The Neuropeptide System and Colorectal Cancer Liver Metastases: Mechanisms and Management

Colorectal cancer (CRC), classified as the third most prevalent cancer worldwide, remains to be a clinical and research challenge. It is estimated that ~50% of CRC patients die from distant metastases, with treatment of this complication still posing significant difficulties. While liver metastasis (LM) cascade is known in the literature, its mechanisms are still unclear and remain studied in different research models. A connection is suggested between nervous system dysfunctions and a range of Neurotransmitters (Nts) (including Neuropeptides, NPs), Neurotrophins (Ntt) and their receptors (Rs) in CRC liver metastasis development. Studies on the role of NP/NP-Rs in the progression and metastasis of CRC, show the complexity of brain–tumor interactions, caused by their different forms of release to the extracellular environment (endocrine, autocrine, paracrine and neurocrine). Many stages of LM are connected to the activity of pro-inflammatory, e.g., Corticotropin-releasing Hormone Receptor 1 (CRHR1), Neuropeptide Y (NPY) and Neurotensin (NT), anti-inflammatory, e.g., Calcitonin Gene-related Peptide (CGRP), CRHR2 and Vasoactive Intestinal Polypeptide (VIP) or dual role neuropeptides, e.g., Substance P (SP). The regulation of the local immunological profile (e.g., CRH/CRHRs), dysfunctions of enteroprotective role of NPs on epithelial cells (e.g., NT/NT-R), as well as structural-functional changes in enteric nervous system innervation of the tumor are also important. More research is needed to understand the exact mechanisms of communication between the neurons and tumor cells. The knowledge on the mechanisms regulating tumor growth and different stages of metastasis, as well as effects of the action of a numerous group of Nts/NPs/Ntt as growth factors, have implications for future therapeutic strategies. To obtain the best treatment outcomes, it is important to use signaling pathways common for many NPs, as well to develop a range of broad-spectrum antagonists. This review aims to summarize the current knowledge on the importance of neuroactive molecules in the promotion of the invasion-metastasis cascade in CRC, as well as the improvements of clinical management of CRC liver metastasis.


Introduction
Colorectal cancer (CRC), classified as the third most prevalent cancer worldwide, after lung and breast/prostate cancer, is also the second leading cause of cancer-associated death [1,2]. Among all the cancers diagnosed in Europe, CRC is the second most widespread (after female breast cancer), also being the second most common cause of cancer-associated death (after lung cancer) [3]. Around 25% of CRC patients exhibit synchronous liver metastases (LM), with around 60% developing metachronous LM during the disease [4][5][6]. The liver is the most common metastasis location in CRC [7][8][9][10] and, most importantly, often the only metastasis-affected site [11]. LM in CRC, as a usually asymptomatic differential expression of the type-1A angiotensin II (ANG II) receptor gene (Agtr1a), which causes a significant increase in plasma levels and in vivo production of GLP1, and PYY release in response to their stimulation with ANG II [61].

Alterations in Large Intestine Innervation during CRC
There is evidence of a direct link between the nervous system and cancer through synapses, non-synapse contacts, or humoral modulation, which contribute to two-way communication and influence cancer metastases. Similar to nerve structures, cancer cells produce Nts/NPs and their receptors [62,63]. In CRC patients, structural and functional changes of large intestine innervation can be observed. Interestingly, in the CRC LM, contrarily to the healthy liver, a lack of autonomic perivascular Protein Gene Product 9.5. (PGP9.5)-and Neuropeptide Y (NPY)-immunoreactive nerves can be observed [64].

