Circulating CD8 lymphocytes predict response to atezolizumab-bevacizumab in hepatocellular carcinoma.

Due to the lack of biomarkers predictive of response to atezolizumab-bevacizumab, the standard of care for advanced HCC, we analyzed baseline and early on-treatment variation of peripheral lymphocyte populations of 37 prospective patients treated by atezolizumab-bevacizumab and in 15 prospective patients treated by sorafenib or lenvatinib (TKIs). RNAseq analysis followed by RT-PCR validation on patients-derived PBMC was also performed. At first imaging, re-evaluation 13 patients receiving atezolizumab-bevacizumab, showed an objective response, 17 stable disease, while 7 were nonresponders. Baseline CD8+ and CD8+PD-L1+ peripheral lymphocytes were lower in responders versus nonresponders (T-test, p = 0.012 and 0.004, respectively). At 3 weeks, 28 of 30 responders displayed a rise of CD8+PD1+ lymphocytes with a positive mean fold change of 4.35 (±5.6 SD), whereas 6 of 7 nonresponders displayed a negative fold change of 0.89 (±0.84 SD). These changes were not observed in patients treated by TKIs. TRIM56, TRIM16, TRIM64, and Ki67 mRNAs were validated as upregulated in responders versus nonresponders after 3 weeks after treatment start, providing possible evidence of immune activation. Baseline CD8+ and CD8+PD-L1+ peripheral lymphocytes and early changes in CD8+PD1+ lymphocytes predict response to atezolizumab-bevacizumab providing noninvasive markers to complement clinical practice in the very early phases of treatment of HCC patients.

Thyroid hormone inhibits hepatocellular carcinoma progression via induction of differentiation and metabolic reprogramming.

BACKGROUND & AIMS: Only limited therapeutic options are currently available for hepatocellular carcinoma (HCC), making the development of effective alternatives essential. Based on the recent finding that systemic or local hypothyroidism is associated with HCC development in humans and rodents, we investigated whether the thyroid hormone triiodothyronine (T3) could inhibit the progression of HCCs. METHODS: Different rat and mouse models of hepatocarcinogenesis were investigated. The effect of T3 on tumorigenesis and metabolism/differentiation was evaluated by transcriptomic analysis, quantitative reverse transcription PCR, immunohistochemistry, and enzymatic assay. RESULTS: A short treatment with T3 caused a shift in the global expression profile of the most aggressive preneoplastic nodules towards that of normal liver. This genomic reprogramming preceded the disappearance of nodules and involved reprogramming of metabolic genes, as well as pro-differentiating transcription factors, including Kruppel-like factor 9, a target of the thyroid hormone receptor ß (TRß). Treatment of HCC-bearing rats with T3 strongly reduced the number and burden of HCCs. Reactivation of a local T3/TRß axis, a switch from Warburg to oxidative metabolism and loss of markers of poorly differentiated hepatocytes accompanied the reduced burden of HCC. This effect persisted 1 month after T3 withdrawal, suggesting a long-lasting effect of the hormone. The antitumorigenic effect of T3 was further supported by its inhibitory activity on cell growth and the tumorigenic ability of human HCC cell lines. CONCLUSIONS: Collectively, these findings suggest that reactivation of the T3/TRß axis induces differentiation of neoplastic cells towards a more benign phenotype and that T3 or its analogs, particularly agonists of TRß, could be useful tools in HCC therapy. LAY SUMMARY: Hepatocellular carcinoma (HCC) represents an important challenge for global health. Recent findings showed that systemic or local hypothyroidism is associated with HCC development. In rat models, we showed that administration of the thyroid hormone T3 impaired HCC progression, even when given at late stages. This is relevant from a translational point of view as HCC is often diagnosed at an advanced stage when it is no longer amenable to curative treatments. Thyroid hormones and/or thyromimetics could be useful for the treatment of patients with HCC.

The metabolic gene HAO2 is downregulated in hepatocellular carcinoma and predicts metastasis and poor survival.

