Axon guidance molecules in liver pathology: Journeys on a damaged passport

The liver is an innervated organ that develops a variety of chronic liver disease (CLD). Axon guidance cues (AGCs), of which ephrins, netrins, semaphorins and slits are the main representative, are secreted or membrane‐bound proteins that can attract or repel axons through interactions with their growth cones that contain receptors recognizing these messengers. While fundamentally implicated in the physiological development of the nervous system, the expression of AGCs can also be reinduced under acute or chronic conditions, such as CLD, that necessitate redeployment of neural networks.


| INTRODUC TION: LIVER NEURONS , WOUND HE ALING PRO CE SS E S AND NERVE S PROUTING
Hepatic innervation accompanies and intensifies during embryonic development, 1 leading to a highly innervated organ, the functions of which are modulated by different nerves.Indeed, the liver is innervated by branches of the splanchnic and vagal nerves, which comprise sympathetic and parasympathetic efferent nerves of the autonomic nervous system (ANS), respectively, and sensory afferent nerves. 2 Their branches innervate the parenchyma via the portal triads, running along the hepatic artery, the portal vein, and bile ducts. 1 The involvement of the ANS in physiological regeneration and wound healing has been repeatedly evidenced over the last decades.Interestingly, on a systemic and developmental basis, most nerves are essential for tissue growth 3 and wound healing, [4][5][6][7] and may in the latter case be accompanied by anarchic nerve sprouting and hyperinnervation, determining long-term pathological sensations close to the site of injury, well-known, for instance, after the amputation of a limb.Of importance, liver regeneration is also dependent on neural output. 8Nerves thus influence tissue repair and regeneration, both phenomena largely linked to carcinogenesis if altered or dysregulated.
There is a permanent connection between the brain and the liver via afferent and efferent nerves.The hepatic branch of the sympathetic system regulates glucose release, hepatic regeneration, 9 fibrosis, 10 angiogenesis, 11 and portal venous tension. 12The parasympathetic system controls glucose storage, hepatic metabolism, 2 and inflammation. 13Importantly, it was shown that after partial hepatectomy, liver regeneration was severely impaired in rats following vagotomy (i.e., the removal of part of the vagus nerves). 14Moreover, a correlation between degeneration of sympathetic nerves and nonalcoholic steatohepatitis (NASH), a causative agent for CLD, was reported. 15These findings suggest that at least a subset of liver nerves rely on axon guidance cues (AGCs) as regulating factors for their own redeployment during CLD, thus modulating disease development.
While AGCs are historically known as neural regulators, 16,17 the link between these factors and liver neurons as part of CLD alterations has seldom been highlighted.In addition to improving our current understanding of the implication of AGCs in liver pathology, this review also proposes to consider liver pathologies as potential neuronregulated targetable conditions.We will first tackle notions pertaining to AGCs in neurology and development.We will then consider these proteins in the context of CLD, before describing their role in HCC.Special attention will be paid to distinguishing correlative and causal data in such datasets in order to streamline data interpretation.We will present all past and ongoing clinical trials involving AGCs, few of which are currently conducted on liver, based on the US Clini calTr ials.govdatabase.
Finally, we will try to describe pertinent future research directions in this interesting field.

| Development
AGCs, including ephrins, netrins, semaphorins, and slits (see Figure 1 for structural features), are crucial for the development of neural circuits linking the 80 billion neurons of the human post-natal central nervous system (CNS). 16,17Most AGCs are bifunctional and determine attractive and repulsive events, navigating the axons through pre-existing tissues to find target cells, in health and disease. 18These signals can be soluble or membrane-bound, operating over large or short distances, and are also restricted by interactions with certain components of the extracellular matrix (ECM) or expressed through gradients with likely diverse spatio-temporal dynamics. 16,17Cs also operate outside the nervous system, where they play an important role in cell migration and cell-cell communication during somitogenesis, vascular development, and organogenesis of various organs, including the heart, liver, kidney, lung, mammary gland and bone. 19,20Moreover, as will be presented below, these proteins are also involved in pathological functions, such as reconfiguring the innervation of diseased organs.

| Adulthood
The following sections will initially present the role of the different types of AGCs in adulthood physiology.Their expression ensures neuronal plasticity, and is triggered following nervous system injury in rodents and during neural pathologies in humans. 18However, studies on intracellular pathways have largely focused on neurons, while the intracellular impact of AGCs on non-neural tissues that display distinct kinetics and functioning from neurons warrants further investigation.

