Search

In the last decade, relevant advances have occurred in the treatment of hepatocellular carcinoma (HCC), with novel drugs entering the clinical practice, among which tyrosine kinase inhibitors (TKIs) such as lenvatinib, cabozantinib and regorafenib, and immune checkpoint inhibitors (ICPIs) either alone or in combination with VEGF inhibitors. Clinical trials have driven the introduction of such novel molecules into the clinics but, at present, no biomarker drives the choice of first-line options, which relies only upon clinical and imaging assessment. Remarkably, clinical and imaging-based evaluations do not consider the huge heterogeneity of HCC and do not allow to realize the potential of personalized treatments. Preclinical research still does not inform the design of clinical trials, even though many animal models mimicking specific subgroups of HCC are available and might provide relevant information. Although animal models directly informing the clinical practice, such as patients-derived xenografts, are not used to help the choice of treatment in advanced HCC, however, the preclinical research can count on a wide range of valuable models. Here we will review some HCC models which might turn informative for specific questions in defined patient subgroups, and we will describe recent preclinical studies for the mechanistic evaluation of immunotherapy-based treatment approaches. To this aim, we will mainly focus on two issues: (i) HCC models informative on NAFLD-NASH HCC and (ii) HCC models helping to elucidate mechanisms underneath immunotherapy. We have chosen these two settings since they represent, respectively, the most rapidly arising cause of chronic liver disease (CLD) and HCC in western countries and the most promising therapeutic option for advanced HCC.

Dermatologic adverse events (DAEs) are associated with a better outcome in patients with hepatocellular carcinoma (HCC) irrespective of the therapeutic agent received. The exact mechanisms associated with the development of DAEs are unknown although several studies point to direct toxicity of tyrosine kinase inhibitors (TKIs) to the skin or an immune-mediated reaction triggered by the oncologic treatment. As is the case in other conditions, individual genetic variants may partially explain a higher risk of DAEs.To evaluate the contribution of several gene variants to the risk of developing DAEs in HCC patients treated with TKIs.We first analyzed 27 single-nucleotide polymorphisms (SNPs) from 12 genes selected as potential predictors of adverse event (AE) development in HCC patients treated with sorafenib [Barcelona Clinic Liver Cancer 1 (BCLC1) cohort]. Three additional cohorts were analyzed forThe BCLC1 cohort showed that patients with arterial hypertension (AHT) (HR = 1.61;DAE development in HCC patients receiving TKIs could be explained by the

ST1: Discovery and validation surgical patient cohorts. ST2: Advanced HCC patient cohort. ST3: Primer sequences for luciferase assay. ST4: Primer sequences for PCR and qPCR. ST5: Antibodies. ST6: Probe sequences for EMSA. ST7: Deregulated miR-30 family members in rat model.

Supplementary Table S1. Characteristics of surgical HCC patients analysed in this study. Supplementary Table S2. Patients assessed for circulating miR-221. Supplementary Table S3. Primer sequences and PCR conditions for the cloning experiment. Supplementary Table S4. Primer sequences and PCR conditions. Supplementary Table S5. Antibodies used in Western blot analysis.

Figure S2. Histopathological images of HCC nodules arisen in DEN-treated rats following Sorafenib administration. Nodules that responded to Sorafenib displayed large necrotic areas.

Microarray analysis, cell transfection and infection, cell proliferation assay, clonogenic and sphere formation assays, TP53 hypothetical binding sites.

Supplementary Data from MicroRNA-221 Targets Bmf in Hepatocellular Carcinoma and Correlates with Tumor Multifocality

This file contains Supplementary Figures. SF1: Correlations between miR-30 family member in human tissues. SF2: Microarray analysis of miRNAs in the rat model. SF3: miR-30e locus and p53 regulation. SF4: MDM2 regulation by miR-30e-3p in HCC cells. SF5: MiR-30e-3p regulates cell growth and cell invasion in HCC cells. SF6: MiR-30e-3p regulates stem cell phenotype in HCC cells. SF7: MiR-30e-3p regulates drug resistance in TP53 deleted HepG2 cells. SF8: MiR-30e-3p regulates drug resistance in TP53 mutated Huh-7 cells. SF9: Circulating miR-30e-3p levels predicts sorafenib response in HCC preclinical models.