BACKGROUND & AIMS: l-2-Hydroxy acid oxidases are flavin mononucleotide-dependent peroxisomal enzymes, responsible for the oxidation of l-2-hydroxy acids to ketoacids, resulting in the formation of hydrogen peroxide. We investigated the role of HAO2, a member of this family, in rat, mouse and human hepatocarcinogenesis. METHODS: We evaluated Hao2 expression by qRT-PCR in the following rodent models of hepatocarcinogenesis: the Resistant-Hepatocyte, the CMD and the chronic DENA rat models, and the TCPOBOP/DENA and TCPOBOP only mouse models. Microarray and qRT-PCR analyses were performed on two cohorts of human hepatocellular carcinoma (HCC) patients. Rat HCC cells were transduced by a Hao2 encoding lentiviral vector and grafted in mice. RESULTS: Downregulation of Hao2 was observed in all investigated rodent models of hepatocarcinogenesis. Interestingly, Hao2 mRNA levels were also profoundly downregulated in early preneoplastic lesions. Moreover, HAO2 mRNA levels were strongly downregulated in two distinct series of human HCCs, when compared to both normal and cirrhotic peri-tumoral liver. HAO2 levels were inversely correlated with grading, overall survival and metastatic ability. Finally, exogenous expression of Hao2 in rat cells impaired their tumorigenic ability. CONCLUSION: Our work identifies for the first time the oncosuppressive role of the metabolic gene Hao2. Indeed, its expression is severely decreased in HCC of different species and etiology, and its reintroduction in HCC cells profoundly impairs tumorigenesis. We also demonstrate that dysregulation of HAO2 is a very early event in the development of HCC and it may represent a useful diagnostic and prognostic marker for human HCC.

Local hypothyroidism favors the progression of preneoplastic lesions to hepatocellular carcinoma in rats.

UNLABELLED: Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that mediate most of the effects elicited by the thyroid hormone, 3,5,3'-L-triiodothyronine (T3). TRs have been implicated in tumorigenesis, although it is unclear whether they act as oncogenes or tumor suppressors, and at which stage of tumorigenesis their dysregulation occurs. Using the resistant-hepatocyte rat model (R-H model), we found down-regulation of TRß1 and TRα1 and their target genes in early preneoplastic lesions and hepatocellular carcinoma (HCCs), suggesting that a hypothyroid status favors the onset and progression of preneoplastic lesions to HCC. Notably, TRß1 and, to a lesser extent, TRα1 down-regulation was observed only in preneoplastic lesions positive for the progenitor cell marker, cytokeratin-19 (Krt-19) and characterized by a higher proliferative activity, compared to the Krt-19 negative ones. TRß1 down-regulation was observed also in the vast majority of the analyzed human HCCs, compared to the matched peritumorous liver or to normal liver. Hyperthyroidism induced by T3 treatment caused up-regulation of TRß1 and of its target genes in Krt-19(+) preneoplastic rat lesions and was associated with nodule regression. In HCC, TRß1 down-regulation was not the result of hypermethylation of its promoter, but was associated with an increased expression of TRß1-targeting microRNAs ([miR]-27a, -181a, and -204). An inverse correlation between TRß1 and miR-181a was also found in human cirrhotic peritumoral tissue, compared to normal liver. CONCLUSION: Down-regulation of TRs, especially TRß1, is an early and relevant event in liver cancer development and is species and etiology independent. The results also suggest that a hypothyroid status of preneoplastic lesions may contribute to their progression to HCC and that the reversion of this condition may represent a possible therapeutic goal to interfere with the development of this tumor.

TP53/MicroRNA Interplay in Hepatocellular Carcinoma.