Key points
• While AGCs are historically identified as neural regulators, links between AGCs and liver neurons are seldom highlighted for interpretation.
• Expression of AGCs often impact fibrosis and immune processes, is of adverse prognosis in the majority of instances, including HCC, suggesting pertinence for further research and in vivo targeting.
• Several clinical trials are ongoing, including in liver pathology.
• Research perspectives include mechanistic and testing in animal models updated to the currently evolving epidemiology of CLD and HCC.

| Physiological roles of EPHs and ephrins
Ephrins (also known as ephrin ligands or eph family receptorinteracting proteins) are a family of proteins that serve as the ligands of the so-called 'EPH' receptor.The eight ephrin ligands identified in humans are divided into two subclasses based on their structure and linkage to the cell membrane.The five GPI-anchored ephrin-A ligands (ephrin A1-5) interact with nine EPHA receptors (EPHA1-8 and EPHA10), and the three transmembrane ephrin-B ligands (ephrin-B1-3) interact with five EPHB receptors (EPHB1-4 and EPHB6) and one EPHA receptor (EPHA4), providing a rich, uncharted, combinatory repertoire of downstream events.Through the contact between receptorexpressing cells and ligand-expressing cells, ephrins and EPHs are capable of eliciting 'bidirectional triggering' where EPHs mediate forward signalling and ephrins reverse signalling, using a flip-flopped head-to-tail configuration with respect to the cell of interest. 21

| Physiological roles of netrins
Orthologues of netrin-1 play a highly conserved role as guidance cues from archaic bilaterians to humans for midline crossing.Three secreted netrins (netrin-1, -3 and -4) have been identified in mammals, in addition to two GPI-anchored membrane proteins, netrin-G1 and -G2.Of note, netrin-1, -3, and -4 bear a laminin VI domain, a notion of interest with respect to ECM-accumulating properties in CLD.Secreted netrins attract axons via the Deleted in Colorectal Cancer (DCC) family of receptors, including DCC and neogenin, but repulse them through the UNC5 family (UNC5A, UNC5B, UNC5C and UNC5D) of receptors. 22The Down Syndrome Cell Adhesion Molecule (Dscam) and the adenosine A2B receptor have also been implicated as additional netrin-1 receptors.Netrin-G1 and netrin-G2, on the other hand, selectively bind to the transmembrane proteins NGL1 and NGL2, respectively.Netrin-1 is mainly associated with membranes and ECM. 23portantly, with respect to CLD, netrins also influence chemotropic F I G U R E 1 General functional scheme implicating axonal guidance cues (AGCs) as mediators between the central nervous system, the autonomic nervous system (ANS) and the liver.The brain, the ANS and the liver signal both ways through the implication of AGCs as functional hubs enabling redeployment of neurons topography and functions during hepatic wound healing processes in chronic liver disease.

