Integrated Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Quantitative Proteomic Analysis Identifies Galectin-1 as a Potential Biomarker for Predicting Sorafenib Resistance in Liver Cancer.

Sorafenib has become the standard therapy for patients with advanced hepatocellular carcinoma (HCC). Unfortunately, most patients eventually develop acquired resistance. Therefore, it is important to identify potential biomarkers that could predict the efficacy of sorafenib. To identify target proteins associated with the development of sorafenib resistance, we applied stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomic approach to analyze differences in protein expression levels between parental HuH-7 and sorafenib-acquired resistance HuH-7 (HuH-7(R)) cells in vitro, combined with an isobaric tags for relative and absolute quantitation (iTRAQ) quantitative analysis of HuH-7 and HuH-7(R) tumors in vivo. In total, 2,450 quantified proteins were identified in common in SILAC and iTRAQ experiments, with 81 showing increased expression (>2.0-fold) with sorafenib resistance and 75 showing decreased expression (<0.5-fold). In silico analyses of these differentially expressed proteins predicted that 10 proteins were related to cancer with involvements in cell adhesion, migration, and invasion. Knockdown of one of these candidate proteins, galectin-1, decreased cell proliferation and metastasis in HuH-7(R) cells and restored sensitivity to sorafenib. We verified galectin-1 as a predictive marker of sorafenib resistance and a downstream target of the AKT/mTOR/HIF-1α signaling pathway. In addition, increased galectin-1 expression in HCC patients' serum was associated with poor tumor control and low response rate. We also found that a high serum galectin-1 level was an independent factor associated with poor progression-free survival and overall survival. In conclusion, these results suggest that galectin-1 is a possible biomarker for predicting the response of HCC patients to treatment with sorafenib. As such, it may assist in the stratification of HCC and help direct personalized therapy.

Activation of phosphatidylinositol 3-kinase/Akt signaling pathway mediates acquired resistance to sorafenib in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most common potentially lethal human malignancies worldwide. Sorafenib, a tyrosine kinase inhibitor, was recently approved by the United States Food and Drug Administration for HCC. In this study, we established two sorafenib-resistant HCC cell lines from Huh7, a human HCC cell line, by long-term exposure of cells to sorafenib. Sorafenib induced significant apoptosis in Huh7 cells; however, Huh7-R1 and Huh7-R2 showed significant resistance to sorafenib-induced apoptosis at the clinical relevant concentrations (up to 10 µM). Thorough comparisons of the molecular changes between Huh7 and resistant cells showed that the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway played a significant role in mediating acquired resistance to sorafenib in Huh7-R1 and Huh7-R2 cells. Phospho-Akt and p85 (a regulatory subunit of PI3K) were up-regulated, whereas tumor suppressor phosphatase and tensin homolog were down-regulated in these resistant cells. In addition, ectopic expression of constitutive Akt in Huh7 demonstrated similar resistance to sorafenib. The knockdown of Akt by RNA interference reversed resistance to sorafenib in Huh7-R1 cells, indicating the importance of Akt in drug sensitivity. Furthermore, the combination of 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one dihydrochloride (MK-2206), a novel allosteric Akt inhibitor, and sorafenib restored the sensitivity of resistant cells to sorafenib-induced apoptosis. In conclusion, activation of PI3K/Akt signaling pathway mediates acquired resistance to sorafenib in HCC, and the combination of sorafenib and MK-2206, an Akt inhibitor, overcomes the resistance at clinical achievable concentrations.

Inhibition of the Wnt/ß-catenin signaling pathway improves the anti-tumor effects of sorafenib against hepatocellular carcinoma.

Sorafenib, a multikinase inhibitor, is currently the only approved drug for advanced hepatocellular carcinoma (HCC). The current study tested the hypothesis whether inhibition of the Wnt/ß-catenin signaling pathway could improve the anti-tumor effects of sorafenib in HCC. ICG-001, a small molecule which blocks the interaction of ß-catenin with its transcriptional coactivator CBP, dose-dependently enhanced the growth-suppressive and apoptosis-induction effects of sorafenib in multiple HCC cell lines. Downregulation of ß-catenin by RNA interference increased sorafenib sensitivity, whereas overexpression of ß-catenin reduced sorafenib sensitivity in Huh7 cells. The sorafenib-sensitization effect of short hairpin RNA (shRNA)-mediated ß-catenin downregulation in Huh7 cells was attenuated by ß-catenin overexpression. Mechanistically, sorafenib combined with ICG-001 or shRNA-mediated ß-catenin downregulation augmented the induction of apoptosis, and resulted in a significant downregulation of Mcl-1 in HCC cells. In Huh7 cell mouse xenograft model, the combination of ICG-001 and sorafenib showed a more significant growth-retarding effect than single agent treatment of sorafenib or ICG-001. Our data indicate that inhibition of the Wnt/ß-catenin signaling pathway improves the antitumor effects of sorafenib against HCC in vitro and in vivo.