The role of microRNAs as oncogenes and tumor suppressor genes has emerged in several cancers, including hepatocellular carcinoma (HCC). The pivotal tumor suppressive role of p53-axis is indicated by the presence of inactivating mutations in TP53 gene in nearly all cancers. A close interaction between these two players, as well as the establishment of complex p53/miRNAs loops demonstrated the strong contribution of p53-effector miRNAs in enhancing the p53-mediated tumor suppression program. On the other hand, the direct and indirect targeting of p53, as well as the regulation of its stability and activity by specific microRNAs, underlie the importance of the fine-tuning of p53 pathway, affecting the cell fate of damaged/transformed cells. The promising results of miRNAs-based therapeutic approaches in preclinical studies and their entrance in clinical trials demonstrate the feasibility of this strategy in several diseases, including cancer. Molecularly targeted drugs approved so far for HCC treatment show intrinsic or acquired resistances with disease progression in many cases, therefore the identification of effective and non-toxic agents for the treatment of HCC is actually an unmet clinical need. The knowledge of p53/miRNA inter-relations in HCC may provide useful elements for the identification of novel combined approaches in the context of the "personalized-medicine" era.

Significance of serum and hepatic microRNA-122 levels in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is believed to be a type of metabolic syndrome. MicroRNA-122 (miR-122) is the most abundant microRNA in the liver and is an important factor for the metabolism of glucose and lipids. In the present study, we examined the correlation between the hepatic and serum miR-122 expression levels and the clinicopathological factors of patients with NAFLD. METHODS: We extracted the total RNA, along with preserved miRNAs, from liver biopsy samples of 67 patients with NAFLD. In 52 of these 67 patients, the total RNA was extracted from serum. The miR-122 that was obtained by quantitative reverse transcription-polymerase chain reaction was quantified using TaqMan MicroRNA assays. RESULTS: A significant correlation was detected between serum and hepatic miR-122 expression (correlation coefficient, 0.461; P=0.005). Patients with mild steatosis (<33%) showed significantly lower levels of hepatic miR-122 compared with patients with severe steatosis (>33%) (hepatic miR-122: mild/severe=2.158±1.786/4.836±7.506, P=0.0473; serum miR-122: mild/severe=0.002±0.005/0.007±0.001, P=0.0491). Moreover, hepatic and serum miR-122 levels were significantly higher in patients with mild fibrosis than in those with severe fibrosis (hepatic miR-122: mild/severe=5.201±7.275/2.394±1.547, P=0.0087; serum miR-122: mild/severe=0.008±0.011/0.002±0.004, P=0.0191). CONCLUSIONS: We found that the hepatic and serum miR-122 levels were associated with hepatic steatosis and fibrosis. The serum miR-122 level can be a useful predictive marker of liver fibrosis in patients with NAFLD.

Noncoding RNAs in Hepatocellular Carcinoma: Potential Applications in Combined Therapeutic Strategies and Promising Candidates of Treatment Response.

The incidence of hepatocellular carcinoma (HCC) is increasing, and 40% of patients are diagnosed at advanced stages. Over the past 5 years, the number of clinically available treatments has dramatically increased for HCC, making patient management particularly complex. Immune checkpoint inhibitors (ICIs) have improved the overall survival of patients, showing a durable treatment benefit over time and a different response pattern with respect to tyrosine kinase inhibitors (TKIs). Although there is improved survival in responder cases, a sizeable group of patients are primary progressors or are ineligible for immunotherapy. Indeed, patients with nonviral etiologies, such as nonalcoholic steatohepatitis (NASH), and alterations in specific driver genes might be less responsive to immunotherapy. Therefore, improving the comprehension of mechanisms of drug resistance and identifying biomarkers that are informative of the best treatment approach are required actions to improve patient survival. Abundant evidence indicates that noncoding RNAs (ncRNAs) are pivotal players in cancer. Molecular mechanisms through which ncRNAs exert their effects in cancer progression and drug resistance have been widely investigated. Nevertheless, there are no studies summarizing the synergistic effect between ncRNA-based strategies and TKIs or ICIs in the preclinical setting. This review aims to provide up-to-date information regarding the possible use of ncRNAs as therapeutic targets in association with molecular-targeted agents and immunotherapies and as predictive tools for the selection of optimized treatment options in advanced HCCs.

