Abstract
Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors.
Highlights
From the ‡Division of Chemotherapy and Clinical Research, National Cancer Center Research Institute, Tokyo, 104-0045 Japan; ¶Department of Pathology, Wan Fan Hospital and Taipei Medical University, Taipei, 11031 Taiwan; ʈBioBusiness Group, Mitsui Knowledge Industry, Tokyo, 164-8555 Japan
Generation of the High-density reverse-phase protein array (RPPA) and Phosphoprotein Profiling—We constructed an RPPA onto which lysates of 95 cell lines derived from eight different types of cancer cultured in the presence and absence of 10% fetal calf serum (FCS) for 17 h and A431 cells untreated or treated with 200 ng/ml EGF for 10 min were randomly plotted
To clarify the molecular mechanism driving the activation of the mammalian target of rapamycin (mTOR) and MAPK pathways in sorafenib-resistant cells, we sequenced the entire exons of 511 kinases in 20 Hepatocellular carcinoma (HCC) cell lines using a next-generation sequencer
Summary
From the ‡Division of Chemotherapy and Clinical Research, National Cancer Center Research Institute, Tokyo, 104-0045 Japan; ¶Department of Pathology, Wan Fan Hospital and Taipei Medical University, Taipei, 11031 Taiwan; ʈBioBusiness Group, Mitsui Knowledge Industry, Tokyo, 164-8555 Japan. Consistent with the RPPA data, intense signals for p-RPS6 S235/236 were detected in the sorafenib-resistant cell lines via immunoblotting (Fig. 2A, lower portion).
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