Radoslav Savic

Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo ☆

BACKGROUND/AIMS The success of sorafenib in the treatment of advanced hepatocellular carcinoma (HCC) has focused interest on the role of Ras signaling in this malignancy. We investigated the molecular alterations of the Ras pathway in HCC and the antineoplastic effects of sorafenib in combination with rapamycin, an inhibitor of mTOR pathway, in experimental models. METHODS Gene expression (qRT-PCR, oligonucleotide microarray), DNA copy number changes (SNP-array), methylation of tumor suppressor genes (methylation-specific PCR) and protein activation (immunohistochemistry) were analysed in 351 samples. Anti-tumoral effects of combined therapy targeting the Ras and mTOR pathways were evaluated in cell lines and HCC xenografts. RESULTS Different mechanisms accounted for Ras pathway activation in HCC. H-ras was up-regulated during different steps of hepatocarcinogenesis. B-raf was overexpressed in advanced tumors and its expression was associated with genomic amplification. Partial methylation of RASSF1A and NORE1A was detected in 89% and 44% of tumors respectively, and complete methylation was found in 11 and 4% of HCCs. Activation of the pathway (pERK immunostaining) was identified in 10.3% of HCC. Blockade of Ras and mTOR pathways with sorafenib and rapamycin reduced cell proliferation and induced apoptosis in cell lines. In vivo, the combination of both compounds enhanced tumor necrosis and ulceration when compared with sorafenib alone. CONCLUSIONS Ras activation results from several molecular alterations, such as methylation of tumor suppressors and amplification of oncogenes (B-raf). Sorafenib blocks signaling and synergizes with rapamycin in vivo, preventing tumor progression. These data provide the rationale for testing this combination in clinical studies.

MicroRNA-Based Classification of Hepatocellular Carcinoma and Oncogenic Role of miR-517a

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) is a heterogeneous tumor that develops via activation of multiple pathways and molecular alterations. It has been a challenge to identify molecular classes of HCC and design treatment strategies for each specific subtype. MicroRNAs (miRNAs) are involved in HCC pathogenesis, and their expression profiles have been used to classify cancers. We analyzed miRNA expression in human HCC samples to identify molecular subclasses and oncogenic miRNAs. METHODS We performed miRNA profiling of 89 HCC samples using a ligation-mediated amplification method. Subclasses were identified by unsupervised clustering analysis. We identified molecular features specific for each subclass using expression pattern (Affymetrix U133 2.0; Affymetrix, Santa Clara, CA), DNA change (Affymetrix STY Mapping Array), mutation (CTNNB1), and immunohistochemical (phosphor[p]-protein kinase B, p-insulin growth factor-IR, p-S6, p-epidermal growth factor receptor, β-catenin) analyses. The roles of selected miRNAs were investigated in cell lines and in an orthotopic model of HCC. RESULTS We identified 3 main clusters of HCCs: the wingless-type MMTV integration site (32 of 89; 36%), interferon-related (29 of 89; 33%), and proliferation (28 of 89; 31%) subclasses. A subset of patients with tumors in the proliferation subclass (8 of 89; 9%) overexpressed a family of poorly characterized miRNAs from chr19q13.42. Expression of miR-517a and miR-520c (from ch19q13.42) increased proliferation, migration, and invasion of HCC cells in vitro. MiR-517a promoted tumorigenesis and metastatic dissemination in vivo. CONCLUSIONS We propose miRNA-based classification of 3 subclasses of HCC. Among the proliferation class, miR-517a is an oncogenic miRNA that promotes tumor progression. There is rationale for developing therapies that target miR-517a for patients with HCC.

Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib.