CDKN1C/P57 is regulated by the Notch target gene Hes1 and induces senescence in human hepatocellular carcinoma.

CDKN1C/P57 is a cyclin-dependent kinase inhibitor implicated in different human cancers, including hepatocellular carcinoma (HCC); however, little is known regarding the role of CDKN1C/P57 and its regulation in HCC. In this study, we show that the down-regulation of Notch1 and Notch3 in two HCC cell lines resulted in Hes1 down-regulation, CDKN1C/P57 up-regulation, and reduced cell growth. In line with these data, we report that CDKN1C/P57 is a target of transcriptional repression by the Notch effector, Hes1. We found that the up-regulation of CDKN1C/P57 by cDNA transfection decreased tumor growth, as determined by growth curve, flow cytometry analysis, and cyclin D1 down-regulation, without affecting the apoptosis machinery. Indeed, the expression of Bax, Noxa, PUMA, BNIP(3), and cleaved caspase-3 was not affected by CDKN1C/P57 induction. Morphologically CDKN1C/p57-induced HCC cells became flat and lengthened in shape, accumulated the senescence-associated ß-galactosidase marker, and increased P16 protein expression. Evaluation of senescence in cells depleted both for Hes1 and CDKN1C/P57 revealed that the senescent state really depends on the accumulation of CDKN1C/p57. Finally, we validated our in vitro results in primary HCCs, showing that Hes1 protein expression inversely correlates with CDKN1C/P57 mRNA levels. In addition, reduced Hes1 protein expression is accompanied by a shorter time to recurrence after curative resection, suggesting that Hes1 may represent a biomarker for prediction of patients with poor prognosis.

Liver tumorigenicity promoted by microRNA-221 in a mouse transgenic model.

UNLABELLED: MicroRNA-221 (miR-221) is one of the most frequently and consistently up-regulated microRNAs (miRNAs) in human cancer. It has been hypothesized that miR-221 may act as a tumor promoter. To demonstrate this, we developed a transgenic (TG) mouse model that exhibits an inappropriate overexpression of miR-221 in the liver. Immunoblotting and immunostaining confirmed a concomitant down-regulation of miR-221 target proteins. This TG model is characterized by the emergence of spontaneous nodular liver lesions in approximately 50% of male mice and by a strong acceleration of tumor development in 100% of mice treated with diethylnitrosamine. Similarly to human hepatocellular carcinoma, tumors are characterized by a further increase in miR-221 expression and a concomitant inhibition of its target protein-coding genes (i.e., cyclin-dependent kinase inhibitor [Cdkn]1b/p27, Cdkn1c/p57, and B-cell lymphoma 2-modifying factor). To validate the tumor-promoting effect of miR-221, we showed that in vivo delivery of anti-miR-221 oligonucleotides leads to a significant reduction of the number and size of tumor nodules. CONCLUSIONS: This study not only establishes that miR-221 can promote liver tumorigenicity, but it also establishes a valuable animal model to perform preclinical investigations for the use of anti-miRNA approaches aimed at liver cancer therapy.

In hepatocellular carcinoma miR-519d is up-regulated by p53 and DNA hypomethylation and targets CDKN1A/p21, PTEN, AKT3 and TIMP2.

MiR-519d belongs to the chromosome 19 miRNA cluster (C19MC), the largest human miRNA cluster. One of its members, miR-519d, is over-expressed in hepatocellular carcinoma (HCC) and we characterized its contribution to hepatocarcinogenesis. In HCC cells, the over-expression of miR-519d promotes cell proliferation, invasion and impairs apoptosis following anticancer treatments. These functions are, at least in part, exerted through the direct targeting of CDKN1A/p21, PTEN, AKT3 and TIMP2. The mechanisms underlying miR-519d aberrant expression in HCC were assayed by genomic DNA amplification, methylation analysis and ChIP assay. The aberrant hypomethylation of C19MC and TP53 were respectively identified as an epigenetic change allowing the aberrant expression of miR-519d and one of the factors able to activate its transcription. In conclusion, we assessed the oncogenic role of miR-519d in HCC by characterizing its biological functions, including the modulation of response to anticancer treatments and by identifying CDKN1A/p21, PTEN, AKT3 and TIMP2 among its targets.

