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1. Introduction

Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) accounts for 80–90% of liver cancers and represents the third leading cause of cancer mortality worldwide. Over the past two decades, the incidence of HCC has doubled in many countries including Europe and United States ^[1]. While approximately 75% of HCCs were associated with hepatitis B or C infection, other major risk factors including aflatoxin B1 (AFB1) exposure, nonalcoholic fatty liver disease (NAFLD), chronic alcohol consumption and obesity are now gaining more and more relevance ^[2]. Among treatment options, surgical resection and percutaneous ablation are considered “curative” modalities; however, they can be performed in a restricted fraction of patients, particularly those detected at early stages. When diagnosed at, or progressed to, intermediate-stage, the standard of care is transarterial chemoembolization ^[3], while large tumors or HCC with vascular invasion can be considered for radioembolization with selective internal radiation treatment (SIRT). When diagnosed at advanced stages or in the presence of extra-hepatic spread, only systemic treatments can be administered. Sorafenib, the first oral multikinase inhibitor (MKI) entering the treatment of HCC, has been the standard of care for almost ten years ^[4]. It inhibits angiogenesis and cell proliferation by targeting platelet-derived growth factor receptor (PDGF-R), vascular endothelial growth factor receptor (VEGFR-2/3), c-Kit, Flt3 and Raf kinases involved in the MAPK/ERK pathway ^[5].The second tyrosine kinase inhibitor to be approved for the first line treatment of advanced HCC is lenvatinib, based on its non-inferiority to sorafenib ^[6]. In the setting of second line treatments, regorafenib and cabozantinib were approved for patients with HCC progression and ramucirumab for patients previously treated with sorafenib, with a serum AFP (Alpha-Fetoprotein) level ≥400 ng/mL ^[7]. Beside these molecularly-targeted agents, immuno-oncology has opened the way to a novel class of drugs modulating the expression of Immune Check Point Inhibitors (ICPI), whose aberrant expression results in the immune escape of cancer cells ^[8]. Programmed cell death protein 1 (PD1), Programmed Death-Ligand 1 (PDL1), Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA4) are the most targeted ICPI in HCC proving to be more effective than Tyrosine Kinase Inhibitors (TKI), especially in combination regimens ^[9]. The most interesting results have been obtained so far with the association of Atezolizumab and Bevacizumab ^[10], however other promising combinations are under clinical investigation. Large clinical trials are however still needed for validation of these effects and for the identification of biomarkers helping the allocation of patients to the most effective treatment option in a personalized perspective. In addition, beside the fraction of patients who are non-responders to ICPIs, others may experience adverse events which prevent the prosecution of treatment. Unfortunately, the molecular classification of HCC has not entered clinical practice so far; thus, genetic and epigenetic factors driving response or resistance to each treatment are still poorly understood.Deregulation of multiple molecular signaling pathways such as Wnt/β-catenin, Ras mitogen-activated protein kinase (Ras/Raf/MAPK), phosphatidylinositol 3-kinase (PI3K), AKT/mammalian target of rapamycin (mTOR), Janus kinase (Jak)-signal transducer activator of transcription factor (Stat) (JAK/STAT) and the Hippo signaling pathway are essential for HCC development and progression ^[11]. Understanding the critical genes and signaling molecules in different HCC subgroups will help to develop tailored therapeutic strategies. Inhibition of a single signaling cascade may induce feedback activation of other pathways; hence, combination of different molecularly targeted agents is expected to show synergistic activity. Notch signaling modulates the development and functions of several immune cell lineages. Among these, Notch1 and Dll4 interaction participates in T cells’ lineage commitment, while Notch2-Dll1 interaction contributes to the development of marginal zone B cells ^[12]. In the peripheral T and B cell compartment, Notch signaling activates T cells’ proliferation and cytokine production, which, in line, are down-regulated by GSI-mediated inhibition of Notch. Similarly, Notch1 and Notch2 activation promotes naive CD8+ T cells to cytotoxic T lymphocytes, which, in turn, drive the antitumoral response and are the targets of ICPIs.Investigations on Notch signaling in T cells functions might lead to a more effective avenue for combined intervention in cancer treatment.The aim of this review is to discuss recent advances in molecular mechanisms involved in Notch signaling regulation that could represent new challenges for HCC therapy. Studies described in this review might contribute to the identification of key molecules responsible for Notch pathway regulation that could represent new therapeutic targets.