All Supplementary Data

The molecular background of hepatocellular carcinoma (HCC) is highly heterogeneous, and biomarkers predicting response to treatments are an unmet clinical need. We investigated miR-30e-3p contribution to HCC phenotype and response to sorafenib, as well as the mutual modulation of TP53/MDM2 pathway, in HCC tissues and preclinical models. MiR-30e-3p was downregulated in human and rat HCCs, and its downregulation associated with TP53 mutations. TP53 contributed to miR-30e-3p biogenesis, and MDM2 was identified among its target genes, establishing an miR-30e-3p/TP53/MDM2 feedforward loop and accounting for miR-30e-3p dual role based on TP53 status. EpCAM, PTEN, and p27 were demonstrated as miR-30e-3p additional targets mediating its contribution to stemness and malignant features. In a preliminary cohort of patients with HCC treated with sorafenib, increased miR-30e-3p circulating levels predicted the development of resistance. In conclusion, molecular background dictates miR-30e-3p dual behavior in HCC. Mdm2 targeting plays a predominant tumor suppressor function in wild-type TP53 contexts, whereas other targets such as PTEN, p27, and EpCAM gain relevance and mediate miR-30e-3p oncogenic role in nonfunctional TP53 backgrounds. Increased circulating levels of miR-30e-3p predict the development of sorafenib resistance in a preliminary series of patients with HCC and deserve future investigations.Significance:The dual role of miR-30e-3p in HCC clarifies how the molecular context dictates the tumor suppressor or oncogenic function played by miRNAs.

Deregulated cell proliferation and apoptosis play a major role in hepatocellular carcinoma (HCC). MicroRNAs participate in the modulation of key molecules linked to hepatocarcinogenesis.Purpose: This study aims to investigate the role of miR-221 in the modulation of Bmf, a proapoptotic BH3-only protein, and to characterize miR-221 contribution to hepatocarcinogenesis through modulation of apoptosis.Experimental Design: Transfection of miR-221 and anti-miR-221 in HCC-derived cell lines and luciferase reporter assay were used to assess Bmf as a target of miR-221. Modulation of miR-221 and Bmf expression contributed to characterize their role in anoikis. Primary HCC tissues were analyzed to assess the clinical relevance of in vitro findings.Results: Enforced miR-221 expression caused Bmf down-regulation, whereas anti-miR-221 induced its up-regulation. A luciferase reporter assay confirmed Bmf as a target of miR-221. Following matrix detachment, miR-221 silencing led to increased apoptotic cell death. The analysis of HCC tissues revealed an inverse correlation between miR-221 and Bmf expression and a direct correlation between Bmf and activated caspase-3, as a marker of apoptosis. High miR-221 levels were associated with tumor multifocality and reduced time to recurrence after surgery.Conclusions: Our results indicate that miR-221, by targeting Bmf, inhibits apoptosis. Moreover, in HCC, miR-221 overexpression is associated with a more aggressive phenotype. These findings, together with the previously reported modulation of CDKN1B/p27 and CDKN1C/p57, show that miR-221 simultaneously affects multiple pro-oncogenic pathways and suggest miR-221 as a potential target for nonconventional treatment against HCC. (Clin Cancer Res 2009;15(16):5073–81)