Purpose: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with active Wnt signaling. Underlying biologic mechanisms remain unclear and no drug targeting this pathway has been approved to date. We aimed to characterize Wnt-pathway aberrations in HCC patients, and to investigate sorafenib as a potential Wnt modulator in experimental models of liver cancer. Experimental Design: The Wnt-pathway was assessed using mRNA (642 HCCs and 21 liver cancer cell lines) and miRNA expression data (89 HCCs), immunohistochemistry (108 HCCs), and CTNNB1-mutation data (91 HCCs). Effects of sorafenib on Wnt signaling were evaluated in four liver cancer cell lines with active Wnt signaling and a tumor xenograft model. Results: Evidence for Wnt activation was observed for 315 (49.1%) cases, and was further classified as CTNNB1 class (138 cases [21.5%]) or Wnt-TGFβ class (177 cases [27.6%]). CTNNB1 class was characterized by upregulation of liver-specific Wnt-targets, nuclear β-catenin and glutamine-synthetase immunostaining, and enrichment of CTNNB1-mutation-signature, whereas Wnt-TGFβ class was characterized by dysregulation of classical Wnt-targets and the absence of nuclear β-catenin. Sorafenib decreased Wnt signaling and β-catenin protein in HepG2 (CTNNB1 class), SNU387 (Wnt-TGFβ class), SNU398 (CTNNB1-mutation), and Huh7 (lithium-chloride-pathway activation) cell lines. In addition, sorafenib attenuated expression of liver-related Wnt-targets GLUL, LGR5, and TBX3. The suppressive effect on CTNNB1 class–specific Wnt-pathway activation was validated in vivo using HepG2 xenografts in nude mice, accompanied by decreased tumor volume and increased survival of treated animals. Conclusions: Distinct dysregulation of Wnt-pathway constituents characterize two different Wnt-related molecular classes (CTNNB1 and Wnt-TGFβ), accounting for half of all HCC patients. Sorafenib modulates β-catenin/Wnt signaling in experimental models that harbor the CTNNB1 class signature. Clin Cancer Res; 18(18); 4997–5007. ©2012 AACR.

New strategies in hepatocellular carcinoma: genomic prognostic markers.

Accurate prognosis prediction in oncology is critical. In patients with hepatocellular carcinoma (HCC), unlike most solid tumors, the coexistence of two life-threatening conditions, cancer and cirrhosis, makes prognostic assessments difficult. Despite the usefulness of clinical staging systems for HCC in routine clinical decision making (e.g., Barcelona-Clinic Liver Cancer algorithm), there is still a need to refine and complement outcome predictions. Recent data suggest the ability of gene signatures from the tumor (e.g., EpCAM signature) and adjacent tissue (e.g., poor-survival signature) to predict outcome in HCC (either recurrence or overall survival), although independent external validation is still required. In addition, novel information is being produced by alternative genomic sources such as microRNA (miRNA; e.g., miR-26a) or epigenomics, areas in which promising preliminary data are thoroughly explored. Prognostic models need to contemplate the impact of liver dysfunction and risk of subsequent de novo tumors in a patient’s life expectancy. The challenge for the future is to precisely depict genomic predictors (e.g., gene signatures, miRNA, or epigenetic biomarkers) at each stage of the disease and their specific influence to determine patient prognosis. Clin Cancer Res; 16(19); 4688–94. ©2010 AACR.

Gene-expression signature of vascular invasion in hepatocellular carcinoma

BACKGROUND & AIMS Vascular invasion is a major predictor of tumor recurrence after surgical treatments for hepatocellular carcinoma (HCC). While macroscopic vascular invasion can be detected by radiological techniques, pre-operative detection of microscopic vascular invasion, which complicates 30-40% of patients with early tumors, remains elusive. METHODS A total of 214 patients with hepatocellular carcinoma who underwent resection were included in the study. By using genome-wide gene-expression profiling of 79 hepatitis C-related hepatocellular carcinoma samples (training set), a gene-expression signature associated with vascular invasion was defined. The signature was validated in formalin-fixed paraffin-embedded tissues obtained from an independent set of 135 patients with various etiologies. RESULTS A 35-gene signature of vascular invasion was defined in the training set, predicting vascular invasion with an accuracy of 69%. The signature was independently associated with the presence of vascular invasion (OR 3.38, 95% CI 1.48-7.71, p=0.003) along with tumor size (diameter greater than 3 cm, OR 2.66, 95% CI 1.17-6.05, p=0.02). In the validation set, the signature discarded the presence of vascular invasion with a negative predictive value of 0.77, and significantly improved the diagnostic power of tumor size alone (p=0.045). CONCLUSIONS The assessment of a gene-expression signature obtained from resected biopsied tumor specimens improved the diagnosis of vascular invasion beyond clinical variable-based prediction. The signature may aid in candidate selection for liver transplantation, and guide the design of clinical trials with experimental adjuvant therapies.