| Physiological roles of semaphorins
Semaphorins (SEMA, 30 members including 21 in mammals) are secreted and membrane-resident proteins that were also originally identified as axon growth cone guidance molecules, signalling attractively or repulsively through multimeric receptor complexes. 27maphorins are divided into eight classes; the first seven are ordered by number, from class 1 to class 7, whereas the eighth group is class V, that is specific to viruses 28 with no obvious link with the liver.Classes 3-7 are found in vertebrates.Each semaphorin is characterized by a specific domain of about 500 amino acids called the 'sema' domain.Semaphorins are ubiquitous 29 and signal through engagement of two main families of receptors, the Neuropilins (NP1 and NP2) and the Plexins (PLEXA1-4, PLEXB1-3, PLEXC1 and PLEXD). 27,29Secreted class 3 semaphorins (except SEMA3E) mainly signal through heterocomplexes of neuropilins and class A and D plexins.Combination of such edifices likely provides specificity for binding and transducing signals from different class 3 semaphorins. 30Membrane-bound class 4-7 semaphorins and SEMA3E bind and directly activate Plexins.The class 7 semaphorin (SEMA7A) can also use integrins as its receptor suggesting its ability to alter cell-ECM crosstalks in ECM accumulating tissues.A variety of neurons, including sympathetic ones respond to semaphorins. 27,29Class 3 semaphorins modulate axon regrowth, re-vascularization, remyelination and the immune response after trauma in the CNS, 31 indicating their potential involvement in other injured innervated tissues, such as the liver.

| Physiological roles of SLITs
SLITS belong to a family of secreted ECM proteins which play an important signalling role in the neural development of most bilaterians (hence including humans), which possess three homologues: SLIT1, SLIT2 and SLIT3. 32SLIT proteins acts as midline repellents, preventing the crossing of longitudinal axons through the midline of the CNS. 33A major feature of SLITS is a β-sandwich domain similar to laminin-G, 34 allowing stable embedding into the ECM, a notion, as for netrins, of interest in CLD.Though non-exclusive, Robos are the principal receptors for SLITs ligands.There are three Robos expressed in neural cells (ROBO1-3), and the fourth Robo receptor (ROBO4) is expressed in endothelial cells.SLIT/ROBO signalling regulates gonadal physiology, pancreatic islet β-cells morphogenesis 35 and function 36 and, of note for this review, peripheral nerve regeneration upon injury. 37portantly, evidence suggests that signalling of ephrin, netrin, semaphorin and slit AGCs intersect to regulate different physiological functions, in organogenesis, but also in angiogenesis, cell proliferation and stem cell regulation, 38 all these functions being relevant to the liver, especially in the context of CLD and HCC.
For several years, different scientific communities have separately been investigating the neural and non-neural roles of AGCs, in the context of development or pathology.Given the richness of the datasets obtained in both settings, it is now possible to provide a more unified view of this knowledge when focusing on a given organ, such as the liver.Accordingly, AGCs would therefore, as many factors high jacked during tumorigenesis, initially play a role as master neural regulators and turn into deleterious signalling cues during CLD and HCC.

| A XON G U IDAN CE MOLECULE S IN CLD
As in many organs during gastrulation, hepatic neurogenesis, although limited in utero, is synchronous to hepatic organogenesis. 1 Hepatic regenerative processes, which are reactivated during CLD and HCC, are functionally related to earlier developmental processes and are accompanied and regulated by neural remodelling. 8,15,39These neural events are known to be regulated by the above mentioned semaphorins, slits, netrins and eph/ephrins [16][17][18] (Figure 2).Hepatic neural ablation highlighted the importance of autonomic innervation in both physiological and pathological contexts, including liver regeneration 2 which further emphasizes the role of neural factors in CLD.Both sympathetic and parasympathetic branches of the ANS influence nutrient metabolism, immune processes, haemodynamics, as well as bloodstream and hormone homeostasis.Their implication in liver repair and regeneration in the context of CLD, including cirrhosis, have been experimentally established. 15,40Indeed, increased sympathetic neuropathy was observed in hepatic steatosis patients, 15 while sympathetic hyperstimulation progressively induced the metabolic syndrome, fatty liver disease and steatohepatitis in mice on normal diet, the latter being reversible by chemical sympathectomy. 41In addition, increased activity of sympathetic fibres in the liver was reported in obese mice fed a high-fat diet, another model of fatty liver disease, and sympathetic disorganization was identified in steatotic mice fed a western diet. 42Interestingly, this sympathetic overactivity was reversible, which suggests the possibility of therapeutic intervention as proposed in the prostate setting. 43patic fibrogenesis is characterized by increased and altered deposition of ECM and represents the most common and pathogenic hallmark of CLD.Although several other cell types may also participate in the onset and development of liver fibrosis, hepatic stellate cells (HSC) are recognized as the primary cells responsible for ECM deposition, and are responsive to neural inputs, 10,44 suggesting a likely contributive if not causal impact of AGCs on the activation of HSCs, perhaps both ways.