Purpose:After 10 years of clinical practice and research studies, there are still no validated prognostic or predictive factors of response to sorafenib for hepatocellular carcinoma (HCC). On the basis of the results of our two retrospective studies, we designed the multicenter INNOVATE study with the aim to validate the role of nitric oxide synthase 3 (NOS3) and ANGPT2 polymorphisms in patients with HCC treated with sorafenib [NCT02786342].Patients and Methods:This prospective multicenter study was conducted at 10 centers in Italy. All eligible patients received a continuous oral treatment with 400 mg of sorafenib twice daily. Genotyping analysis was performed for NOS3 (rs2070744) and ANGPT2 SNPs (rs55633437). The primary outcome was progression-free survival (PFS), whereas secondary outcomes included overall survival (OS) and disease-control rate.Results:A total of 165 patients were enrolled between March 2016 and June 2018. NOS3 rs2070744 CC/CT genotypes were significantly associated with a higher median PFS (5.9 months vs. 2.4 months; HR = 0.43; P = 0.0007) and OS (15.7 months vs. 8.6 months; HR = 0.38; P < 0.0001) compared with TT genotype. There was no statistically significant association between ANGPT2 rs55633437 TT/GT genotypes and PFS (2.4 months vs. 5.7 months; HR = 1.93; P = 0.0833) and OS (15.1 months vs. 13.0 months; HR = 2.68; P = 0.55) when compared with the other genotype. Following adjustment for clinical covariates, multivariate analysis confirmed NOS3 as an independent prognostic factor for PFS (HR = 0.50; P = 0.0128) and OS (HR = 0.29; P = 0.0041).Conclusions:The INNOVATE study met the primary endpoint, confirming that patients with advanced HCC with NOS3 rs2070744 CC/CT genotypes had a better prognosis with respect to TT genotype patients.

Purpose: The aberrant expression of miR-221 is a hallmark of human cancers, including hepatocellular carcinoma (HCC), and its involvement in drug resistance, together with a proved in vivo efficacy of anti-miR-221 molecules, strengthen its role as an attractive target candidate in the oncologic field. The discovery of biomarkers predicting the response to treatments represents a clinical challenge in the personalized treatment era. This study aimed to investigate the possible role of miR-221 as a circulating biomarker in HCC patients undergoing sorafenib treatment as well as to evaluate its contribution to sorafenib resistance in advanced HCC.Experimental Design: A chemically induced HCC rat model and a xenograft mouse model, together with HCC-derived cell lines were employed to analyze miR-221 modulation by Sorafenib treatment. Data from the functional analysis were validated in tissue samples from surgically resected HCCs. The variation of circulating miR-221 levels in relation to Sorafenib treatment were assayed in the animal models and in two independent cohorts of patients with advanced HCC.Results: MiR-221 over-expression was associated with Sorafenib resistance in two HCC animal models and caspase-3 was identified as its target gene, driving miR-221 anti-apoptotic activity following Sorafenib administration. Lower pre-treatment miR-221 serum levels were found in patients subsequently experiencing response to Sorafenib and an increase of circulating miR-221 at the two months assessment was observed in responder patients.Conclusions: MiR-221 might represent a candidate biomarker of likelihood of response to Sorafenib in HCC patients to be tested in future studies. Caspase-3 modulation by miR-221 participates to Sorafenib resistance. Clin Cancer Res; 23(14); 3953–65. ©2017 AACR.

Supplementary Table 2 from MiR-199a-3p Regulates mTOR and c-Met to Influence the Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

Supplementary Table 4 from Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

Supplementary Table 3 from MiR-199a-3p Regulates mTOR and c-Met to Influence the Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

Gene promoter methylation causes loss of tumor suppressor genes function in human cancer. Here, we show that the CDH4 gene, a member of the cadherin family encoding for R-cadherin, contains a CpG island located at the 5′ of the first exon, which functions as a promoter element and is frequently affected by methylation in human cancer. By using methylation-specific PCR and reverse transcription-PCR in human cancer cell lines, promoter methylation could be directly linked to loss of gene expression. After treatment with the demethylating agent 5-aza-2-deoxycytidine, expression could be restored. Analysis of human primary tumors revealed that the CDH4 gene is methylated in 78% (38 of 49) of colorectal and 95% (20 of 21) of gastric carcinomas. CDH4 methylation was not detected in nonneoplastic colonic (0 of 10) and stomach (0 of 10) tissues or in peripheral blood (0 of 17). CDH4 methylation was detected in histologically normal tissues located in proximity of the neoplasms, indicating that CDH4 methylation is an early event in gastrointestinal tumor progression. We also proved that CDH4 methylation can be revealed in the peripheral blood of cancer patients. Our results indicate that CDH4 may act as a tumor suppressor gene in human gastrointestinal tumors and can potentially be used as an early diagnostic marker for gastrointestinal tumorigenesis.

