Liver cell circuits and therapeutic discovery for advanced liver disease and cancer

Hepatocellular carcinoma (HCC) is a major global health challenge with rising incidence. Despite the previous approval of several novel therapeutic approaches, HCC remains the second common cause of cancer-related death worldwide. The vast majority of HCCs arises in the context of chronic fibrotic liver diseases caused by viral or metabolic etiologies. In patients with advanced liver disease the risk of HCC persists even after viral cure or control of infection. Moreover, given the change in the lifestyle and increase of obesity and metabolic disorders, HCC incidence is predicted to drastically augment in the next decade. Early detection, improvement of the screening method in patient at-risk and development of chemopreventive strategies are therefore urgently needed to reduce HCC risk. This review summarizes the major challenges in the identification of patient at risk for HCC and the emergent strategies for HCC prevention to improve patients’ outcome.


Introduction
Advanced liver diseases and hepatocellular carcinoma (HCC), the major type of liver cancer, are a major challenge for global health affecting more than 20% of the EU population. HCC is the second leading and fastest rising cause of cancer-related death worldwide and the leading cause of death among cirrhotic patients [1]. The European Association for the Study of the Liver (EASL) reported that HCC is responsible for 70,000 deaths/year in Western countries. It is estimated that 85-95% of the HCCs arise in the context of chronic fibrotic liver diseases, mainly due to chronic hepatitis B and C, alcoholic liver disease and non-alcoholic steatohepatitis (NASH), the most severe form of non-alcoholic fatty liver disease (NAFLD) [1,2]. In Europe, 70% of the cases can be attributed to chronic hepatitis C. Despite the development and widespread use of direct-acting antivirals (DAAs) which have revolutionary improved the management of hepatitis C virus (HCV) infection, the risk of HCC development in patient with advanced fibrosis persists even after a decade of viral cure [3]. Moreover, given the change in the lifestyle and the increasing of metabolic disorders, obesity and diabetes, NAFLD and NASH are predicted to be a major cause for HCC in the future [2]. HCC incidence will therefore keep increasing in the coming decades. are often diagnosed in advanced stage with poor liver function. Moreover, about 70% of the patients experience tumor recurrence within 5 years after surgical resection of ablation [1]. While several new compounds for HCC treatment have been recently approved, the overall response rate remains limited. The most recent combination of Vascular endothelial growth factor (VEGF)-targeting agents with immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) is characterized by response rates remaining at less than 30% [4]. Despite emerging molecular targeted therapies for HCC, identification of novel targets has been daunting tasks due to the complexity and heterogeneity of cancer initiation and development mechanisms [5]. Prevention of HCC development and progression in patients at risk has therefore emerged as a promising strategy to decrease the overall HCC disease burden. In this review, we summarize the challenge to identify patients at risk for HCC, review current HCC chemoprevention approaches and discuss the challenges and limitations in HCC prevention.

HCC screening and prediction
Given the limited therapeutic options currently available for HCC, prevention and early detection in at-risk individuals remain the most important strategy. Despite many advances, some challenges and roadblocks remain. Clinical practice guidelines recommend biannual HCC screening in at-risk patients for early tumor detection using ultrasound with or without α-fetoprotein (AFAP) detection [6]. Meta-analysis studies showed that early tumor detection is significantly associated with a higher probability of benefiting from a curative treatment and so a higher survival probability in patients. Early detection can also help to decrease the economic burden of HCC [7,8]. However, it appears that HCC screening is underutilized, with a rate lower than 20%, because of patient and provider-related barriers [6]. Given the rising numbers of patients with advanced liver fibrosis, the vast number of patients to be screened and the emergence of novel populations at-risk (i.e. NASH patients and HCVcured patients) constitute another major barrier [9]. Therefore, prediction of HCC risk in patient may help to improve HCC screening by distinguishing high-risk populations eligible for HCC chemoprevention from low-risk populations for long term vigilance. Nowadays, reliable tools to predict HCC risk, tumor recurrence and treatment response are still absent. Only the Barcelona Clinic Liver Cancer (BCLC) prognostic system was validated by EASL clinical guidelines, which classifies patients and recommend treatment accordingly [10]. However, this system has some limitations due to intermediate profiles making the choice of the best treatment often challenging.
To address this unmet medical need, several biomarker candidates have been developed based on genome wide expression profiling of patient liver tissues to predict the clinical outcome and HCC risk in patient with advanced liver disease. The most widely studied biomarker is a pan-etiology 186-gene clinical prognostic liver signature (PLS) in diseased liver tissues robustly predicting liver disease progression, patient outcome, HCC risk and tumor recurrence in multiple patient cohorts [11][12][13][14][15]. A reduced version of the PLS, comprising 32 bioinformatically selected and clinically validated genes, was recently implemented in an FDA-approved diagnostic platform for clinical use as Laboratory Developed Test (LDT) [16]. Moreover, a blood-based non-invasive signature comprising 8 soluble proteins, the PLSsec, has been developed as a surrogate of the PLS to accurately predict HCC risk in patient with advanced fibrosis [17]. Another signature based on genome wide epigenetic changes in the liver induced by viral and metabolic liver disease has been recently suggested to predict HCC risk [18,19]. Jühling et al. uncovered a 25 gene prognostic epigenetic signature termed PES robustly predicting HCC risk and survival in patients with metabolic and viral liver disease [19]. The PES reflects the presence of epigenetic dysregulation appearing in patients with chronic liver disease which drive hepatocarcinogenesis by altering gene expression [18,19].
In association with clinical scoring systems and biomarkers (i.e. AFAP), the PLS, the PLSec and the PES provide a perspective to improve patient care, HCC risk stratification and surveillance. Moreover, they may also help clinical testing of chemopreventive compounds by enrolling high-risk patients and at the same time decreasing clinical trial costs.