Low-Baseline PD1+ Granulocytes Predict Responses to Atezolizumab-Bevacizumab in Hepatocellular Carcinoma.

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.

MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma.

BACKGROUND: Metabolic reprogramming is a well-known marker of cancer, and it represents an early event during hepatocellular carcinoma (HCC) development. The recent approval of several molecular targeted agents has revolutionized the management of advanced HCC patients. Nevertheless, the lack of circulating biomarkers still affects patient stratification to tailored treatments. In this context, there is an urgent need for biomarkers to aid treatment choice and for novel and more effective therapeutic combinations to avoid the development of drug-resistant phenotypes. This study aims to prove the involvement of miR-494 in metabolic reprogramming of HCC, to identify novel miRNA-based therapeutic combinations and to evaluate miR-494 potential as a circulating biomarker. METHODS: Bioinformatics analysis identified miR-494 metabolic targets. QPCR analysis of glucose 6-phosphatase catalytic subunit (G6pc) was performed in HCC patients and preclinical models. Functional analysis and metabolic assays assessed G6pc targeting and miR-494 involvement in metabolic changes, mitochondrial dysfunction, and ROS production in HCC cells. Live-imaging analysis evaluated the effects of miR-494/G6pc axis in cell growth of HCC cells under stressful conditions. Circulating miR-494 levels were assayed in sorafenib-treated HCC patients and DEN-HCC rats. RESULTS: MiR-494 induced the metabolic shift of HCC cells toward a glycolytic phenotype through G6pc targeting and HIF-1A pathway activation. MiR-494/G6pc axis played an active role in metabolic plasticity of cancer cells, leading to glycogen and lipid droplets accumulation that favored cell survival under harsh environmental conditions. High miR-494 serum levels associated with sorafenib resistance in preclinical models and in a preliminary cohort of HCC patients. An enhanced anticancer effect was observed for treatment combinations between antagomiR-494 and sorafenib or 2-deoxy-glucose in HCC cells. CONCLUSIONS: MiR-494/G6pc axis is critical for the metabolic rewiring of cancer cells and associates with poor prognosis. MiR-494 deserves attention as a candidate biomarker of likelihood of response to sorafenib to be tested in future validation studies. MiR-494 represents a promising therapeutic target for combination strategies with sorafenib or metabolic interference molecules for the treatment of HCC patients who are ineligible for immunotherapy.

MicroRNAs at the Crossroad between Immunoediting and Oncogenic Drivers in Hepatocellular Carcinoma.

Treatments aimed to reverse the tumor-induced immune tolerance represent a promising approach for advanced hepatocellular carcinoma (HCC). Notwithstanding, primary nonresponse, early, and late disease reactivation still represent major clinical challenges. Here, we focused on microRNAs (miRNAs) acting both as modulators of cancer cell hallmarks and immune system response. We outlined the bidirectional function that some oncogenic miRNAs play in the differentiation and program activation of the immune system development and, at the same time, in the progression of HCC. Indeed, the multifaceted spectrum of miRNA targets allows the modulation of both immune-associated factors and oncogenic or tumor suppressor drivers at the same time. Understanding the molecular changes contributing to disease onset, progression, and resistance to treatments might help to identify possible novel biomarkers for selecting patient subgroups, and to design combined tailored treatments to potentiate antitumor approaches. Preliminary findings seem to argue in favor of a bidirectional function of some miRNAs, which enact an effective modulation of molecular pathways driving oncogenic and immune-skipping phenotypes associated with cancer aggressiveness. The identification of these miRNAs and the characterization of their 'dual' role might help to unravel novel biomarkers identifying those patients more likely to respond to immune checkpoint inhibitors and to identify possible therapeutic targets with both antitumor and immunomodulatory functions. In the present review, we will focus on the restricted panel of miRNAs playing a bidirectional role in HCC, influencing oncogenic and immune-related pathways at once. Even though this field is still poorly investigated in HCC, it might represent a source of candidate molecules acting as both biomarkers and therapeutic targets in the setting of immune-based treatments.