Supplementary Table 1 from MiR-199a-3p Regulates mTOR and c-Met to Influence the Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

Supplementary Table 2 from MiR-122/Cyclin G1 Interaction Modulates p53 Activity and Affects Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

We investigated the role of microRNAs (miRNAs) in the pathogenesis of human hepatocellular carcinoma (HCC). A genome-wide miRNA microarray was used to identify differentially expressed miRNAs in HCCs arisen on cirrhotic livers. Thirty-five miRNAs were identified. Several of these miRNAs were previously found deregulated in other human cancers, such as members of the let-7 family, mir-221, and mir-145. In addition, the hepato-specific miR-122a was found down-regulated in ∼70% of HCCs and in all HCC-derived cell lines. Microarray data for let-7a, mir-221, and mir-122a were validated by Northern blot and real-time PCR analysis. Understanding the contribution of deregulated miRNAs to cancer requires the identification of gene targets. Here, we show that miR-122a can modulate cyclin G1 expression in HCC-derived cell lines and an inverse correlation between miR-122a and cyclin G1 expression exists in primary liver carcinomas. These results indicate that cyclin G1 is a target of miR-122a and expand our knowledge of the molecular alterations involved in HCC pathogenesis and of the role of miRNAs in human cancer. [Cancer Res 2007;67(13):6092–9]

Supplementary Figure 3 from MiR-122/Cyclin G1 Interaction Modulates p53 Activity and Affects Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

Supplementary Figure 1 from MiR-122/Cyclin G1 Interaction Modulates p53 Activity and Affects Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

Supplementary Table 5 from Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

Supplementary Material and Methods, Results, 1 figure, and 1 table

Supplementary Figure 4 from MiR-122/Cyclin G1 Interaction Modulates p53 Activity and Affects Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

Supplementary Table 2 from Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

Supplementary Figure Legends 1-4 from MiR-122/Cyclin G1 Interaction Modulates p53 Activity and Affects Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

Supplementary Table 1 from MiR-122/Cyclin G1 Interaction Modulates p53 Activity and Affects Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

Supplementary Table 3 from Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

Supplementary Figure 2 from MiR-122/Cyclin G1 Interaction Modulates p53 Activity and Affects Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

Supplementary Table 1, Figures 1-4 from Oncogenic Role of miR-483-3p at the IGF2/483 Locus

Supplementary Table 1 from Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

Supplementary Figure 1 from MiR-199a-3p Regulates mTOR and c-Met to Influence the Doxorubicin Sensitivity of Human Hepatocarcinoma Cells

Introduction: Immune check point inhibitors have recently entered the armamentarium of advanced hepatocellular carcinoma (HCC) treatment. Among them, the combination of atezolizumab plus bevacizumab has pushed it a step forward; however, a number of patients still present primary non-responses without any biomarker to predict responses to different options. Here, we aimed to identify a putative baseline biomarker to predict the response to atezolizumab–bevacizumab, by investigating whether baseline PD1+ and PD-L1+ peripheral granulocyte percentages might offer a non-invasive, cheap, and easily feasible assay. Methods: A prospective Italian cohort of 34 patients treated by atezolizumab–bevacizumab was tested to assay the baseline percentage of peripheral granulocytes and their PD1 and PD-L1 expression. The neutrophil to lymphocyte ratio (NLR) was also considered, and all data were compared with the clinical course of patients. Results: A low-baseline PD1+ peripheral granulocyte percentage turned out to predict responder patients (mean ±SD of PD1+ granulocyte percentage in responders versus non-responders: 9.9 ± 9.1 vs. 29.2 ± 17.6; student’s t-test, p < 0.01). In line, patients identified by a low PD1+ granulocyte percentage displayed a longer TTP (log-rank test, p < 0.0001). A lower granulocyte percentage on total white blood cells, irrespective of PD1 or PD-L1 expression, is also associated with responses to atezolizumab–bevacizumab (log-rank test, p < 0.05). No predictive value was observed for either the PD-L1+ granulocyte percentage or NLR. Conclusions: A low-baseline PD1+ peripheral granulocyte percentage is associated with responses to atezolizumab–bevacizumab treatment in advanced HCC. These findings encourage evaluating this minimally invasive, cheap, and easy test in further independent cohorts and outlining the relevance of innate immunity in the response to immune-checkpoint inhibitors.

