Christophe Louandre

EGFR activation is a potential determinant of primary resistance of hepatocellular carcinoma cells to sorafenib

Sorafenib is currently the medical treatment of reference for hepatocellular carcinoma (HCC), but it is not known whether sorafenib is equally active in all HCC. Here, our aim was to explore intrinsic differences in the response of HCC cells to sorafenib, to identify potential mechanisms leading to primary resistance to this treatment. We analyzed a panel of six human HCC cell lines and compared the activity of the main oncogenic kinase cascades, their clonogenic potential, proliferation and apoptosis upon exposure to sorafenib. We report that HCC cells present important differences in their response to sorafenib, and that some cell lines are more resistant to the actions of sorafenib than others. We identify the activated epidermal growth factor receptor (EGFR) as a parameter that promotes the resistance of HCC cells to sorafenib. In resistant cells, the efficacy of sorafenib was increased when EGFR was inhibited, as was demonstrated using two chemical inhibitors (erlotinib or gefitinib), a monoclonal antibody directed against EGFR (cetuximab), and RNA interference directed against EGFR. A combination of EGFR inhibitors and sorafenib affords a better control over HCC proliferation, most likely through an improved blockade of the RAF kinases. Our findings therefore confirm the importance of RAF kinases as therapeutic targets in HCC, and identify EGFR as a determinant of the sensitivity of HCC cells to sorafenib. Our findings bear possible implications for the improvement of the efficacy of sorafenib in HCC, and might be useful for the identification of predictive biomarkers in this context.

Iron-dependent cell death of hepatocellular carcinoma cells exposed to sorafenib

The multikinase inhibitor sorafenib is currently the treatment of reference for advanced hepatocellular carcinoma (HCC). In our report, we examined the cytotoxic effects of sorafenib on HCC cells. We report that the depletion of the intracellular iron stores achieved by using the iron chelator deferoxamine (DFX) strikingly protects HCC cells from the cytotoxic effects of sorafenib. The protective effect of the depletion of intracellular iron stores could not be explained by an interference with conventional forms of programmed cell death, such as apoptosis or autophagic cell death. We also found that DFX did not prevent sorafenib from reaching its intracellular target kinases. Instead, the depletion of intracellular iron stores prevented sorafenib from inducing oxidative stress in HCC cells. We examined the possibility that sorafenib might exert a cytotoxic effect that resembles ferroptosis, a form of cell death in which iron‐dependent oxidative mechanisms play a pivotal role. In agreement with this possibility, we found that pharmacological inhibitors (ferrostatin‐1) and genetic procedures (RNA interference against IREB‐2) previously reported to modulate ferroptosis, readily block the cytotoxic effects of sorafenib in HCC cells. Collectively, our findings identify ferroptosis as an effective mechanism for the induction of cell death in HCC. Ferroptosis could potentially become a goal for the medical treatment of HCC, thus opening new avenues for the optimization of the use of sorafenib in these tumors.

The retinoblastoma (Rb) protein regulates ferroptosis induced by sorafenib in human hepatocellular carcinoma cells

Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC), the most frequent form of primary liver tumour. The loss of function of the retinoblastoma (Rb) protein is an important event during liver carcinogenesis, but it is unclear whether the Rb status modulates the response of HCC cells to sorafenib. Here, we examined this question in HCC cells with reduced levels of Rb achieved through stable RNA interference. We show that HCC cells with reduced levels of Rb exhibit a two- to threefold increase in cell death induction upon exposure to sorafenib compared with controls. Sorafenib treatment of Balb/c nude mice that received tumour xenografts derived from HCC cells with reduced Rb levels resulted in complete tumour regression in 50% of the animals treated, compared with tumour stabilization in mice that received control cells. We show that, upon exposure to sorafenib, the Rb-negative status of HCC cells promotes the occurrence of ferroptosis, a form of oxidative necrosis. The findings highlight the role of Rb in the response of HCC cells to sorafenib and the regulation of ferroptosis.

BAD, a proapoptotic member of the BCL2 family, is a potential therapeutic target in hepatocellular carcinoma.