Morphological Changes in Innervation and Neuropeptide Panel in CRC
Structural changes in CRC innervation mostly concern ENS, occurring most commonly in the form of gradual reduction, leading to the total destruction of the nerve structures [65][66][67]. Atrophy of submucosal and MPs within close proximity to the tumor occurs [67,68]. Among the NPs, a decrease in CGRP+ neurons and nerves was observed in both plexus types in the transitional zone between cancerous area and unchanged tissue. The decrease also concerned SP+ nerve fibers in all intramural plexuses [65] and NPY-ergic neurons, as well as the density of nerve fibers in both plexuses [66]. Interestingly, there were no significant quantitative differences in the numbers of SP+, SM+, Vasoactive Intestinal Polypeptide (VIP)-ergic and Pituitary Adenylate Cyclase-activating Peptide (PACAP)-ergic neurons, as well as SM+ nerve fibers in cancer, compared with healthy regions [65,66]. Lower numbers of VIP-ergic and PACAP-ergic nerve fibers were observed in submucosal and MPs than in control sections [66]. In turn, an unchanged density of galanin (Gal)-positive nerve fibers was observed, while the percentage of Gal+ neurons was higher in CRC (46%) than the healthy intestine (35%) [68]. A reduction in the size of Gal+ MPs in the vicinity of the tumor was also reported, as compared with unchanged tissue [67]. Mean Gal content in tumor was lower (9.38 ng/g) than in the morphologically unaltered intestine (12.27 ng/g) [68]. Ultrastructural changes in CRC patients include an increase in the mass of extracellular matrix (ECM), occurrence of myelin-like structures, numerous apoptotic cells, as well as the presence of mast and plasma cells in MPs in the tumor surrounding area [69].

The Perineural Invasion (PNI) in CRC
There are ongoing studies on the involvement of perineural invasion (PNI) of cancer cells in the modulation of tumorigenesis [62,63,70]. PNI might be an underestimated mode of metastasis spread, acting in combination with lymphatic and vascular invasion [70][71][72][73], as well as on its own [70]. In CRC,~16-40% of the patients exhibited PNI characterized by neoplastic invasion of nerves, with altered molecular determinants of the process [70]. It is debated if nerve ablation can delay/inhibit the formation of tumors and/or reduce metastaticity [63].
The markers closely associated with PNI include Nts (e.g., ACh, NE and their receptors: AChR, NE-R), Ntt (NGF, Brain-Derived Neurotrophic Factor (BDNF), Glial Cell line-derived Neurotrophic Factor (GDNF) and their receptors: Neurotrophic Receptor Tropomyosin-related Kinase B (TrKB)), as well as typical NPs (e.g., SP, Gal, NPY/CGRP) [63,70]. PNI is a multistep process, during which a major role is played by the so-called perineural niche, together with numerous signaling molecules (including NPs/NP-Rs) [70]. There is a lack of detailed studies on the mechanisms of nerve-tumor interactions in PNI in CRC, as most of the research concerns different types of cancer (e.g., prostate and gastric cancers, pancreatic ductal adenocarcinoma) [63,70]. However, a prognostic role of PNI was proven in CRC. Defining PNI as a presence of cancer cells inside the nerve sheath, or at least 33% of the nerve periphery surrounded by cancer cells in CRC, shorter 5-year survival rates were observed compared with negative PNI. Additionally, positive correlations between PNI and lymph node metastases, tumor grade depth of invasion, clinical-stage, vessel invasion and tumor growth pattern were observed [74]. PNI was indicated as an independent bad prognostic factor in CRC [70,72,74], affecting both overall survival (OS) [74], cancer-specific survival (CSS) and disease-free survival (DFS) [72]. PNI is also an independent factor in CRC recurrence, points to a more malignant tumor phenotype and, as an important parameter, should be considered in pathological classification of CRC [70]. Recently, a large cohort study indicated that PNI is also more commonly observed in colitis-associated (90%) than in sporadic CRC [73].