Recombinant Human Acid Sphingomyelinase as an Adjuvant to Sorafenib Treatment of Experimental Liver Cancer

Background Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third leading cause of cancer death worldwide. The only approved systemic treatment for unresectable HCC is the oral kinase inhibitor, sorafenib. Recombinant human acid sphingomyelinase (rhASM), which hydrolyzes sphingomyelin to ceramide, is an orphan drug under development for the treatment of Type B Niemann-Pick disease (NPD). Due to the hepatotropic nature of rhASM and its ability to generate pro-apoptotic ceramide, this study evaluated the use of rhASM as an adjuvant treatment with sorafenib in experimental models of HCC. Methodology/Principal Findings In vitro, rhASM/sorafenib treatment reduced the viability of Huh7 liver cancer cells more than sorafenib. In vivo, using a subcutaneous Huh7 tumor model, mouse survival was increased and proliferation in the tumors decreased to a similar extent in both sorafenib and rhASM/sorafenib treatment groups. However, combined rhASM/sorafenib treatment significantly lowered tumor volume, increased tumor necrosis, and decreased tumor blood vessel density compared to sorafenib. These results were obtained despite poor delivery of rhASM to the tumors. A second (orthotopic) model of Huh7 tumors also was established, but modest ASM activity was similarly detected in these tumors compared to healthy mouse livers. Importantly, no chronic liver toxicity or weight loss was observed from rhASM therapy in either model. Conclusions/Significance The rhASM/sorafenib combination exhibited a synergistic effect on reducing the tumor volume and blood vessel density in Huh7 xenografts, despite modest activity of rhASM in these tumors. No significant increases in survival were observed from the rhASM/sorafenib treatment. The poor delivery of rhASM to Huh7 tumors may be due, at least in part, to low expression of mannose receptors. The safety and efficacy of this approach, together with the novel findings regarding enzyme targeting, merits further investigation.

Lymphotoxins: new targets for hepatocellular carcinoma.

In this issue of Cancer Cell, Haybaeck et al. unravel the role of lymphotoxin pathway in the development of hepatocellular carcinoma (HCC). Aberrant activation of this cascade in mice livers recapitulates the stages of fibrosis and inflammation that precedes human liver cancer, providing a novel family of potential therapeutic targets.

Use of acid sphingomyelinase for cancer therapy.

Acid sphingomyelinase (ASM) is a lipid hydrolase that cleaves the sphingolipid, sphingomyelin, into ceramide. Mutations in the ASM gene (SMPD1) result in the rare lysosomal storage disorder, Niemann-Pick disease (NPD). In addition to its role in NPD, over the past two decades, the importance of sphingolipids, and ASM in particular, in normal physiology and the pathophysiology of numerous common diseases also has become known. For example, altered sphingolipid metabolism occurs in many cancers, generally reducing the levels of the pro-apoptotic lipid, ceramide, and/or elevating the levels of the proliferative lipid, sphingosine-1-phosphate (S1P). These changes likely contribute to the tumorigenicity and/or metastatic capacity of the cancer. In addition, many cancer therapies induce ceramide-mediated death, and cancer cells have evolved novel mechanisms to overcome this effect. In the present review, we discuss sphingolipid metabolism in cancer, and specifically the potential for pharmacological modulation using ASM. Of note, recombinant human ASM (rhASM) has been produced for human use and is being evaluated as a treatment for NPD. Thus, its use for cancer therapy could be rapidly evaluated in the clinic after appropriate animal model studies have been completed. As this enzyme was initially studied in the context of NPD, we start with a brief overview of the history of ASM and NPD, followed by a discussion of the role of ASM in cancer biology, and then summarize emerging preclinical efficacy studies using rhASM as an adjunct in the treatment of solid tumors.