Animal Models of Hepatocellular Carcinoma: Current Applications in Clinical Research.

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.

Aflatoxin B1 DNA-Adducts in Hepatocellular Carcinoma from a Low Exposure Area.

Aflatoxin B1 (AFB1) is a class 1 carcinogen with an ascertained role in the development of hepatocellular carcinoma (HCC) in high exposure areas. Instead, this study aimed to assay whether chronic/intermittent, low-dose AFB1 consumption might occur in low-exposure geographical areas, ultimately accumulating in the liver and possibly contributing to liver cancer. AFB1-DNA adducts were assayed by immunostaining in liver tissues from three Italian series of twenty cirrhosis without HCC, 131 HCC, and 45 cholangiocarcinoma, and in an AFB1-induced HCC rat model. CD68, TP53 immunostaining, and TP53 RFLP analysis of R249S transversion were used to characterize cell populations displaying AFB1-DNA adducts. Twenty-five HCCs displayed AFB1-adducts both in neoplastic hepatocytes and in cells infiltrating the tumor and non-tumor tissues. Nuclear immunostaining was observed in a few cases, while most cases showed cytoplasmic immunostaining, especially in CD68-positive tumor-infiltrating cells, suggestive for phagocytosis of dead hepatocytes. Similar patterns were observed in AFB1-induced rat HCC, though with higher intensity. Cholangiocarcinoma and cirrhosis without HCC did not displayAFB1-adducts, except for one case. Despite not providing a causal relationship with HCC, these findings still suggest paying attention to detection and control measures for aflatoxins to ensure food safety in low exposure areas.

Notch Signaling Regulation in HCC: From Hepatitis Virus to Non-Coding RNAs.

The Notch family includes evolutionary conserved genes that encode for single-pass transmembrane receptors involved in stem cell maintenance, development and cell fate determination of many cell lineages. Upon activation by different ligands, and depending on the cell type, Notch signaling plays pleomorphic roles in hepatocellular carcinoma (HCC) affecting neoplastic growth, invasion capability and stem like properties. A specific knowledge of the deregulated expression of each Notch receptor and ligand, coupled with resultant phenotypic changes, is still lacking in HCC. Therefore, while interfering with Notch signaling might represent a promising therapeutic approach, the complexity of Notch/ligands interactions and the variable consequences of their modulations raises concerns when performed in undefined molecular background. The gamma-secretase inhibitors (GSIs), representing the most utilized approach for Notch inhibition in clinical trials, are characterized by important adverse effects due to the non-specific nature of GSIs themselves and to the lack of molecular criteria guiding patient selection. In this review, we briefly summarize the mechanisms involved in Notch pathway activation in HCC supporting the development of alternatives to the γ-secretase pan-inhibitor for HCC therapy.

Elucidating the Molecular Basis of Sorafenib Resistance in HCC: Current Findings and Future Directions.

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.