Functional Innervation Disorders in CRC
Functional disorders in CRC and colitis concern mostly changes in interactions between large intestine innervation and the immune system [52,67]. Interestingly, such alterations occur on the level of NP-Rs, present on most of the immune cells. Anti-inflammatory roles of VIP and CGRP, as well as pro-inflammatory effects of serotonin and NPY, are also often underlined. In turn, SP has both anti-and pro-inflammatory effects. Apart from neurons, the production of Nts: ACh, choline acetyltransferase (ChAT), acetylcholinesterase, and both muscarinic/cholinergic and nicotinic ACh receptors, was also demonstrated on numerous immune cells (e.g., T and B cells, dendritic cells, macrophages), potentially extending the anti-inflammatory action of ACh in the large intestine [75].
Influence of some ANS Nts (e.g., NE, ACh) and their co-transmitters (e.g., NPY, adenosine triphosphate and/or VIP) on the proliferation of Intestinal Epithelial Stem Cells (IESCs) is also often underlined, despite little knowledge on the mechanisms of that process [52,76]. It seems that regulation of IESCs proliferation occurs with the participation of both branches of ANS, independently of ENS. Due to more numerous IESCs in the deeper regions of intestinal crypts, SNS and Nts can regulate the proliferation of these cells. ACh is also a PNS mediator, initiating a signaling cascade resulting in suppression of cyclin D1 and a downstream decrease in cell proliferation [76]. The role of ANS-IESC interactions is also considered in the context of differentiation of some kinds of colon cancers from somatic SCs, as well as maintenance of IESC-like properties under neoplastic conditions [76,77].