Proteins of the BCL2 family are key regulators of apoptosis. Their expression levels are frequently altered in cancers, enabling tumor cells to survive. To gain insight into the pathogenesis of hepatocellular carcinoma (HCC), we performed a comprehensive survey of the expression of the members of the BCL2 family in samples obtained from surgically resected HCCs. Here, we report the occurrence of a new molecular anomaly, consisting of a strong reduction in the expression of the proapoptotic protein BAD in HCC compared with surrounding nontumoral tissue. We investigate the function of BAD in a panel of HCC cell lines. Using gene overexpression and RNA interference, we show that BAD is involved in the cytotoxic effects of sorafenib, a multikinase blocker, which is currently the sole therapeutic drug effective for the treatment of HCC. Finally, we report that ABT-737, a compound that interacts with proteins of the BCL2 family and exhibits a BAD-like reactivity, sensitizes HCC cells toward sorafenib-induced apoptosis. Collectively, our findings indicate that BAD is a key regulator of apoptosis in HCC and an important determinant of HCC cell response to sorafenib. Mol Cancer Res; 8(8); 1116–25. ©2010 AACR.

Upregulation of BAD, a pro-apoptotic protein of the BCL2 family, in vascular smooth muscle cells exposed to uremic conditions.

Chronic kidney disease (CKD) has recently emerged as a major risk factor for cardiovascular pathology. CKD patients display accelerated atherosclerotic process, leading to circulatory complications. However, it is currently not clear how uremic conditions accelerate atherosclerosis. Apoptosis is an important homeostatic regulator of vascular smooth cells under pathological conditions. In the present study, we explored the regulation of apoptosis in cells of the vascular wall in the uremic context. We analysed the expression and regulation of the proteins of the BCL2 family that play an essential role in apoptosis. Our results, obtained in mice and primary human smooth muscle cells exposed to two uremic toxins, point to the existence of an alteration in expression and function of one pro-apoptotic member of this family, the protein BAD. We explore the regulation of BAD by uremic toxins and report the sensitization of vascular smooth muscle cells to apoptosis upon BAD induction.

The kinase-inhibitor sorafenib inhibits multiple steps of the Hepatitis C Virus infectious cycle in vitro.

Hepatitis C Virus (HCV) chronic infection is a major cause of hepatocellular carcinoma. Sorafenib is the only medical treatment that has been approved for the treatment of this cancer. It is a multikinase inhibitor with anti-tumor activity against a wide variety of cancers. Sorafenib blocks angiogenesis and tumor cell proliferation through inhibition of kinases, such as VEGFR2, PDGFR, or the serine/threonine kinases RAF. Previous studies have reported an anti-HCV effect of sorafenib in vitro, but various mechanisms of action have been described. The aim of this study was to clarify the action of sorafenib on the complete HCV infectious cycle. In order to examine the action of sorafenib on all steps of the HCV infectious cycle, we used a combination of validated cell culture models, based on the HuH-7 reference cell line and primary human hepatocytes. We found that sorafenib blocks HCV infection by altering the viral entry step and the production of viral particles. Moreover, we observed that treatment with sorafenib lead to a modification of Claudin-1 expression and localization, which could partly be responsible for the anti-HCV effect. Collectively, our findings confirm the anti-HCV effect of sorafenib in vitro, while highlighting the complexity of the action of sorafenib on the HCV infectious cycle.

Metallothionein-1 as a biomarker of altered redox metabolism in hepatocellular carcinoma cells exposed to sorafenib

BackgroundSorafenib, a kinase inhibitor active against various solid tumours, induces oxidative stress and ferroptosis, a new form of oxidative necrosis, in some cancer cells. Clinically-applicable biomarkers that reflect the impact of sorafenib on the redox metabolism of cancer cells are lacking.MethodsWe used gene expression microarrays, real-time PCR, immunoblot, protein-specific ELISA, and gene reporter constructs encoding the enzyme luciferase to study the response of a panel of cancer cells to sorafenib. Tumour explants prepared from surgical hepatocellular carcinoma (HCC) samples and serum samples obtained from HCC patients receiving sorafenib were also used.ResultsWe observed that genes of the metallothionein-1 (MT1) family are induced in the HCC cell line Huh7 exposed to sorafenib. Sorafenib increased the expression of MT1G mRNA in a panel of human cancer cells, an effect that was not observed with eight other clinically-approved kinase inhibitors. We identified the minimal region of the MT1G promoter that confers inducibility by sorafenib to a 133 base pair region containing an Anti-oxidant Response Element (ARE) and showed the essential role of the transcription factor NRF2 (Nuclear factor erythroid 2-Related Factor 2). We examined the clinical relevance of our findings by analysing the regulation of MT1G in five tumour explants prepared from surgical HCC samples. Finally, we showed that the protein levels of MT1 increase in the serum of some HCC patients receiving sorafenib, and found an association with reduced overall survival.ConclusionThese findings indicate that MT1 constitute a biomarker adapted for exploring the impact of sorafenib on the redox metabolism of cancer cells.