Target and drug discovery
Another major challenge is the identification of clinically relevant targets. Development of chemopreventive strategies has been daunting tasks due to (i) the complexity of cancer initiation and development mechanisms (ii) the difficulty to validate targets in patients (iii) the absence of reliable and simple models reflecting the cell circuits relevant for HCC development [1,9].
A "reverse-engineering" approach has been recently developed in order to identify relevant targets in clinical cohorts with completed long-term followup, before validation in experimental models [15]. Advancing this concept to a platform for drug and target discovery, Crouchet et al. developed a simple and robust human cell culture system that models the clinical PLS (described above) in an inducible and reversible manner (cPLS system). This system recapitulates the cell circuits relevant for liver disease progression and HCC development in a simplified manner for all the major etiologies [19][20][21]. In contrast to other models, this system is based on a clinically relevant readout and multitarget approach (186 genes). It offers the opportunity to validate clinical targets and to discover anti-fibrotic compounds for HCC chemoprevention by reversing the PLS from a poor-to a -good-prognosis. This model was recently used as a novel and simple drug discovery platform by screening computationally prioritized candidate compounds currently approved for long-term clinical use without severe adverse effects. The cPLS model, followed by validation in animal models, allowed the discovery of nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of fibrotic liver disease and HCC chemoprevention [21]. Mechanistic studies demonstrated that nizatidine prevents HCC by decreasing liver inflammation, preventing fibrosis development and through direct anti-cancer properties. Discovery of nizatidine as a novel anti-fibrotic compound for HCC prevention demonstrates the validity and translatability of the cPLS model [21].
As described above, chronic liver disease induces epigenetic reprogramming in patients associated with hepatocarcinogenesis. Epigenetic changes, mainly consisting in hypermethylation or acetylation of histones, results in perturbation of gene transcription. It was demonstrated that epigenetic changes are correlated with induction of the poor-prognosis PLS associated with HCC risk in patients [19]. The bromodomain and motif extraterminal (BET) proteins are chromatin readers which bind histone acetylation and promote oncogene upregulation by recruiting transcription complexes [22] ( Figure  1). BET proteins are overexpressed in several solid tumors and play a key role in hepatocarcinogenesis [22]. Interestingly, the BET inhibitor JQ1 has been shown to reverse both the poor-prognosis PLS and PES in the cPLS model [19]. Moreover, JQ1 had a marked impact on fibrosis in rodent model, restored transcriptional program in the liver and prevented HCC development [19]. BET inhibitors were therefore suggested as a novel strategy for HCC chemoprevention [23] (Figure 1).
Validation of anti-fibrotic compounds for chemoprevention also requires robust and easy to use patient-derived models to validate therapeutic approaches predicting clinical success. To address this challenge, Thomas Baumert's laboratory developed a patient-derived spheroid system harboring the main liver cell compartments and reflecting the liver micro-environment, in which, the clinical PLS can be modeled and reversed by candidate compounds [21]. In the future, we can imagine implementing cost-effective clinical testing of candidate compounds by combining target and drug discovery pipeline with reverseengineering approaches and HCC biomarkers to risk-stratify the patients.