2. Notch Genes

In 1911, Morgan and colleagues described a Drosophila with a notch on a wing margin caused by a heterozygous deletion of a gene located on the X chromosome that was consequently named “NOTCH” ^[13]. Many years later an initial description of Notch structure and functions was provided by Wharton and colleagues and it was shown to be a conserved intracellular pathway involved in a variety of cellular processes ^[14]. Despite the simplicity of its activation, a fine regulation of Notch signaling is required to avoid pathological effects including cancer development. Four Notch receptors (Notch1, Notch2, Notch3, Notch4) have been identified in humans. These receptors transduce signals by interacting with transmembrane ligands of the Delta-like (DLL1, 3 and 4) and Jagged (Jagged1 and 2) protein family on neighboring cells ^[15]. Once processed in the Endoplasmic Reticulum and in the Golgi, Notch receptors migrate to the cell membrane where they are activated by ligand interaction ^[16][17]. Upon ligand binding, the Notch receptor is cleaved by the protease ADAM10 or by TACE. The resulting fragment, called Notch extracellular truncation (NEXT), is then cleaved by γ-secretase and the Notch Intra Cellular Domain (NICD) is translocated to the nucleus allowing the transcription of Notch target genes including the Hairy enhancer of split homologs’ transcription factors (Hes and Hey) ^[18][19] (Figure 1). Low levels of NICD are sufficient to activate transcription because it acts as a transcriptional coactivator on the RBP-JK factor constantly bound to the promoter of target genes ^[20]. The activation of Notch signaling is finely regulated in different tissues driving organ morphogenesis during development ^[21].Figure 1. Notch signaling pathway.Notch receptors are heterodimeric cell membrane proteins containing an extracellular subunit and a fragment that includes a transmembrane and an intracellular domain. Upon binding with ligand expressed on the surface of neighboring cells, two proteolytic cleavages occur. The first one takes place outside the trans-membrane domain by metalloproteinase TACE/ADAM10. The resultant Notch fragment, called Notch extracellular truncation (NEXT), is necessary for the second cleavage performed by γ-secretase within the trans-membrane region. This last proteolytical event releases Notch intracellular domain (NICD) that translocates to the nucleus, interacts with DNA binding proteins CSL (CBF1 Suppressor of Hairless Lag1) otherwise known as RBP-JK and Mastermind (MAML) transactivating target genes.

Notch Signaling in HCC

In the liver, hepatoblasts are the precursors of hepatocytes and cholangiocytes. Notch signaling can inhibit the differentiation of hepatoblasts into hepatocytes, allowing cholangiocyte formation ^[22][23]. Both the loss and gain of Notch functions may contribute to liver cancer development including both cholangiocarcinoma (CC) and HCC and different Notch receptors have different functions during liver cancer development ^[24]. Jagged1 plays a central role in the differentiation of hepatocyte progenitor cells (HPCs) and in hepatocyte proliferation during rat liver regeneration suggesting its possible involvement in HCC development ^[25][26]. Moreover, Jagged1 DNA copy number variation is associated with poor survival after liver cancer surgical resection ^[27] and its gene expression is comparable to Notch1 expression, suggesting an activation of Notch signaling that might be responsible for HCC development ^[27][28]. Occurrence of HCC in mice was reported after constitutive activation of NICD. Using bigenic the AFP-Notch Intracellular Domain (AFP-NICD) mice, in which Cre-mediated recombination in embryonic hepatoblasts results in the expression of a constitutively active form of Notch1 in >95% of cholangiocytes and hepatoblasts, Villanueva et al. showed HCC development in 100% of mice ^[29]. The analysis of differentially expressed genes revealed significant up-regulation of Notch target genes such as Hes1, and Hey2 and the activation of Notch signaling correlated with the transcriptional induction of insulin-like growth factor 2 (IGF2), an HCC promoter gene ^[30]. Aberrant Notch expression is frequently found in HCC tissue ^[31][32][33] where it participates in almost all the hallmarks of cancer, including resistance to anticancer treatments ^[33]. In line with this, several strategies inhibiting Notch increase treatment efficacy in preclinical models. Among these, γ-secretase inhibition (GSI) represents one the first approaches tested, including in clinical trials ^[34]. GSIs are a class of small molecules able to prevent the proteolytic cleavage of Notch receptors and the subsequent release of the Notch intracellular domain (NICD), which results from ligand interaction. Remarkably, gamma secretases are membrane-bound proteases involved in the cleavage of several other substrates including the beta-amyloid precursor, which accounts for their use in Alzheimer’s disease. Due to the pan-inhibition nature of GSIs, adverse effects have been described in clinical trials on advanced tumors, such as gastro-intestinal side effects, resulting from the blocking of the high constitutive expression of Notch in the GI tract. The poor results obtained in clinical trials in terms of tumor shrinkage or overall-survival (OS) might be related both to the absence of molecularly-based inclusion criteria and to the blockade of all four Notch receptors irrespective of their ligands. Indeed, experimental evidence shows that each Notch receptor is involved in specific and different functions. In addition, the downstream effects of Notch activation are strongly influenced by the interaction with specific ligands. Thus, alternative strategies have been considered, such as monoclonal antibodies targeting single Notch receptors or specific ligands. Many reviews have already summarized the current knowledge of Notch signaling in HCC development and have outlined the therapeutic potential of targeting Notch signaling in HCC ^[35][36][37]. Importantly, Notch plays pleomorphic roles in HCC, which relies both on the aberrant expression of specific Notch receptors and on their activation by specific ligands. A precise knowledge on the deregulated expression of each Notch receptor and ligand, coupled with phenotypic changes, is still lacking in HCC. This hampers the development of molecularly-targeted approaches. Notwithstanding, given the relevance of Notch aberrant activation in HCC, also in terms of resistance to treatments, this pathway is very attractive when hypothesizing combined treatment. Thus, in this review we highlighted the mechanisms involved in Notch signaling pathway regulation which might deserve attention when planning therapeutic strategies modulating Notch.