| EPHs and ephrins as involved in hepatic viral and pro-fibrotic events
Although mostly expressed during development, the ephrin-A/ephA duos are reactivated in adulthood in pathological contexts.Indeed, ephrins and ephs regulate fundamental biological processes, such as cell migration, myofibroblast activation, angiogenesis and tissue remodelling, which are involved in fibrosis.Increased expression of ephrin-B2 was observed in patients with cardiac, 45 kidney, 46 lung, 47 skin 48 and in malaria-and CCl 4 -related liver fibrosis. 49,50In the liver, platelet-derived growth factor signalling through ephrin-B2 encourages hepatic angiogenesis. 51Ephrin-B2 was shown to modulate HSC activation in vitro and enhance pathological sinusoidal remodelling and portal hypertension in vivo by stimulating VEGF production by HSC. 49,50Concomitantly, downregulation of ephrin-B2 in patients with nodular regenerative hyperplasia of the liver was observed. 52ese data introducing a possible proliferation repressive activity of this protein.5][56] Finally, ephA2, regulated transcriptionally via the receptor TR4, 57 was identified as a host factor for the entry of hepatitis C virus, 58 providing evidence of the important role of this type of protein in chronic hepatitis C development, a major aetiology of CLD.Conversely, neural consequences of the increased occupancy rate of ephA2 by HCV particles are unknown.

| Netrin-1 as a near universally induced AGC in CLD
Netrin-1, besides its AGC status, was also characterized as interacting with a dependence receptor in cancer, that is a receptor transducing death signals when left unbound. 59Several studies document the equivocal role of netrin-1 in the liver.Netrin-1 is the sole netrin member for which information is found in CLD.In primary biliary cholangitis, NTN1 is correlated with immune response pathways that influence pathogenesis of primary biliary cholangitis, 60 though perturbation data were lacking in this study.In patients, netrin-1 was F I G U R E 2 Structural features of human axonal guidance cues.Ephrins-A and -B encode for a receptor-binding domain linked to a GPI anchor or a transmembrane domain, respectively, ensuring their attachment to the plasma membrane.Secreted netrins encode for a laminin domain followed by three EGF-like repeats upstream of an NTR domain, endowed with zinc metalloproteinase inhibitory function.Vertebrate semaphorins encode for several common sequences across all types: their specific Sema domain linked to a PSI domain, a cysteine-rich module frequently found in extracellular fragments of signalling proteins, itself attached to a immunoglobulin-like C2-type domain.Other domains that are closer to the plasma membrane foster attachment to the 'mother' cell in most cases.SLITs encode for several leucine-rich repeats prior to several EGF-like repeats in which is intercalated a laminin G-like domain.A C-terminal cystine knot domain terminates the chain.upregulated and its receptor UNC5A was downregulated in HBVand HCV-positive hepatic lesions at all stages of fibrosis and in HCC, 61,62 providing HCV with a feedforward loop, beneficial for both the virus and the related hepatitis. 61A study showed the liverprotective and anti-inflammatory action of the neuroimmune netrin-1 through its receptor A2B. 63 Netrin-1 also attenuated hepatic steatosis via UNC5b/PPARγ-mediated suppression of inflammation and ER stress. 64In non-steatotic conditions, hepatic inflammation triggered by viral and bacterial motifs elicits production of netrin-1 through an atypical mechanism, that is exclusive activation of translation, where netrin-1 displays macrophage-dependent proinflammatory activity. 65Netrin-1 also protects hepatocytes against cell death through sustained translation during the hepatic unfolded protein response (UPR), 66 a hallmark of CLD that fosters, on a longterm basis resistance to cell death.Consistently, and in line with its neurotrophic status, netrin-1 promotes liver regeneration possibly by facilitating vagal nerve repair after partial hepatectomy in mice. 67,68While netrin-1 seems to be induced, in several instances translationally through STAU1 and LARP1, 61,65 in a variety of liver conditions and in vivo models, it is still challenging to unequivocally assign to netrin-1 a specific impact on a chronic basis on liver histology itself, aside from its anti-apoptotic and regenerative roles, which could have carcinogenic consequences in genetically unstable and histologically injured livers.While in rare cases formally introduced as AGC in liver studies, 63 hepatic netrin-1 has to date mainly been considered as a mere pro-survival agent, following cancer-oriented views.