Abstract 2996: Overexpression of potential oncogenic microRNAs defines a new molecular subclass of hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third cause of cancer-related death worldwide. Its incidence is increasing mainly due to hepatitis C virus (HCV) infection. A molecular classification of HCC is still lacking. microRNAs (miRNAs) are small non-coding RNA involved in HCC pathogenesis. Their expression profiling represents a powerful tool to classify cancers. Objectives: (1) To provide a miRNA-based molecular classification of HCC and, (2) To investigate the function of potential oncogenic miRNAs in HCC models. Methods: Expression of 358 miRNAs was analyzed in 89 HCV-related HCCs using a bead-based miRNA expression profiling method. Integrative analysis including miRNA profiling, gene expression (Affymetrix U133 2.0 ® ), DNA changes (Affymetrix STY Mapping Array ® ), IHC (p-Akt, p-IGF-IR, p-S6, p-EGFR, β-catenin) and mutation analysis (β-catenin) was performed. Expression of selected miRNA was validated in a validation set (n=167) by qRT-PCR. Methylation-specific PCR, FISH and SNP-array analyses were performed to identify mechanisms of miRNA deregulation. The function of miRNAs of interest was investigated in vitro by analyzing cell proliferation (thymidine incorporation), migration and invasion (trans-well migration and invasion assays, wound healing assay). Tumor development and growth following direct injection of luciferase-expressing cells stably transfected with specific miRNAs into the liver of nude mice were monitored to investigate their function in vivo. The bioluminescent signal emitted by luciferase-expressing cells was used as indicator of tumor growth. Results: Three classes of HCC patients were identified and defined by activation of different pathways: Wnt signalling (32/89, 36%), IFN-related genes (29/89, 33%) and proliferative cascades (IGF, Akt/mTOR) (28/89, 31%). A subgroup within the proliferative class (8/89, 9% overexpressed a cluster of miRNAs on 19q13.42 (median fold change: 8.8). Hypomethylation of CpG island upstream the miRNA cluster (2/8, 25%) and copy number gains (1/8, 12.5%) were detected. Their overexpression was confirmed in a validation set (17/167, 10.2%). Members of the cluster family significantly increased proliferation (p Conclusions: Overexpression of 19q miRNA family and activation of proliferative pathways defined a subclass of HCC. Members of 19q miRNA family increased cell proliferation, migration and invasion in vitro and promoted tumor development in vivo suggesting their role as novel potential oncogenic drivers in HCC. 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 2996.

Expression-based drug screening of neural progenitor cells from individuals with schizophrenia

A lack of biologically relevant screening models hinders the discovery of better treatments for schizophrenia (SZ) and other neuropsychiatric disorders. Here we compare the transcriptional responses of 8 commonly used cancer cell lines (CCLs) directly with that of human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs) from 12 individuals with SZ and 12 controls across 135 drugs, generating 4320 unique drug-response transcriptional signatures. We identify those drugs that reverse post-mortem SZ-associated transcriptomic signatures, several of which also differentially regulate neuropsychiatric disease-associated genes in a cell type (hiPSC NPC vs. CCL) and/or a diagnosis (SZ vs. control)-dependent manner. Overall, we describe a proof-of-concept application of transcriptomic drug screening to hiPSC-based models, demonstrating that the drug-induced gene expression differences observed with patient-derived hiPSC NPCs are enriched for SZ biology, thereby revealing a major advantage of incorporating cell type and patient-specific platforms in drug discovery.Unbiased large scale screening of small molecules for drug discovery in psychiatric disease is technically challenging and financially costly. Here, Readhead and colleagues integrate in silico and in vitro approaches to design and conduct transcriptomic drug screening in schizophrenia patient-derived neural cells, in order to survey novel pathologies and points of intervention.