NPs and Their Mechanisms of Action in Pre-Cancerous Alterations and Colonic Inflammation
Up to 3% of CRC arise as a consequence of IBDs [78]. The source of many acute and chronic IBDs, caused by disorders of the brain-gut mucosa axis, are stress factors [50,52,58,79,80]. The main effects of stress on intestinal physiology are already the subject of many excellent reviews [62,80] and the participation of NPs, e.g., Corticotropin-releasing Hormone/Factor (CRH/CRF), NT, SP and VIP, in the pathogenesis of IBD is underlined [52,58,79,[81][82][83][84].
The role of NTs/NPs in pathogenesis of inflammatory-associated CRC was also proven [52,62,75,[84][85][86][87][88][89]. It needs to be noted that the action of NPs produced typically in the upper GIT portions (e.g., CGRP, NT, SP, VIP), studied in different models of colitis or on non-transformed colonocytes, can have both pro-inflammatory (NT, NPY, SP) and anti-inflammatory (CGRP, VIP) effects [52,89]. The role of fat-colonic mucosa interactions in IBD, with the participation of SP and its receptors: neurokinin 1 (NK-1R) and NK-2R, is described by some interesting research examples. An increase in production of mRNA of both SP receptors was observed in IBD mesenteric fat preadipocytes, while the levels of SP mRNA rose in ulcerative colitis (UC) preadipocytes. Moreover, the action of SP via NK-1R resulted in a release of interleukin (IL)-17 in Crohn's disease (CD) and UC preadipocyte and IL-17R in IBD colon biopsies [83].
An especially important role in the development of acute intestinal inflammation is attributed to the neurotensinergic system (NT/NTSRs) [58,[87][88][89][90][91][92]. Studies of the mechanism of NP action in colonic inflammation point out that both NT [87] and SP [86] mediate the acute phase of intestinal inflammation in vivo, inducing production of pro-inflammatory IL-8 cytokine by colonocytes. Furthermore, NT stimulates the expression in mesenteric fat depots [89]. An increase in NT/NTSR1 mRNA was also detected in the mesenteric fat of mice with chemically induced colitis [93]. The actions of SP and NT occur through a mechanism of phosphorylation and degradation of Nuclear Factor-kappaB (NF-κB), as well as phosphorylation of p65, with the participation of conventional Protein Kinase C (PKC) [87], or in the case of SP-isoform PKC delta [86]. It was also proven that the pro-inflammatory signaling pathway mediated by NT/NTSR1, with the participation of Matrix Metalloproteinase (MMP)-mediated release of Tumor Necrosis Factor (TNF)-α, transactivates Epidermal Growth Factor Receptor (EGFR), followed by the Mitogen-activated Protein Kinase (MAPK, MAP) pathway, increasing the expression of IL-8 in human non-transformed colonic epithelial NCM460 cells [90]. Studies on an animal model (rats) reported that NT, as a pro-inflammatory factor in the large intestine, also takes part in SP-dependent mast cell degranulation and is a key NP in the pathogenesis of Clostridium difficile-induced colonic inflammation [85,90]. Other research points to transactivation of the Insulin-like Growth Factor Receptor Type I (IGF1R) pathway by NT, resulting in combined effects of serine-threonine protein kinase AKT (AKT1) phosphorylation and NF-κB activation. These pathways participate both in pro-inflammatory and tissue repair signaling in colonic epithelial cells and can also play a role in IBD pathogenesis [94]. NT participates in the regulation of various enteric interactions with other systems, such as the CNS and neuroendocrine system. In intestinal carcinogenesis, the enterotrophic and enteroprotective effect of NT/NTSR system dysfunction on epithelial cells was also considered [89].
An important role in chronic stress and regulation of persistent inflammation, hence colon carcinogenesis, is played by the CRH (CRF) family of NPs. These group of peptides also includes urocortin I (Ucn1), II (Unc2), III (Unc3), CRH-binding protein (CRH-bp) and two distinct CRH receptors (CRHR1 and CRHR2), belonging to the GPCRs family [79,82,84]. Local expression of all of the CRH family components was observed in normal and inflamed colon and rectum. It was mostly localized in cells of MPs, submucosal ganglia, glial cells and nerve fibers, as well as non-neuronal components (e.g., smooth muscle cells, endothelial cells (ECs), mononuclear cells of lamina propria mucosae, and goblet cells in colonic epithelium). The role of the CRH family in the inflammatory process in vivo imitates changes resulting from stress exposure (e.g., colonic transit, motility, proto-oncogene Fos expression in myenteric neurons, visceral hypersensitivity and defecation), while treatment of in vitro cultured cells with CRF peptides confirms their participation mostly in secretomotor and mucosal alterations [79,84]. The mechanism of CRH family action is mostly based on the regulation of the local immunological profile [84]. Many authors point at the dual role of the CRH-CRHR signaling pathway in colon cancer tumorigenesis, progression and metastasis, with the role of both pro-and anti-inflammatory pathways [95][96][97][98]. CRHR2/Ucn2 signaling inhibits tumor growth and metastasis through downregulation of endogenous IL-6/IL-6R expression, reduction of STAT3 phosphorylation mediated by this cytokine, as well as inhibition of STAT3 in CRC cells, resulting in a block of expression of genes regulated by STAT3, as well as inhibition of cell cycle and EMT [96,98]. Additionally, the expression of Fas ligand in CRC cells was correlated with a loss of CRHR2 mRNA, poor tumor differentiation and high risk for distant metastases (including LM) [96]. CRHR2/Unc2 signaling turned out to also be a negative regulator of cell resistance to Fas/FasL-apoptosis in CRC via targeting the miR-7/YY1/Fas [98]. More importantly, activation of the CRH/CRHR1 most often leads to the promotion of inflammation, and upregulation of CRHR2-to an opposite response (anti-inflammatory). The pro-inflammatory action of the CRH system in the intestine occurs through a direct influence on immune cells (adaptive immunity), as well as cytokine production. The anti-inflammatory actions of CRH, Ucn1 and Ucn2 may also occur as a result of Toll-like Receptor 4 (TLR4) expression regulation, as is a part of the innate immunity [84].

Proliferation, Migration, and Invasion of CRC cells
The first step in the invasion-metastasis cascade is the local invasion of tumor cells into the surrounding matrix, with the last step being the colonization of distant organs [21]. Uncontrolled proliferation, excessive migration and invasion of cancer cells are critical phenomena in the first step of CRC metastasis. Only a limited number of cancer cells can migrate to vital organs (e.g., liver, lungs and brain). Hence, the study of factors and mechanisms affecting the metastatic potential of these cells is also of great importance in relation to CRC LM [99].