Combinations of mTORC1 inhibitor RAD001 with gemcitabine and paclitaxel for treating non-Hodgkin lymphoma.

Single-agent mammalian target of rapamycin complex 1 (mTORC1) inhibitors have recently been reported as effective salvage treatment in non-Hodgkin lymphoma (NHL). The combined effect of mTORC1 inhibitor, RAD001, with chemotherapeutic agents used for relapsed or refractory NHL was examined. Synergistic interactions were observed for RAD001 plus gemcitabine or paclitaxel in six NHL cell lines; enhanced gemcitabine- and paclitaxel-induced caspase-dependent apoptosis associated with down-regulation of mTOR signaling was detected. Synergistic interactions were also observed with RAD001 plus gemcitabine and paclitaxel. In conclusion, synergistic cytotoxicity was observed with RAD001 plus gemcitabine and paclitaxel in NHL cells. Combination therapy with these three drugs should be examined in patients with refractory or relapsed NHL.

Induction of DNA damage-inducible gene GADD45beta contributes to sorafenib-induced apoptosis in hepatocellular carcinoma cells.

Markers that could accurately predict responses to the general kinase inhibitor sorafenib are needed to better leverage its clinical applications. In this study, we examined a hypothesized role in the drug response for the growth arrest DNA damage-inducible gene 45ß (GADD45ß), which is commonly underexpressed in hepatocellular carcinoma (HCC) where sorafenib may offer an important new therapeutic option. The anticancer activity of sorafenib-induced GADD45ß expression was tested in a panel of HCC cell lines and xenograft models. We found that GADD45ß mRNA and protein expression were induced relatively more prominently in HCC cells that were biologically sensitive to sorafenib treatment. GADD45ß induction was not found after treatment with either the mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 or the Raf inhibitor ZM336372, suggesting that GADD45ß induction by sorafenib was independent of Raf/MEK/ERK signaling activity. However, c-Jun NH2-terminal kinase (JNK) kinase activation occurred preferentially in sorafenib-sensitive cells. Small interfering RNA-mediated knockdown of GADD45ßor JNK kinase limited the proapoptotic effects of sorafenib in sorafenib-sensitive cells. We defined the -339/-267 region in the GADD45ß promoter containing activator protein-1 and SP1-binding sites as a crucial region for GADD45ß induction by sorafenib. Together, our findings suggest that GADD45ß induction contributes to sorafenib-induced apoptosis in HCC cells, prompting further studies to validate its potential value in predicting sorafenib efficacy.

OSU-03012, a novel celecoxib derivative, induces reactive oxygen species-related autophagy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Systemic treatments for HCC have been largely unsuccessful. OSU-03012 is a derivative of celecoxib with anticancer activity. The mechanism of action is presumably 3-phosphoinositide-dependent kinase 1 (PDK1) inhibition. This study investigated the potential of OSU-03012 as a treatment for HCC. OSU-03012 inhibited cell growth of Huh7, Hep3B, and HepG2 cells with IC(50) below 1 mumol/L. In Huh7 cells, OSU-03012 did not suppress PDK1 or AKT activity. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and flow cytometry analysis indicated that OSU-03012 did not induce cellular apoptosis. Instead, morphologic studies by light and electron microscopy, as well as special biological staining with monodansylcadaverine, acridine orange, and microtubule-associated protein 1 light chain 3, revealed OSU-03012-induced autophagy of Huh7 cells. This OSU-03012-induced autophagy was inhibited by 3-methyladenine. Moreover, reactive oxygen species (ROS) accumulation was detected after OSU-03012 treatment. Blocking ROS accumulation with ROS scavengers inhibited autophagy formation, indicating that ROS accumulation and subsequent autophagy formation might be a major mechanism of action of OSU-03012. Daily oral treatment of BALB/c nude mice with OSU-03012 suppressed the growth of Huh7 tumor xenografts. Electron microscopic observation indicated that OSU-03012 induced autophagy in vivo. Together, our results show that OSU-03012 induces autophagic cell death but not apoptosis in HCC and that the autophagy-inducing activity is at least partially related to ROS accumulation.