Purpose: Patients with liver cirrhosis or hepatocellular carcinoma (HCC) have decreased serum insulinlike growth factor (IGF)-1 levels. We evaluated whether IGF-1 levels were associated with the outcomes of patients with advanced HCC treated with systemic antiangiogenic therapy.Experimental Design: The study was based on patients with advanced HCC who were enrolled in two clinical trials evaluating first-line combination antiangiogenic therapy. Serum samples were collected before treatment and four to six weeks after the start of treatment. The levels of IGF-1, IGF-2, and IGF-binding protein-3 (IGFBP3) were analyzed for their associations with treatment outcomes.Results: A total of 83 patients were included in the study. Patients who had high (!the median level) baseline IGF-1 levels had significantly higher disease control rate (DCR) than patients who had low (

CCN family protein 2 (CCN2), also known as connective tissue growth factor, is a secreting protein that modulates multiple cellular events. We previously demonstrated the metastasis-suppressive effect of CCN2 in lung cancer cells. In this study, we investigate the role of CCN2 in anoikis, a form of programmed cell death that is critical in suppressing cancer metastasis. CCN2 binds to the epidermal growth factor receptor (EGFR) and triggers ubiquitination by inhibiting the formation of the β-pix/Cbl complex, resulting in the degradation of EGFR. Binding of CCN2 to EGFR suppresses the phosphorylation of c-Src and extracellular signal-regulated kinase but increases the expression of death-associated protein kinase, which leads to anoikis. Overall, our findings provide evidence validating the use of CCN2 as an anti-metastatic therapy in lung cancer patients, and prospect a potential therapeutic synergy between CCN2 and the anti-EGFR antibody for the treatment of lung cancer.

Sorafenib, a multikinase inhibitor targeting Raf kinase and vascular endothelial growth factor receptor, is currently the only approved agent for hepatocellular carcinoma (HCC). However, the majority of HCC patients are inherently resistant to sorafenib treatment. To study molecular mechanisms underlying the resistance to sorafenib in vivo, we studied immunocompromised mice implanted with xenografts of human HCC cells subcutaneously, and identified tumors that showed primary resistance to sorafenib. Primary cultured cells were established from sorafenib-resistant xenografts, and verified for in vitro and in vivo sensitivity to sorafenib. The differentially expressed genes of the resistant cells versus control cells were analyzed by cDNA microarray. The expressions of candidate markers were confirmed by Western blotting, and their significances were verified by specific pathway inhibitors. A subline of Huh7 cells was established by primary culture from a sorafenib-resistant HCC-xenograft (designated as Huh7.1.SR). Compared to Huh7 cells primarily cultured from HCC-xenograft treated with vehicle alone (designated as Huh7.1.v), Huh7.1.SR had a moderate increase of resistance to sorafenib in vitro, with 50%-inhibitory concentration (IC50) increased from 5 to 10 μM. Subcutaneous xenograft studies confirmed that Huh7.1.SR retained the phenotype of in vivo resistance to sorafenib. The IPA (ingenuity pathway analysis) of differentially expressed genes of Huh7.1.SR versus Huh7.1.v cells revealed several pathway networks centering at transforming growth factor beta receptor (TGFBR), hepatocyte nuclear factors, NOTCH, pro-inflammatory cytokines, and nuclear factor kappa B. Huh7.1.SR had an increased expression of TGF-beta and p-Smad2, indicating that the TGF-beta pathway was activated. Huh7.1.SR cells expressed phenotypic features of epithelial to mesenchymal transition (EMT), including higher expression of vimentin, lower expression of E-cadherin, and increased cell migration. The expressions of Slug and Twist, two well-known transcriptional factors of inducing EMT, also increased in Huh7.1.SR cells. Treatment of SD-208, an inhibitor of TGF-beta receptor, partially reversed the phenotypic features of EMT in Huh7.1.SR cells. Moreover, concomitant treatment of SD-208 improved the sensitivity to sorafenib in Huh7.1.SR cells. Our data indicate that activation of TGF-beta signaling pathway is an important mechanism mediating the resistance to sorafenib in HCC in vivo. (The study was supported by the grant NSC 100-2325-B-002-043- from National Science Council of Taiwan) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1904. doi:1538-7445.AM2012-1904

Sorafenib, a multikinase inhibitor against Raf kinase and vascular endothelial growth factor receptor (VEGFR), was recently approved for the treatment of advanced hepatocellular carcinoma (HCC). However, the majority of HCC are inherently resistant to sorafenib. A global proteomic approach was applied to delineate the molecular mechanisms affecting the effectiveness of sorafenib in HCC cells. HCC cells (Huh7, SKHep1, and PLC5) were treated with sorafenib and analyzed by the SILAC (Stable Isotope Labeling with Amino acids in Cell culture) methodology. A total of 354 phosphoproteins were identified from untreated Huh7 cells (labeled with light amino acid, designated as L) and sorafenib-treated cells (labeled with heavy amino acid, designated as H). Forty-five proteins with high L/H ratio, indicating decreased expression after sorafenib treatment, were recognized. Among them, downstream proteins of Raf kinase, which are expected to be downregulated by sorafenib, were found. On the other hand, 30 proteins with low L/H ratio, indicating increased expression after sorafenib treatment, were recognized. Biological interaction network analysis of the phosphoproteome using IPA (ingenuity pathway analysis) method revealed that several signaling pathways including PI3K/AKT were significantly altered in sorafenib-treated Huh7 cells. To validate the relevance of these pathway alterations, the expression of p-AKT (Ser 473) was examined and was found significantly upregulated by sorafenib. Downregulation of AKT by siRNA attenuated the upregulation of p-AKT induced by sorafenib and enhanced the cytotoxicity of sorafenib in Huh7 cells. Combination of MK-2206, an allosteric inhibitor of AKT, abolished sorafenib-induced p-AKT upregulation, and resulted in a synergistic anti-proliferative effect in HCC cells. In conclusion, an unbiased and global phosphoproteomic approach by SILAC proves useful in identifying molecules altered by sorafenib in HCC cells; and by using this approach we showed that targeting AKT is a potentially useful strategy to improve the efficacy of sorafenib in advanced HCC. (The study was supported by the grant NSC 97-2628-B-002-004-MY3 from National Science Council of Taiwan and a research grant from Merck & Co., Inc.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1652.