HCC chemoprevention strategies
Cancer prevention strategies are based on different interventions: primary, secondary, and tertiary preventions ( Figure 2). Primary prevention mainly consists in limiting the risk factors. It is estimated that 40 to 45% of cancers are attributable to preventable risk factors including tobacco, alcohol, physical inactivity, and unbalanced diet. While the incidence of cancer is increasing worldwide, prevention appears as a key strategy to reduce this burden [24,25]. Primary prevention of HCC mainly focuses on lifestyle modification and HBV vaccination. Secondary prevention is the identification and treatment of premalignant or malignant processes through screening, early detection, and effective treatment in patient already exposed to etiological agents. Tertiary prevention aims to reduce cancer recurrence after treatment or de novo carcinogenesis in the pro-carcinogenic milieu [5,24] (Figure 2).

HCC prevention in metabolic liver disease
About 25% of the global population suffers from NAFLD. NAFLD is strongly associated with metabolic syndrome including obesity, dyslipidemia and type 2 diabetes mellitus [2]. Obesity and high body-mass index (BMI) are directly associated with HCC risk by driving steatosis, chronic inflammation, insulin resistance and oxidative stress [2]. AASLD guidelines recommend HCC surveillance in NAFLD patient every 6 months. However, abdominal ultrasound in obese patients is challenging [6]. Mounting evidence have shown that targeting metabolic abnormalities by either nutritional or pharmaceutical intervention may be an effective strategy to prevent HCC in obese and NAFLD patients. The primary preventive strategy for NAFLD patient consists in lifestyle changes through diet and exercise, as they benefit the full pathogenic spectrum of NAFLD and reduce progression of liver damage. Pharmaceutical intervention is mainly focused on modulation of metabolic pathways and inflammation using metformin and statins [2,26]. Metformin is an inhibitor of the AMP-activated protein kinase used in patient with diabetes. It has been associated with a reduction of HCC risk most likely by reducing oxidative stress and hyperinsulinemia [27,28]. Statins are broadly used as cholesterol lowering compounds. However, several studies have suggested an anti-HCC effect independent of this effect. Statins were shown to inhibit hepatocarcinogenesis by targeting different HCC drivers (i.e. Myc, NF-κB, Akt) by decreasing inflammation and by reducing fibrogenesis [5]. Statins may therefore have a HCC protective effect in at-risk patient regardless of the etiology but can also be used as tertiary intervention to prevent recurrence.

Chronic hepatitis B and persistence of HBV cccDNA
Chronic HBV infection accounts for the development of more than 50% of the HCCs in the world. HBV vaccination program has successfully reduced the number of chronic HBV carriers and is effective as a primary HCC prevention. However, chronic HBV infection is still a major cause of liver disease in unvaccinated patients, in particular in Southeast Asia and sub-Saharan Africa [29]. According to the World Health Organization (WHO), more than 240 million people are chronically infected with HBV. These individuals are at risk for HCC and represent a target population for secondary prevention. Antiviral therapies targeting HBV replication have been evaluated for HCC prevention. Retrospectives studies reported HCC risk reduction after suppression of HBV replication with the new generation of nucleot(s)ide analogs (i.e. entecavir and tenofovir) [30,31]. However, despite significant reduction, the HCC risk cannot be eliminated. The surveillance in patient with chronic HBV infection is therefore mandatory. HBV DNA integration into the host genome as well as the viral covalently closed circular DNA (cccDNA) are unique features of the HBV life cycle likely contributing to carcinogenesis. Next generation therapeutic approaches aiming to completely eradicate HBV including elimination of the viral cccDNA may help to improve prevention of HCC development in the future [32].

Chronic hepatitis C and persistent HCC risk post DAA cure
Chronic HCV infection leads to fibrosis, cirrhosis and associated complication including liver failure and HCC. It corresponds to the major HCC etiology in Western countries and Japan [1]. Globally, 71 million persons are infected. The "baby boomers" represent the specific population with the highest rates of chronic HCV infection and related-HCC mortality because most of the individuals were infected before the discovery of HCV and the development of the modern detection techniques [1]. Despite the development of the DAAs which have enabled an improvement of HCV clearance, the HCV-related HCC incidence is predicted to dramatically increase in the next decades. DAAs use is still hampered by limited access and chronic hepatitis C is often undiagnosed because chronic disease progresses during decades without symptoms. Moreover, studies in large clinical cohorts have shown that despite decrease in HCC risk after sustained virologic response, patients with advanced liver fibrosis remain at high risk for HCC [3]. Recent studies published by Hamdane et al. have shown that chronic HCV infection induces epigenetic alterations associated HCC risk which persist even after DAA cure [18]. Epigenetic alterations are therefore attractive targets to prevent HCC in at-risk patients treated for HCV infection. Moreover, in a proof-of-concept study, Jühling et al. showed that inhibition of the chromatin reader BRD4 using a small molecule JQ1 prevents liver fibrosis progression toward HCC Europe PMC Funders Author Manuscripts by restoring transcriptional reprogramming of the genes epigenetically altered in patients [19] (Figure 1). HCV-induced HCC is often diagnosed at a late stage due to the long and asymptomatic progression of chronic liver disease. As the development of a prophylactic HCV vaccine is still challenging [33], identifying the patients with HCC high risk is likely to improve management of the patients and prevention of HCC.

