Nicolas Charette

Salirasib inhibits the growth of hepatocarcinoma cell lines in vitro and tumor growth in vivo through ras and mTOR inhibition

BackgroundDysregulation of epidermal growth factor and insulin-like growth factor signaling play important roles in human hepatocellular carcinoma (HCC), leading to frequent activation of their downstream targets, the ras/raf/extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) pathways. Salirasib is an S-prenyl-cysteine analog that has been shown to block ras and/or mTOR activation in several non hepatic tumor cell lines. We investigated in vitro the effect of salirasib on cell growth as well as its mechanism of action in human hepatoma cell lines (HepG2, Huh7, and Hep3B) and its in vivo effect in a subcutaneous xenograft model with HepG2 cells.ResultsSalirasib induced a time and dose dependent growth inhibition in hepatocarcinoma cells through inhibition of proliferation and partially through induction of apoptosis. A 50 percent reduction in cell growth was obtained in all three cell lines at a dose of 150 μM when they were cultured with serum. By contrast, salirasib was more potent at reducing cell growth after stimulation with EGF or IGF2 under serum-free conditions, with an IC50 ranging from 60 μM to 85 μM. The drug-induced anti-proliferative effect was associated with downregulation of cyclin A and to a lesser extent of cyclin D1, and upregulation of p21 and p27. Apoptosis induction was related to a global pro-apoptotic balance with caspase 3 activation, cytochrome c release, death receptor upregulation, and a reduced mRNA expression of the apoptosis inhibitors cFLIP and survivin. These effects were associated with ras downregulation and mTOR inhibition, without reduction of ERK and Akt activation. In vivo, salirasib reduced tumour growth from day 5 onwards. After 12 days of treatment, mean tumor weight was diminished by 56 percent in the treated animals.ConclusionsOur results show for the first time that salirasib inhibits the growth of human hepatoma cell lines through inhibition of proliferation and induction of apoptosis, which is associated with ras and mTOR inhibition. The therapeutic potential of salirasib in human HCC was further confirmed in a subcutaneous xenograft model.

Salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis through DR5 and survivin-dependent mechanisms.

Ras activation is a frequent event in human hepatocarcinoma that may contribute to resistance towards apoptosis. Salirasib is a ras and mTOR inhibitor that induces a pro-apoptotic phenotype in human hepatocarcinoma cell lines. In this work, we evaluate whether salirasib sensitizes those cells to TRAIL-induced apoptosis. Cell viability, cell death and apoptosis were evaluated in vitro in HepG2, Hep3B and Huh7 cells treated with DMSO, salirasib and YM155 (a survivin inhibitor), alone or in combination with recombinant TRAIL. Our results show that pretreatment with salirasib sensitized human hepatocarcinoma cell lines, but not normal human hepatocytes, to TRAIL-induced apoptosis. Indeed, FACS analysis showed that 25 (Huh7) to 50 (HepG2 and Hep3B) percent of the cells treated with both drugs were apoptotic. This occurred through activation of the extrinsic and the intrinsic pathways, as evidenced by a marked increase in caspase 3/7 (five to ninefold), caspase 8 (four to sevenfold) and caspase 9 (eight to 12-fold) activities in cells treated with salirasib and TRAIL compared with control. Survivin inhibition had an important role in this process and was sufficient to sensitize hepatocarcinoma cells to apoptosis. Furthermore, TRAIL-induced apoptosis in HCC cells pretreated with salirasib was dependent on activation of death receptor (DR) 5. In conclusion, salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis by a mechanism involving the DR5 receptor and survivin inhibition. These results in human hepatocarcinoma cell lines and primary hepatocytes provide a rationale for testing the combination of salirasib and TRAIL agonists in human hepatocarcinoma.

Kinetics of angiogenic changes in a new mouse model for hepatocellular carcinoma

BackgroundThe increasing incidence of hepatocellular carcinoma in Western countries has led to an expanding interest of scientific research in this field. Therefore, a vast need of experimental models that mimic the natural pathogenesis of hepatocellular carcinoma (HCC) in a short time period is present. The goal of our study was (1) to develop an efficient mouse model for HCC research, in which tumours develop in a natural background of fibrosis and (2) to assess the time-dependent angiogenic changes in the pathogenesis of HCC.MethodsWeekly intraperitoneal injections with the hepatocarcinogenic compound N-nitrosodiethylamine was applied as induction method and samples were taken at several time points to assess the angiogenic changes during the progression of HCC.ResultsThe N-nitrosodiethylamine-induced mouse model provides well vascularised orthotopic tumours after 25 weeks. It is a representative model for human HCC and can serve as an excellent platform for the development of new therapeutic targets.