| Semaphorins as fibrotic regulators in CLD
Causal hepatic data on semaphorins are scanter than those on netrins, albeit in favour of a pathogenic role in CLD.SEMA3C, a new marker of HSC activation, was overexpressed in fibrotic patient livers and exacerbates TGFβ-mediated myofibroblast activation and liver fibrosis in vivo. 69In addition, inhibition of its receptor NRP2 reduced liver fibrosis. 69Interestingly, serum concentrations of SEMA3C, SEMA5A and SEMA6D were associated with pathogenesis of viral hepatitis and progression of fibrosis in HCV genotype 1 and 3-infected patients.Moreover, causal status of viral replication was assessed after antiviral treatments, and verified. 70SEMA3C and SEMA5A were overexpressed in HCV cirrhotic liver and HCC tissues. 70Of note, SEMA3E was showed to play a causal role in HSC activation, LSEC pathological regeneration, and the progression of liver fibrosis in CCl 4 -treated mice. 71Finally, SEMA7A was correlated with hepatic steatosis 72 and evolutive fibrosis in mice and patients. 73

| Slits as profibrotic or angiogenic factors in CLD
SLIT2/ROBO1-2 couples were shown to be overexpressed in patients with liver fibrosis.ROBO2 was detected in septa of fibrotic livers and, interestingly, on the surface of HSCs in experimental models of fibrosis in vivo, where SLIT2 increases the expression of pro-fibrotic mediators, such as TGF-β1, CTGF, as well as collagen-1, a major ECM component accumulating in CLD, in vitro 74 and in vivo. 75IT2, which activates HSCs in vivo, 76 also mediates profibrotic and angiogenic effects of liver myofibroblasts, the conditioned medium of which promoted survival of HSCs and tubulogenesis of endothelial cells. 77Ductular reaction that promotes periductular fibrosis and inflammatory cell recruitment in CLD, also enhances intrahepatic angiogenesis through SLIT2-ROBO1 signalling. 78Of note, as was the case with netrin-1 on inflammation 65 and SEMA3E on fibrosis, 71 an anti-ROBO1 neutralizing antibody was reported to inhibit the progression of liver injury and fibrosis in the mouse CCl 4 model. 75summary of the functions of axon guidance molecules in liver pathology is provided in Table 1.Such data were enriched by a search performed on single cell RNAseq and IHC databases, indicating frequent RNA expression in fibroblasts (likely activated HSCs) and antibody-validated evidence for protein expression (Table 2).
Of note, despite the wealth of available data described above, regulation of AGC transcripts was found limited to SEMA4C only upon comparison between chronic hepatitis and normal livers (GSE89377 dataset, gathering a continuum of samples from normal liver to HCC), suggesting that frequent and unadressed post-transcriptional regulations may account for this unexpected result.Taken together, datasets pertaining to all four classes of AGCs highlight their HSC activating and ECM participating roles, and prompt for further investigations on their targetability in CLD in the clinic.