Francesca Fornari,1,2 Catia Giovannini,1,3 Fabio Piscaglia,4,5 Laura Gramantieri4 1Centre for Applied Biomedical Research - CRBA, University of Bologna, St. Orsola Hospital, Bologna, Italy; 2Department for Life Quality Studies, University of Bologna, Rimini, Italy; 3Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, 40138, Italy; 4Division of Internal Medicine, Hepatobiliary and Immunoallergic Diseases, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 5Department of Medical and Surgical Sciences, University of Bologna, Bologna, ItalyCorrespondence: Francesca Fornari; Laura Gramantieri Tel/Fax +390512143902+390512142579Email francesca.fornari2@unibo.it; laura.gramantieri@aosp.bo.itAbstract: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. Sorafenib is the first multi-tyrosine kinase inhibitor approved for HCC and it has represented the standard of care for advanced HCC for almost 10 years, offering a survival benefit when compared to placebo. However, this benefit is limited, showing rare objective responses and a disease control rate approaching 50– 60%, with most patients experiencing disease progression at 6 months. These scant results dictate the urgent need for strategies to overcome both primary and acquired resistance. Herein we report several mechanisms supporting resistance to sorafenib in HCC patients, including activation of oncogenic pathways. Among these, the AKT/mTOR pathway plays a crucial role being at the crossroad of multiple driving events. Autophagy, multidrug-resistant phenotype, hypoxia-related mechanisms and endoplasmic reticulum stress are gaining more and more relevance as crucial events driving the response to anticancer drugs, including sorafenib. Several HCC-specific miRNAs take part to the regulation of these cellular processes. Remarkably, molecularly targeted strategies able to overcome resistance in these settings have also been reported. So far, the vast majority of data has been derived from laboratory studies, which means the need for an extensive validation. Indeed, most of the possible drug associations displaying promising effects in improving sorafenib efficacy herein described derive from preclinical explorations. Notably, data obtained in animal models can be inconsistent with regard to the human disease for efficacy, safety, side effects, best formulation and pharmacokinetics. However, they represent the necessary preliminary step to improve the management of advanced HCC.Keywords: HCC, sorafenib, microRNA, autophagy

Serena De Matteis,1 Martina Ghetti,2 Laura Gramantieri,3 Giorgia Marisi,2,&ast; Andrea Casadei-Gardini4,&ast; 1Department of Specialistic, Diagnostic and Experimental Medicine, University of Bologna, Bologna, Italy; 2Biosciences Laboratory, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola (FC), Italy; 3Center for Applied Biomedical Research, St. Orsola-Malpighi University Hospital, Bologna, Italy; 4Department of Medical Oncology, San Raffaele Scientific Institute IRCCS, Milan, Italy&ast;These authors contributed equally to this workCorrespondence: Martina GhettiBiosciences Laboratory, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, via Piero Maroncelli 40, Meldola (FC), 47014, ItalyTel/Fax +39-0543-739977Email martina.ghetti@irst.emr.itAbstract: Hepatitis B- and C-virus (HBV and HCV) infections contribute to hepatocellular carcinoma (HCC) development through several different mechanisms. In addition to a diverse molecular background, HCC subtypes also show differences in their metabolic profiles, suggesting that prevention and treatment might require the integration of multiple different approaches. We here analyzed the response of two HCC cell lines representative of different virus-related etiology, namely Hep3B (HBV+) and HUH7 (permissive to HCV replication) to sorafenib treatment. Our findings suggest that virus-related specificities influence treatment response in HCC, along with molecular, metabolic and microenvironmental factors. These differences have to be taken into account in the design of future clinical trial aimed to improve HCC patients’ outcome.Keywords: hepatocellular carcinoma, hepatitis virus, kinase inhibitor