MicroRNAs as Modulators of Tumor Metabolism, Microenvironment, and Immune Response in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality. Molecular heterogeneity and absence of biomarkers helping patient allocation to the best therapeutic option contribute to poor prognosis in advanced stages. MicroRNAs' (miRNAs) deregulated expression contributes to tumor development and progression and influences drug resistance in HCC. Accordingly, miRNAs have been extensively investigated as both biomarkers and therapeutic targets. The diagnostic and prognostic roles of circulating miRNAs have been ascertained, though with some inconsistencies across studies. From a therapeutic perspective, miRNA-based approaches demonstrated safety profiles and antitumor efficacy in HCC animal models. Nevertheless, caution should be used when transferring preclinical findings to the clinic, due to possible molecular inconsistency between animal models and the heterogeneous patterns of human diseases. A wealth of information is offered by preclinical studies exploring the mechanisms driving miRNAs' aberrant expression, the molecular cascades triggered by miRNAs and the corresponding phenotypic changes. Ex-vivo analyses confirmed these results, further shedding light on the intricacy of the human disease often overcoming pre-clinical models. This complexity seems to be ascribed to the intrinsic heterogeneity of HCC, to different risk factors driving its development, as well as to changes across stages and previous treatments. Preliminary findings suggest that miRNAs associated with specific risk factors might be more informative in defined patients' subgroups. The first issue to be considered when trying to envisage a possible translational perspective is the molecular context that often drives different miRNA functions, as clearly evidenced by "dual" miRNAs. Concerning the possible roles of miRNAs as biomarkers and therapeutic targets, we will focus on miRNAs' involvement in metabolic pathways and in the modulation of tumor microenvironment, to support their exploitation in defined contexts.

Hepatic Cancer Stem Cells: Molecular Mechanisms, Therapeutic Implications, and Circulating Biomarkers.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers. HCC is associated with multiple risk factors and is characterized by a marked tumor heterogeneity that makes its molecular classification difficult to apply in the clinics. The lack of circulating biomarkers for the diagnosis, prognosis, and prediction of response to treatments further undermines the possibility of developing personalized therapies. Accumulating evidence affirms the involvement of cancer stem cells (CSCs) in tumor heterogeneity, recurrence, and drug resistance. Owing to the contribution of CSCs to treatment failure, there is an urgent need to develop novel therapeutic strategies targeting, not only the tumor bulk, but also the CSC subpopulation. Clarification of the molecular mechanisms influencing CSC properties, and the identification of their functional roles in tumor progression, may facilitate the discovery of novel CSC-based therapeutic targets to be used alone, or in combination with current anticancer agents, for the treatment of HCC. Here, we review the driving forces behind the regulation of liver CSCs and their therapeutic implications. Additionally, we provide data on their possible exploitation as prognostic and predictive biomarkers in patients with HCC.

Direct Antiviral Treatments for Hepatitis C Virus Have Off-Target Effects of Oncologic Relevance in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: HCV eradication by direct-acting antiviral agents (DAAs) reduces de novo hepatocellular carcinoma (HCC) incidence in cirrhosis; however, contrasting evidence about beneficial or detrimental effects still exists in patients who have already developed HCC. METHODS: we investigated whether sofosbuvir and daclatasvir modulate cell proliferation, invasion capability and gene expression (RNA-seq) in HCC-derived cell lines, hypothesizing possible off-target effects of these drugs. Results observed in HCC cell lines were validated in non-HCC cancer-derived cell lines and a preliminary series of human HCC tissues by qPCR and IHC. RESULTS: DAAs can affect HCC cell proliferation and migration capability by either increasing or reducing them, showing transcriptomic changes consistent with some unexpected drug-associated effects. Off-target gene modulation, mainly affecting ribosomal genes, mitochondrial functions and histones, points to epigenetics and proliferation as relevant events, consistent with matched phenotypic changes. A preliminary validation of in vitro findings was performed in a restricted cohort of HCC patients previously treated with DAAs, with immunohistochemical correlations suggesting DAA-treated HCCs to be more aggressive in terms of migration and epidermal-to-mesenchymal transition. CONCLUSIONS: Our findings suggested the possible occurrence of off-target effects ultimately modulating cell proliferation and/or migration and potentially justified previous findings showing some instances of particularly aggressive HCC recurrence as well as reduced incidence of recurrence of HCC following treatment with DAAs.