Extracellular Matrix Dynamics in Hepatocarcinogenesis: a Comparative Proteomics Study of PDGFC Transgenic and Pten Null Mouse Models

Abstract

We are reporting qualitative and quantitative changes of the extracellular matrix (ECM) and associated receptor proteomes, occurring during the transition from liver fibrosis and steatohepatitis to hepatocellular carcinoma (HCC). We compared two mouse models relevant to human HCC: PDGFC transgenic (Tg) and Pten null mice, models of disease progression from fibrosis and steatohepatitis to HCC. Using mass spectrometry, we identified in the liver of both models proteins for 26 collagen-encoding genes, providing the first evidence of expression at the protein level for 16 collagens. We also identified post-transcriptional protein variants for six collagens and lysine hydroxylation modifications for 14 collagens. Tumor-associated collagen proteomes were similar in both models with increased expression of collagens type IV, VI, VII, X, XIV, XV, XVI, and XVIII. Splice variants for Col4a2, Col6a2, Col6a3 were co-upregulated while only the short form of Col18a1 increased in the tumors. We also identified tumor specific increases of nidogen 1, decorin, perlecan, and of six laminin subunits. The changes in these non-collagenous ECM proteins were similar in both models with the exception of laminin β3, detected specifically in the Pten null tumors. Pdgfa and Pdgfc mRNA expression was increased in the Pten null liver, a possible mechanism for the similarity in ECM composition observed in the tumors of both models. In contrast and besides the strong up-regulation of integrin α5 protein observed in the liver tumors of both models, the expression of the six other integrins identified was specific to each model, with integrins α2b, α3, α6, and β1 up-regulated in Pten null tumors and integrins α8 and β5 up-regulated in the PDGFC Tg tumors. In conclusion, HCC–associated ECM proteins and ECM–integrin networks, common or specific to HCC subtypes, were identified, providing a unique foundation to using ECM composition for HCC classification, diagnosis, prevention, or treatment.

Author Summary

The microenvironment can have a profound influence on cellular behavior and survival and on growth of developing tumor cells. We present the first comprehensive analysis of the extracellular matrix (ECM) and associated receptor proteomes, applied here to the study of hepatocellular carcinoma (HCC). This study demonstrates the utility of mass spectrometry-based approaches to characterize, at the protein level, gene families with extensive sequence homology, post-transcriptional regulations, and post-translational regulations. This is also the first study to analyze and compare liver proteome changes occurring during the transition from fibrosis and steatohepatitis, common preneoplastic conditions in humans, to HCC, using two mouse models. This approach identifies ECM and integrin components, which could play an important role in the early steps of hepatocarcinogenesis, and provides a path to identifying ECM–tumor cell networks that may contribute to the heterogeneous features of HCC.