HCC chemopreventive approaches: examples of clinical studies
Chronic inflammation and liver fibrosis are well established drivers of HCC and therefore constitute attractive targets for HCC chemoprevention [1]. However, there is an enormous unmet medical need for liver fibrosis and antifibrotic therapies are not yet available. Major efforts in drug and target discovery are underway to treat fibrotic liver disease and prevent HCC development. Several generic molecules targeting inflammation and fibrosis entered in clinical trials and are also evaluated for HCC prevention (for a review, see [9]). Recently, the use of non-steroidal anti-inflammatory drugs such as aspirin in patients was shown to be associated with a decrease of hepatocarcinogenesis and HCC risk [34]. This anticancer effect may be due to the anti-inflammatory properties of aspirin though inhibition of the NF-κB signaling. Different studies also suggested a direct inhibition of P4HA2, a key enzyme of the collagen synthesis. However, other studies have shown an antiplatelet effect of aspirin limiting its clinical application in patients with chronic liver disease [34,35].
More studies are needed to decipher the exact mechanism of action.
Multiple signaling pathways have been identified as drivers of hepatocarcinogenic and cell proliferation and became attractive for generic HCC prevention. As example, the inhibitors or the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway, sirolimus or everolimus are now investigated for HCC recurrence in phase III clinical trials [36]. Erlotinib, the inhibitor of the epidermal growth factor receptor (EGFR) is also entering in phase I clinical trial following preclinical studies which demonstrated that erlotinib prevents HCC development in vivo and reverses the poor-prognosis PLS associated with HCC risk [9,37]. All these strategies may be of utmost interest to prevent preneoplastic lesions but also to prevent HCC recurrence after resection or transplantation by targeting the pro-carcinogenic milieu, which persists in the injured liver, even after viral cure.

Conclusions and perspectives
HCC is a major cause of cancer-related death worldwide which is predicted to increase in the next decades. Despite tremendous progress, curative treatments are still unsatisfactory. The vast majority of HCC patients are diagnosed at an advanced stage because the populations at risk are not clearly defined. Improvement of patient surveillance and screening as well as development of HCC chemopreventive strategies will therefore ameliorate patient management and prognosis in the future. Promotion of lifestyle change by healthcare professionals may significantly decrease HCC incidence. Moreover, combining predictive gene signature and/or HCC biomarkers will help to risk stratify the patient and establish effective and accurate therapeutic strategy for HCC prevention. The development of target discovery pipeline and patient-derived models coupled with reverse-engineering approaches will help to develop novel chemopreventive strategies. Discovery of safe generic limitées actuellement disponibles pour le CHC, la prévention et la détection précoce chez les personnes à risque restent la stratégie la plus intéressante. Malgré de nombreuses avancées, certains défis et obstacles subsistent. Les directives de pratique clinique recommandent un dépistage semestriel du CHC chez les patients à risque pour une détection précoce de la tumeur par échographie avec ou sans détection del'α-foetoprotéine (AFAP) [6]. Des études de méta-analyse ont montré que la détection précoce de la tumeur est significativement associée à une probabilité plus élevée de bénéficier d'un traitement curatif et donc à une probabilité de survie plus élevée chez les patients. La détection précoce peut également contribuer à diminuer la charge économique liée au CHC [7,8]. Cependant, il semble que le dépistage du CHC soit sous-utilisé, avec un taux inférieur à 20 %, tant à cause des patients qu'aux praticiens [6]. Étant donné le nombre croissant de patients atteints de fibrose hépatique avancée, le nombre conséquent de patients à dépister et l'émergence de nouvelles populations à risque (c'est-à-dire les patients atteints de NASH et les patients guéris d'une hépatite C chronique) constituent un autre obstacle majeur [9].