| A XON G U IDAN CE MOLECULE S IN H CC
AGCs triggered interest as cancer contributors and anticancer targets for the first time in the years 2000.Many AGCs were also shown to control the development of the vasculature and may thus control angiogenesis in tumours, 96 even prompting a general review on the topic, entitled 'The brain within the tumor'. 97Given their implication in physiological and pathological conditions in various organs, neuronal guidance molecules have thus been the focus of research in oncology, 98,99 as well as in HCC through the studies depicted below.
However, as already mentioned, their original functions remain seldom considered in this field, in which theoretical prerequisites linking AGCs to cancers tend to be more directly cell death-related. 98

| EPHs and ephrins as HCC aggressiveness factors
The 'eph' receptor tyrosine kinase was identified from a HCC cell line in 1987. 100EphA1, subject to ADAM12-mediated cleavage, 101 and ephA2 were found overexpressed in HCC patient samples and associated with the absence of tumour capsule, portal vein invasion, lower differentiation, 63 advanced TNM stage and poor prognosis of HCC 102 as did ephrin-A3. 82Ephrin-A1 expression gradually increases from normal liver to cirrhosis to HCC.It is strongly correlated with α-foetoprotein (r = 0.87, a value rarely encountered in pathology). 79Ephrin-A1 also contributes to several malignant characteristics of HCC cells. 79Ephrin-A1-induced EphA1 activation was reported to promote SDF-1 secretion and chemotaxis of endothelial progenitor cells to HCC through the SDF-1/CXCR4 signalling pathway.Perturbation studies abolished tube formation in vitro and decreased tumour size and angiogenesis due to inhibition of endothelial progenitor cell homing to the tumour tissue. 80hrin-A1/ephA1 inhibition in vitro attenuated neo-angiogenesis and tumour growth in HCC in vitro and in vivo. 80The ephrin-A3/ ephA2 axis is known to regulate cellular metabolic plasticity to enhance cancer stemness in hypoxic HCC.Mechanistically, the ephrin-A3/ephA2 axis promote maturation of the SREBP1 transcription factor.Expression of its target, ACLY, was significantly associated with the expression of ephrin-A3 and hypoxia markers in clinical cohorts. 82Conversely, the TR4 nuclear receptor suppresses HCC cell invasion via downregulating EphA2 expression. 85Analysis of tumour tissue from 304 patients with HCC having undergone surgical resection generated a methylationbased prognostic signature in which ephrin-B2 was also considered a candidate 'epi' driver of HCC, 103 although causality was not checked.Ephrin-A2 promotes tumorigenicity through the Rac1/ Akt/NF-κB signalling pathway 81 and is a potential biomarker for HCC. 63Ephrin-A5, conditioned by miR-96 and -182 regulation, 104 participates in a kinase complex that sustains the viability of HCC cells through downstream protein kinase B-dependent, extracellular signal-regulated kinase-dependent, and p38-dependent signalling pathways. 83Peritumoral small ephrin-A5 isoform level predicts postoperative survival in hepatocellular carcinoma, 105 suggesting that subtle isoform complexities may enrich the diversity of actions of AGCs.Finally, expression of ephrin-B1 in HCC cells enhanced in vivo aggressiveness and endothelial migration and proliferation in vitro, 84 an interesting finding due to HCC's targetability by VEGF inhibitors and susceptibility to microvascular invasion, a strong and adverse event.

| Netrin-1 as an anti-apoptotic factor in HCC
Netrin-1 is the sole netrin member for which information is found in HCC.Interestingly, data featuring cirrhosis-and HCC-associated depletion of the netrin-1 and the death receptor Uncoordinated Phenotype-5A (UNC5A) 62 also featured its causal role in skewing the hepatic UPR towards pro-survival outcomes. 86Netrin-1 promotes the collective migration of liver cancer cells in a 3D cell culture model 87 as well as the promotion of cell migration and invasion by down-regulation of BVES (a novel adhesion molecule enabling tight TA B L E 2 AGC/AGC receptors expressing cell types and their regulation from cirrhosis-to-HCC.Data were retrieved from the Single Cell RNAseq portal (Broad Institute), the ProteinAtlas database (Karolinska Institute) and the publicly available GEO bulk RNAseq dataset GSE124535 (n = 35 F4/HCC pairs, Log 2 fold-changes of TPM normalized values are shown after GEO2R analyses using default settings).For IHC, ProteinAtlas staining certainty scores increase from uncertain to supported to approved to enhanced.Although some occasional data pertaining to netrin-1 in cancer may identify this factor as endowed with anticancer properties, 107 all of the literature in the liver indicate that netrin-1 and its receptors (through their inhibition 59 as dependence receptors) promote or aggravate the pathogenic features of HCC cells.Nevertheless, to our knowledge, documented clinical correlative data and in vivo causal data determining the actual ability of netrin-1 to trigger similar phenotypes are to date lacking.