Neurotransmitters (Nts)
Among the classic Nts, the most studied for a potential role in the first step of CRC metastasis are: acetylcholine (ACh), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT). The promigratory effect of NE on the migration of the human SW480 colon carcinoma cells (CCcs) was mediated by numerous β2-adrenoceptors (β2-AR) via tyrosine kinase activity [100]. In the case of ACh, increased proliferation of CCcs was mediated by transactivation of EGFRs, as well as through MRs (CHRM) [99,101,102]. Particular attention is paid to the role of the CHMR in CCc proliferation, survival, migration, invasion and metastasis. Detailed studies on H508 CCcs (expressing solely M3Rs) and SNU-C4 (expressing solely EGFR) showed that the mechanisms of this transactivation, resulting in increased proliferation of CCcs, are based on MMP7-catalyzed release of Heparin-binding EGF-like Growth Factor (HB-EGF). ACh-induced activation of EGFR and downstream Extracellular Signal-regulated Kinase (ERK) signaling also regulates transcriptional activation of MMP7 [101]. Other research reports that incubation of H508 cells with ACh resulted in a three-fold increase in cell migration, similar to that invoked by EGF action. Furthermore, ACh-induced HT-29 cell invasion was blocked by atropine [99]. Studies of CHRM agonist action in HT-29 and H508 cells, and in vivo models, proved their common functional interactions with CHRM through the stimulation of MMP1 expression, and deoxycholyltaurine-induced cell invasion [102]. Later research also indicated a role of ACh in post-M3R signaling pathways, increasing MMP1 expression and driving CCcs invasion. It was shown that ACh stimulated robust phosphorylation of p38 MAPK, which was EGFR-independent and could be blocked by PKC-α inhibition [103].
It has been reported that 5-HT is a key mediator of the growth of colon carcinoma allografts in vivo [104]. Studies on 5-HT receptor 3A (HTR3A) knockdown in the six CCc lines showed inhibition of their proliferation and colony formation, resulting in cell cycle arrest and the promotion of cell apoptosis [105]. Other studies confirm the role of serotonin and its receptors (5-HT(1B), 5-HT(3), 5-HT(4)) in enhancing CRC growth. Serotonin and its receptors' agonists increase proliferation and growth of cells, and with the use of selective antagonists, inhibits proliferation and promotes HT-29 cells apoptosis [106,107]. In relation to mechanisms connected with CRC distant metastases, it was reported that tumor invasion occurs through the activation of the Axin1/β-catenin/MMP7 signaling pathway and 5-HT(1D)R. This receptor, by targeting Axin1 and dissociated β-catenin from the complex, activated β-catenin/Lymphoid Enhancer-binding Factor 1(LEF1)/T-cell Factor 4 (TCF4)/MMP7 signaling in CRC metastasis. Activation of 5-HT(1D)R directly induces cell invasion and migration both in vitro and in vivo, while its inhibition has a potent anti-metastatic effect via the Wnt pathway [108].

Neuropeptides
Angiotensin II (ANG II) In vitro research indicated promotion of growth, invasion and anti-apoptotic effect after the stimulation of CCc lines with ANG II (HT-29 and CT26) or angiotensinogen (ATG) (HT-29). It was also proven that hyperglycaemia induced ANG II activation in CCcs. Additionally, it was observed that a decrease in ATG production in the liver, mediated by cholesterol-conjugated antisense S-oligodeoxynucleotide, suppressed LM of HT-29 cells [24]. Other authors showed an increase in migration of other human CRC cells (DLD-1 and LIM2405) as a result of ANG II action via both receptor types (AT1R and AT2R) [109].

Endothelins (ETs)
Endothelins (ETs) include ET-1, ET-2, and ET-3, ET-A and ET-B receptors (ETAR and ETBR) (GPCRs family) with clearly defined functions, and upstream processing enzymes such as ET converting enzyme (ECE) [110]. ET-1 is an NP in the human colon with binding sites on neural plexuses and mucosa. In the 1990s, specific ET-1 binding sites were localized in the stromal tissues, including tumor vessels, fibroblasts and nerve fibers in human CRC. Hence, it has been suggested that ET-1 might play the roles of both mitogens and neurotransmitters through paracrine action [111]. For many years, ET-1 was investigated in the context of its impact on cancer progression (including CRC), as well as a potential antagonist of ET receptors in the therapy of various diseases [112,113]. A role of the ET/ETAR axis was also proven in the process of cancer spread and metastasis (including CRC LM) [112,114,115]. ET-1 production was detected in many human CCc lines [116]. In a rat LM model, a role of this NP in the promotion of tumor growth via ETAR was reported [117]. In vitro research (SW480 and SW620 cells) confirmed the role of ETAR in promotion of CRC LM. The effects of ET-1 on CCc survival, invasion and MMP-2 expression, occur through a phosphatidyl-inositol-3-kinase (PI3K)-mediated mechanism [115]. It has been recently demonstrated that enhanced proliferation, migration and survival of multiple CCcs via ET-1 also occurs through ETAR, in the mechanism of activation of YAP/TAZ, two transcription coactivators of the Hippo tumor suppressor signaling pathway [118].
High expression in the mouse colonic epithelial cells, both in healthy animals and those with experimental-induced colitis, also concerns ET-2. This NP was also observed in nerve fibers and MPs of the muscle layer, co-expressing with VIP [119]. The role of ET-2 and ET-3 in CRC progression is less recognized. However, it was reported that increased expression of both these peptides significantly weakens migration and invasion of human CCcs [110].

Galanin (Gal)
Gal/galanin receptor 1 (GalR1), as a novel receptor-ligand system that regulates CRC cell survival and drug resistance, was reported [120]. It was also suggested that at least a part of CRC cells with high Gal expression is more malignant and probably responsible for tumor recurrence. The positive correlation between high Gal expression and tumor metastasis, together with the aggressive behavior of CRC cells with high NP expression, might indicate the potential role of Gal in the spread of cancer stem cells (CSCs) in stage II CRC [121].

Gastrin/Progastrin
Progastrin (PG), gastrin and CCK act through the cholecystokinin-2 receptor (CCK2R, CCK-BR, CCK-B). Activation of CCK2R by gastrin stimulates a rapid tyrosine phosphorylation of the Focal Adhesion Kinase (FAK) pathway in CCcs (Colo320) [122]. Further studies confirmed the role of CCK2R in the regulation of invasiveness and motility of CRC cells [123]. The mouse research model also showed that autocrine/paracrine secretion of PG can promote proliferation of colonic epithelial cells indirectly due to stimulation of colonic myofibroblasts for production of IGF2 [124]. A pioneering study on the immature PG-derived peptide called Glycine-extended Gastrin (G17-Gly) reported that it can stimulate the invasiveness of CCcs. G17-Gly administration significantly enhanced the LoVo cells migration [125]. Other research isolated a novel splice variant of CCK-BR (CCK-BRi4sv) regulating intracellular free Ca +2 and CCcs proliferation though a gastrin-independent mechanism [126].
The potential role in CRC cell invasion and metastasis was also reported in studies on Colo320WT cells with mature G17. This peptide increased β-catenin expression [127] and activated the β-catenin/TCF-4 pathway, which leads to high expression of c-Myc and cyclin D1 [128]. Stimulation of HT-29 cells by G17 also caused an increase in phosphorylation of ERK1/ERK2 and AKT, increased Cyclooxygenase-2 (COX-2) expression, Prostaglandin E2 (PGE2) production and DNA synthesis, which resulted in cell growth [129]. Enhanced proliferation of colonic cells in vivo by non-amidated G17-Gly, as well as a second immature PG-derived peptide, C-terminal flanking peptide (CTFP), was confirmed in mice model of liver metastasis. However, CTFP does not seem to influence xenograft growth or the incidence of LM [130]. In turn, in the case of mouse colon cancer stem/progenitor cells in vitro, an increased proliferation through PG/G protein-coupled receptor 56 (GPR56) and PG/CCK2R systems was reported [131].
Normal human and mouse colonocytes do not express GRP/GRPR, but both components of this system exhibit differential expression in CRC and CCc lines [133][134][135]142]. GRP alone shows mitogen as well as morphogen activity, whereas the GRP/GRPR system regulates the histological progression of CRC in mice by promoting a well-differentiated phenotype [133]. The dependence of heterochromatin protein 1Hsβ expression on GRP/GRPR signaling was also shown. Inhibition of 1Hsβ caused increased invasiveness of human CCcs [143]. In vitro studies (Caco-2 and HT-29 cells) of the mechanisms of tumor cell metastasis showed that GRP promotes tumor cell motility and attachment to ECM, as a result of upregulation of Intercellular Adhesion Molecule-1 (ICAM-1) via FAK [144]. The mechanism increasing CRC migration via GRP/GRPR activity also includes Gα13-PRG-RhoA-ROCK, as well as Cox-2/PGE2 signaling pathways [145]. The nonamidated derivatives with pro-GRP C-terminus also manifested the pro-proliferative activity in the colonic mucosa in in vitro (DLD-1, HCT15, HCT116, HT-29), as well as in vivo conditions [146].
The Nm family also includes components of the neurotensinergic system (NT/NTSRs), the role of which in various cancer tumorigenesis (including CRC) is undisputed and reviewed [147][148][149][150][151]. The role of this system in CRC LM is less known. However, a role of the endocrine form of NT was described in the stimulation of growth of many CCc lines (SW480, SW620, HT-29, HCT116 and CI.19A), characterized with NTSR1 expression [152]. In HT-29 cells, the involvement of a complex of two structurally different receptors: NTR1 (GPCR family) and NTSR3 (type I receptor with a single transmembrane domain), was proven in the modulation of the MAPK pathway after NT stimulation and phosphoinositide (PI) turnover mediated by the NTSR1 [153]. Interestingly, it was proven in HT-29 and HCT116 cell lines that NT stimulates MAPK phosphorylation and cell growth through a pathway which does not involve EGF and IGFR systems [154]. Wang et al. showed that administration of sodium butyrate (NaBT), a Histone Deacetylase Inhibitor (HDACi), prevented NT-mediated induction of genes promoting cell proliferation and invasion (e.g., c-Myc, COX-2 and IL-8) [155]. Mouse model studies also showed promotion of tumor growth through stimulating action of NT/NTSR1 on the expression of miR-21 and miR-155 in colonocytes via AKT and NF-κB signaling [156]. Kim et al. showed differential expression of NT/NTSR1, lack of NTSR2 mRNA expression and consistent NTSR3 mRNA expression in all examined CCc lines, as well as promoter methylation of NTSR1/2. The role of NTSR1 in CCcs proliferation and migration was confirmed using an NTSR1 antagonist (SR48692) [157].
In relation to NPY, it was proven that the proliferation of intestinal epithelial cells is promoted via PI3K/β-catenin signaling and downregulation of miR-375-dependent apoptosis in these cells [137].
Almost two decades ago, it was reported that the proliferation of normal human colonic cells in response to SP is a result of NK1R activation via proinflammatory cytokines (IFN-γ, TNF-α and IL-1β). This effect was reversed by an NK1R antagonist (Spantide 1) [140]. Similarly, in human CCcs (LiM6 and DLD1), a block of NK1R using another antagonist (the clinical drug aprepitant) also caused significant growth inhibition. Blockage of the SP/NK1R signaling resulted in inhibition of canonical Wnt signaling [138].
A potential role in CRC growth has also been attributed to PACAP, which increases the number of viable cells and regulates FasR expression in HCT8 cells [159].