The Ras inhibitor farnesylthiosalicyclic acid (FTS) prevents nodule formation and development of preneoplastic foci of altered hepatocytes in rats.

BACKGROUND Aberrant activation of oncogenes, such as Ras, likely contributes to the development of hepatocarcinoma (HCC). AIMS/METHODS We evaluated in vivo the effect of intraperitoneal injections of the Ras inhibitor S-trans, trans-farnesylthiosalicyclic acid (FTS) on Ras activation and the development of preneoplastic liver lesions in rats receiving weekly diethylnitrosamine (DEN) injections for 16weeks. Western blotting, quantitative PCR, immunohistochemistry, Tunel and caspase activity assays were used. RESULTS FTS prevents liver nodule formation and reduces foci expressing the tumour marker GSTp. FTS abrogates DEN-induced Ras membrane activity, increases Tunel positive cells in transformed, GSTp-expressing hepatocytes, up-regulates caspase 3 and 8 activity, induces Fas, Fas ligand and JNK phosphorylation that occurs independently of TNFalpha and Trail. Cytochrome C release, Bax, Bcl2, Bcl-xl, Ki67 and nuclear cyclin D expression is not affected by FTS. CONCLUSIONS FTS inhibits Ras activation and prevents preneoplastic liver nodule development by inducing apoptosis in transformed hepatocytes through activation of the Fas/Fas ligand system. FTS might be new molecule for HCC treatment.

Tumor reoxygenation following administration of Mitogen-Activated Protein Kinase inhibitors: A rationale for combination with radiation therapy

BACKGROUND AND PURPOSE The relevance of Mitogen Activated Protein Kinase (MAPK) inhibitors as co-treatments for radiation therapy is investigated, with special focus on a potential link between the MAPK pathway and tumor hypoxia, which is a critical determinant for response to therapy. MATERIALS AND METHODS The effects of two MAPK inhibitors, Sorafenib and PD0325901, were monitored daily using in vivo EPR (Electron Paramagnetic Resonance) oximetry in FSaII and TLT tumor models in order to identify a window of reoxygenation, during which tumor blood flow, oxygen consumption and radiation sensitivity were assessed. RESULTS Reoxygenation was shown after two days of treatments with Sorafenib or PD0325901 in two tumor models, which was further successfully exploited with Sorafenib for improving the radiation response of FSaII tumors by a factor of 1.5. The increase in tumor oxygenation was shown to be the result of two major factors: (i) an increase in blood flow for Sorafenib, that might be linked to its anti-angiogenic effect (vascular normalization), and (ii) a decrease in oxygen consumption for Sorafenib and PD0325901, due to an alteration of the mitochondrial activity. CONCLUSION We evidenced tumor reoxygenation in vivo following MAPK inhibition and suggest a rationale for the combination of radiation therapy with Sorafenib.

Ras inhibition in hepatocarcinoma by S-trans-trans-farnesylthiosalicyclic acid: Association of its tumor preventive effect with cell proliferation, cell cycle events, and angiogenesis

Activation of Ras and its downstream signaling pathways, likely contribute to the development of hepatocarcinoma. We have previously shown that intraperitoneal injections of the Ras inhibitor S‐trans, trans‐farnesylthiosalicyclic acid (FTS) blocks Ras activation and prevents heptocarcinoma development in rats receiving weekly injections of the carcinogene diethylnitrosamine (DEN) for 16 wk. Using this in vivo model, we evaluated the relationship between the tumor preventive effect of Ras inhibition and activation of downstream signaling pathways, cell proliferation, cell cycle events, and angiogenesis. Western blotting, quantitative PCR, immunohistochemistry, and transcription factor activity assays were used. DEN‐induced activation of NFkB and Stat3 was abrogated by FTS treatment. FTS treatment showed no effect on DEN‐induced elevation of TNFα, interleukin 6 and TLR4, known activators of these transcription factors. FTS significantly reduced phosphorylation of the MAPkinase p38 and of the p70S6 kinase, a surrogate marker for mTor activation, without affecting ERK and AKT phosphorylation. These events were associated with reduced c‐myc and cyclin D expression as well as reduced cell proliferation in transformed, GSTp‐positive hepatocytes. Moreover, FTS treatment shifted cell proliferation from transformed hepatocytes to apparently normal, GSTp negative hepatocytes. FTS treatment did not down‐regulate expression of angiogenesis markers HIFα, VEGF, VEGF receptor1, and placenta growth factor. FTS treatment inhibits important signaling pathways involved in cellular proliferation leading to strongly reduced proliferation of transformed hepatocytes without affecting normal hepatocytes. This re‐adjustment of the proliferation balance likely contributes to the tumor preventive of FTS in the context of Ras inhibition in hepatocarcinogenesis.

