Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient

Significance Elucidation of evasive resistance to targeted therapies is a major challenge in cancer research. As a proof-of-concept study, we describe quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated hepatocellular carcinoma (HCC) patient. This approach reveals signaling pathway activity in a tumor and how it evades therapy. The described method will allow precision medicine based on phenotypic data. In particular, application of the method to a patient cohort will potentially identify new biomarkers, drug targets, and signaling pathways that mediate evasive resistance. Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine.