cells derived from lung and liver were more sensitive to R428, a small molecule inhibitor to Axl receptor tyrosine kinase in vivo mouse model, than mice xenografted with cells derived from peritoneum. C, five representative immunohistochemistry images of Axl staining on matched primary and metastatic pancreatic cancers from five different patients are depicted (A31, A29, A55, A89, and A92). A31 is an example of low expression in the primary and matched metastasis. The arrow in A31 primary indicates strong positive labeling of endothelium within a small capillary, as compared with negative labeling of the neoplastic cells. The negative labeled cancer cells are circled with a dashed line in A31 metastasis. A29 is an example of strong positive labeling in the primary carcinoma and weak labeling in the metastasis. By contrast, A55 is an example of weak labeling in the primary and strong labeling in the metastasis. A89 and A92 are examples of strong positive labeling in both the primary and the matched metastasis. The arrow in the A89 metastasis indicates a negative labeled normal bile duct.