Ras in digestive oncology: from molecular biology to clinical implications.

Purpose of review The modalities of Ras mutation detection, its role as a predictive biomarker, mechanisms of wild-type Ras activation, and the role of Ras-directed targeted therapies will be discussed mainly in colorectal cancer. Recent findings RAS genotype is generally considered to be highly concordant between primary colorectal tumours and metastases. However, recent data show significant discordance between primary tumours and specific metastatic sites, but also heterogeneity within primary tumours. Moreover, the mechanisms of Ras activation expand far beyond mutations through altered expression or function of physiological Ras activators and inhibitors. Accordingly, genomic signatures of Ras or epidermal growth factor receptor (EGFR) activation are being developed and are potential predictive biomarkers of response to anti-EGFR antibodies. Finally, several recent clinical trials targeting Ras or its downstream signalling with statins or Raf inhibitors have shown promising activity in chemorefractory metastatic colorectal cancer. Summary RAS mutation remains an important biomarker predicting response to anti-EGFR therapies and perhaps clinical outcomes after surgery for metastatic colorectal cancer, but new techniques including genomic signatures need to be validated to take into account the complexity of Ras activation. The importance of Ras signalling as a therapeutic target has recently been outlined by successful clinical trials with Raf inhibitors.

873 ANGIOGENIC CHANGES IN A NEW MOUSE MODEL FOR HEPATOCELLULAR CARCINOMA ASSESSED WITH STATE-OF-THE-ART IMAGING TECHNOLOGY

Background: The increasing incidence of hepatocellular carcinoma in Western countries has led to an expanding interest in this field. A vast need of experimental models that mimic the natural pathogenesis of hepatocellular carcinoma in a short time period is present. The goal of our study was (1) to develop an efficient mouse model for hepatocellular carcinoma research, (2) to assess time-dependent angiogenic changes and (3) to investigate tumour growth and neo-vascularisation using state-of-the-art imaging techniques. Methods: 5-week-old male mice received weekly intraperitoneal injections with N-nitrosodiethylamine (DEN) (35 mg/kg bodyweight) and samples were taken at several time points. Histology, ELISA and immunohistochemical stainings were used to identify the HCC-lesions and to quantify angiogenic factors VEGF and PlGF. HCC livers (25W) were perfused with Batson’s n°17 solution to produce vascular casts (arterial and venous). A state-of-the-art multimodal microPET/CT was used for in vivo detection of HCC-lesions with [18F]-fluoromethylcholine ([18F]FMCH) and for 3D-reconstruction of the vascular casts. Results: After 16W of DEN-injections a mild fibrosis (F1-F2) and dysplastic lesions appear, resulting in a pre-malignant environment. An increase of angiogenic factors VEGF and PlGF takes place, but not explicit enough to induce an increase in endothelial cells, which were upregulated after 20W. After 25W of DEN-injections, the dysplastic lesions have progressed to vascularised exophytic tumours which are macroscopically visible and give rise to a further increase in angiogenic factors, leading to the formation of new blood vessels. HCC-lesions were characterised by an increased uptake of [18F]FMCH, allowing visualisation of these hotspots (>3mm) with microPET/CT (figure 1). The vascular casts of HCC-livers clearly revealed the chaotic pattern and hierarchically disorganisation of tumour induced blood vessels (figure 2). Arteries formed a circumferential mantle around the hepatic tumours, while the central tumour regions showed a lower arterial density (figure 3). Electron microscopy revealed several angiogenic spots, with mostly sprouting angiogenesis, yet intussusceptive angiogenesis was also seen (figure 3). Conclusion: While most DEN-induced models take at least one year to develop tumours, weekly injections with DEN give rise to tumour occurrence after 25W. The well vascularised orthotopic tumours are a representative model for HCC and can serve as an excellent platform for the development of new therapeutic targets. The CT-reconstructions of vascular casts can be used as an interesting tool to study angiogenesis in animal models, especially for testing angiogenesis-inhibiting therapies. Furthermore, [18F]FMCH PET imaging may be clinically relevant for the visualization of HCC-lesions.