| Semaphorins as pro-or anti-HCC players
Apart from ephrins and netrin-1, semaphorin-3A seems to represent the only well documented pathogenic type of semaphorin with respect to HCC.However, other semaphorins such as semaphorin-3B and 3F seem to bear antitumoral properties, suggesting functional switches between them during disease progression.The semaphorin receptor neuropilin-1 (NRP1) is upregulated in HCC compared to normal liver tissue in a miR-148b-dependent manner. 108To evaluate the therapeutic potential of targeting NRP1 in HCC, SV40-driven HCC transgenic mice were treated with peptide N, an NRP1-binding recombinant protein and competitive inhibitor, enabling the authors to demonstrate that this semaphorin receptor promotes tumour growth and vascular remodelling. 93The expression of SEMA3A was also elevated in HCC patients and conditioned by the microRNA miR-192-5p. 109SEMA3A promotes HCC growth, invasion, and metastasis in vivo, by increasing galectin-3, enolase-2 and EpCAM. 89 another study, SEMA3A was shown to promote tumour progression and was identified as a factor of poor prognosis. 110Importantly, SEMA3A also binds to NRP-plexA receptor complexes on tumourassociated macrophages, guiding them to hypoxic regions of the tumour, where they further promote angiogenesis and tumour growth. 89This phenomenon was notably reversed via SEMA3A RNA interference or macrophage-specific NRP inactivation in vitro and in vivo in lung cancer. 111A study demonstrated that SEMA3D was able to restrain the progression of HCC by inactivating PI3K/AKT signalling. 90In an in silico study using the TCGA cohort, SEMA3F was correlated with invasiveness, metastasis and activation of focal adhesion kinase transcripts, 91 while SEMA4C was instrumental to the HCC-promoting lncRNA CYTOR to exert its pathogenic role. 92

| Slits are candidate contributors to HCC through their ROBO receptors
Few causal orthotopic data exist on SLIT proteins in HCC, prompting the authors to consider their ROBO receptors.ROBO1 is overexpressed in HCC patient samples compared to normal and adjacent peri-tumoral tissues, with a strong enrichment in poorly differentiated HCC compared to well-differentiated and moderately differentiated samples. 112ROBO1 promotes tumour angiogenesis and tumour growth in HCC in vivo and its targeting with a neutralizing monoclonal antibody showed anti-tumour activity in a HCC xenograft model. 94Considering now SLIT, under epigenetic regulation in HCC, 113 and ROBO GTPase-activating proteins (SRGAPs), one study reported their strong correlation with HCC onset in public databases, as well as its ability to trigger EMT in vitro. 95though warranting more basic investigations on the role of AGC in HCC, these informations are in favour of a pathogenic reactivation of AGC-related processes in primary liver cancer.Causally identified functions of axon guidance molecules in HCC are provided in Table 1.Most pathophysiologically relevant data are depicted in Figure 3. Cells of origin of AGCs or of their receptor(s) together with experimental evidence for protein expression and localization in liver tissues are provided in Table 2 using publicly available databases and datasets.

| A XON G U IDAN CE MOLECULE S A S THER APEUTI C TARG E TS IN C AN CER : P OTENTIAL AVEN UE S IN H CC
Analysing cirrhosis/HCC paired tissues, the majority of AGCs and receptors transcripts, except Ephrin-A5, were found up-regulated from F4 to HCC (  Hence, one can propose that in addition to their known impact on tumour cells, HCC data and current clinical trials targeting ephrins, netrin-1, semaphorins and SLITs in other conditions than HCC would also benefit from being reinterpreted in light of their ability to target them as axon guidance molecules in the next future.