The Bcl-2 Homology Domain 3 (BH3) Mimetic ABT-737 Reveals the Dynamic Regulation of Bad, a Proapoptotic Protein of the Bcl-2 Family, by Bcl-xL

The proteins of the B-cell lymphoma 2 (Bcl-2) family are important regulators of apoptosis under normal and pathological conditions. Chemical compounds that block the antiapoptotic proteins of this family have been introduced, such as 4-[4-[(4′-Chloro[1,1′-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide (ABT-737), a BH3-mimetic that neutralizes Bcl-2 and Bcl-xL. In this study, we used ABT-737 to explore the dynamic regulation of Bcl-2 proteins in living cells of different origins. Using ABT-737 as well as RNA interference or the application of growth factors, we examined the impact of the functional availability of the antiapoptotic proteins Bcl-2 and Bcl-2-extra large (Bcl-xL) on the Bcl-2 network. We report that ABT-737 increases the expression of Bcl-2-associated death promoter (Bad), a proapoptotic partner of the proteins Bcl-2 and Bcl-xL. Our observations indicate that Bad overexpression induced by ABT-737 results from the control of its normally rapid protein turnover, leading to the stabilization of this protein. We demonstrate the relevance of Bad post-translational regulation by Bcl-xL to the physiological setting using RNA interference against Bcl-xL as well as the application of epidermal growth factor, a growth factor that promotes the dissociation of Bad from Bcl-xL. Our results highlight a new facet of the mode of action of the antiapoptotic proteins Bcl-2 and Bcl-xL consisting of the regulation of the stability of the protein Bad. Finally, our results shed light on the mode of action of ABT-737, currently the best characterized inhibitor of the antiapoptotic proteins of the Bcl-2 family, and bear important implications regarding its use as an anticancer drug.

Alpha-fetoprotein is a biomarker of unfolded protein response and altered proteostasis in hepatocellular carcinoma cells exposed to sorafenib

Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC). A decrease in the serum levels of Alpha-fetoprotein (AFP) is reported to be the biological parameter that is best associated with disease control by sorafenib. In order to provide a biological rationale for the variations of AFP, we analyzed the various steps of AFP production in human HCC cell lines exposed to sorafenib. Sorafenib dramatically reduced the levels of AFP produced by HCC cells independently of its effect on cell viability. The mRNA levels of AFP decreased upon sorafenib treatment, while the AFP protein remained localized in the Golgi apparatus. Sorafenib activated the Regulated Inositol-Requiring Enzyme-1α (IRE-1α) and the PKR-like ER Kinase (PERK)-dependent arms of the Unfolded Protein Response (UPR). The inhibition of IRE-1α partially restored the mRNA levels of AFP upon treatment with sorafenib. The inhibition of both pathways partially prevented the drop in the production of AFP induced by sorafenib. The findings provide new insights on the regulation of AFP, and identify it as a biomarker suitable for the exploration of HCC cell proteostasis in the context of therapeutic targeting.

Oxidized low density lipoprotein induces the cyclin-dependent kinase inhibitor p21waf1 and the tumor suppressor Rb

Oxidized low density lipoprotein (OxLDL) is known to be cytotoxic towards different cell types of the arterial wall, leading to progression of an atherosclerotic plaque. We previously reported that OxLDL activates the tumor suppressor p53 in human fibroblasts [Biochem. Biophys. Res. Commun. 276 (2000) 718]. In the present work, we demonstrate that OxLDL increased intracellular levels of the kinase inhibitor p21(waf1) (p21) and of the tumor suppressor Rb. Concomitantly, level of the hypophosphorylated active form of Rb (HypoP-Rb) was also enhanced. Cycloheximide prevented the OxLDL-induced increase in p21, Rb, and HypoP-Rb, whereas okadaic acid had no effect. This increase was also prevented by the antioxidant vitamin E. In addition, the lipid extract of OxLDL, which includes the lipid peroxidation products, reproduced the action of the OxLDL particle itself. OxLDL and its lipid extract induced an oxidative stress, as assessed by the intracellular levels of reactive oxygen species and lipid peroxidation products. Finally, OxLDL induced a dose-dependent inhibition of DNA synthesis as assessed by thymidine incorporation. These results demonstrate that OxLDL or its lipid peroxidation products, by generation of an oxidative stress, enhances the expression of p21 and Rb genes, leading to an accumulation of the Hypo-P active form of the tumor suppressor Rb. This phenomenon is in accordance with the fact that p21 is a mediator of p53-dependent cell-cycle arrest in G1 and is most probably involved in the cytotoxicity of OxLDL.