| FUTURE PER S PEC TIVE S
Despite growing evidence on the likely pathogenic involvement of guidance cues in the liver, little is known about how they influence their environment.
To provide a better comprehensive overview of AGC impact on

14783231, 2023, 9 ,
Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/liv.15662by Cochrane France, Wiley Online Library on [20/11/2023].See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions)on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensejunction formation) expression in human HCC cells.106Finally, while able to protect hepatocytes from cell death upon the UPR, a HCC relevant phenomenon, as depicted earlier, 66 netrin-1 also induces epithelial-mesenchymal transition and promotes invasiveness of HCC cells 88 in a context where the epidermal growth factor receptor (EGFR) is also activated by netrin-1 following HCV infection.61 solid tumours, NCT02575261 in glioma patients, NCT00364780 in lung cancer patients), netrin-1 (NCT02977195 in another panel of solid tumours) and semaphorins (NCT04573543, NCT02820285 in NASH) have been considered as potential targets in several clinical trials.SLIT targeting does not seem to have entered clinical trials so far.The reason for which no data pertaining to clinical trials on most axonal guidance cues and HCC could be identified so far is unknown.
fibrosis, CLD-to-HCC transition and HCC progression a panel of studies will be needed to (i) elucidate the ligand/receptor couples at play in various pathological contexts, (ii) identify the cell types targeted by the different cues as well as their cell of origin, (iii) assess the consequences of changes in their expression in in vivo models pertinent to the current changes in the epidemiology of CLD, (iv) determine their cognate signalling pathways, and (v) examine the crosstalk between the main representatives of the four axon guidance families.Interestingly, most ligands and receptors show prognostic value.Therefore, they should be studied in large cohorts of CLD and HCC patients, in correlation with disease stage, liver function and clinical outcome, if possible at the protein level given the secreted character of ligands and hence their likely post-transcriptional regulation.Datasets described herein indicate that most axon guidance molecules are likely pathogenic in CLD and HCC.Linking their historical functions of neural regulators and spatiotemporal orienteers of neurons to the phenotypes caused by these proteins in the liver may open new avenues for more integrated research.This review raises questions such as: Do AGCs mediate or dampen neural control of the diseased liver?Is this neural control pathogenic or compensatory with respect to liver disease progression?What are the neural targets of each class of AGCs-namely, do they preferably interact with sympathetic or parasympathetic branches of the liver ANS? Potentially novel therapies targeting either axon guidance ligands or receptors are mostly being studied at early clinical phases I and II, evaluating their tolerance and safety profile.More data are therefore expected to arise from such studies focusing on these intriguing molecules that constitute the interface between neurons and their liver parenchymal targets, which are seldom recognized as such in hepatology.AUTH O R CO NTR I B UTI O N S Ievgeniia Chicherova provided initial drafts of the main text and of some figures.Charlotte Hernandez enriched the manuscript and F I G U R E 3 Main HCC-relevant processes regulated by axonal guidance cues.14783231, 2023, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/liv.15662by Cochrane France, Wiley Online Library on [20/11/2023].See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions)on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License F I G U R E 4 Prognostic orientation and associated value of axonal guidance cues (AGCs) transcripts in HCC TCGA RNA samples and Protein Atlas data samples.(A) AGCs and and AGCs receptors transcripts prognostic values at 5-year mortality rate (FPKM levels, Log-rank test, p < 0.05, derived from TCGA data).(B, D) Distribution of unfavourable and favourable prognostic markers.(C, E) Associated degree of certainty (p value) of favourable and unfavourable AGCs.Were included only statistically significant markers.Panels (B-E) were derived from www.prote inatl as.org (accessed 7 February 2023).
Functions of AGCs in liver pathology.

AGC and receptor names Positivity by ssRNAseq in CLD Evidence for protein signal in HCC Frequency of medium or strong staining in HCC by IHC Expression changes (F4 to HCC) Log 2 FC; p adj < 0.05
Abbreviations: AGC, axonal guidance cue; HCC, hepatocellular carcinoma; ns, non-significant.