Corticotropin-Releasing Hormone/Factor (CRH/CRF)
A dual role of CRH/CRHR signaling was described in CRC and metastasis [96,97]. CRHR2/Ucn2 signaling inhibits CRC cell proliferation, migration, invasion and colony formation. Thus, CRHR2 downregulation is associated with a higher risk of distant metastases (including LM) [96]. By contrast, regarding CRH/CRHR1 signaling, a pro-inflammatory and, therefore, tumor-promoting effect, was observed in colitis-associated cancer [161]. Research on the detailed mechanisms of this process showed that CCc proliferation occurs through an IL-6/JAK2/STAT3-dependent mechanism and VEGF-induced tumor angiogenesis [97].
Glucagon (GCG) and Glucagon-Like Peptide 1 (GLP1) and GLP2 GCG promotes the proliferation of human and CRC cells in vitro and in vivo through binding to GCG receptor (GPCRs family). The downstream signals of GCGR include an activator of AMP-activated protein kinase (AMPK) and MAPK pathways, governing the development and progression of CRC [162].
GCG is cleaved into GLP1, GLP2 and other small peptides in intestinal L cells and brain neurons. Both of the peptides function in small intestine contractility and growth, while GLP2 stimulates mucosal enterocyte proliferation [60,163]. The use of GLP1 receptor (GLP1R) antagonist (exendin-4, Ex-4) resulted in a reduction of growth and survival in mouse CT26 CCcs via an increase of intracellular cAMP levels and inhibition of GSK3 and ERK1/2 signaling. Additionally, Ex-4 induced apoptosis, inhibited proliferation and caused changes in the morphology of cultured cells [164]. A weaker proliferation of the same mouse cells after Ex-4 application was confirmed by recent studies. However, in the human CCc lines (Colo320, Caco-2, HT-29, SW480 and LoVo), it was proven that this agonist did not enhance the proliferation and migration of these cells [165].
GLP2, as a main nutrient-responsive NP, functions in promoting cell proliferation and survival through CRC-related molecular pathways [166][167][168]. It was reported that GLP2 promotes an increase of mucosal colonic neoplasm in mice [169]. Activation of GLP2R directly stimulates protein synthesis by activation of the PI3K/AKT-mTOR pathway. GLP2 action on proliferation and growth of the intestinal epithelial cells appears to be indirect, being dependent on IGF1R signaling [166] and occurring through increased IGF1/2 expression in myofibroblasts [170].

Somatostatin (SM)
SM is produced by the CNS and PNS, EECs, inflammatory and immune cells, as well as many cancer cells. It has an inhibitory effect on cell motility and proliferation (G1 cell cycle arrest) and induces apoptosis [171][172][173]. SM acts through 5 receptor subtypes (sst1-sst5) (GPCR family) expressed by many normal and malignant cells [56,171]. In a study on Caco-2, HT-29 and HCT116 cells, expression of sst3/4/5, sst3/5 and sst2/3/5 respectively, was detected. The inhibitory effect of SM on the proliferation of CCcs was based on COX-2 downregulation via activation of two receptors: sst3 or sst5 [129]. Interesting studies on the interactions between SM-positive EECs and adjacent colonic SCs in crypt stem cell niche, indicated that the sub-population of aldehyde dehydrogenase (ALDH)-positive SCs is regulated by sst1 via a paracrine mechanism [174]. The impact of SM on uncontrolled CCcs proliferation and the potential role in CRC progression (including LM) can be explained by the genetic/epigenetic changes of the SM gene in CRC (which will be discussed later).
Numerous studies on the mechanisms of Ntt action in CRC progression (including the first step of metastasis) are focused on TrKB [176,[180][181][182][183][184]. It was proven that TrKB mediates production of endogenous BDNF with some differences, depending on cell lines (WiDr, SW480, SW620 and Colo205), inducing proliferation and cell survival, as well as inhibiting apoptosis [180]. Ntt activate Ras, PI3K, phospholipase C-γ1 and MAPK pathways [175,176]. It was shown that both receptor types (TrKB and TrKC) induce cell growth and invasion, as well as function as anti-apoptotic factors [182]. Similarly, BDNF itself increased viability, migration, invasion and inhibited anoikis (detachment-induced apoptosis) in the CCcs [183].
The schematic diagrams of the main signaling pathways regulated by Nts/Nps/Ntt, as well as downstream signaling of their activated receptors, which are correlated with proliferation, cell cycle progression, migration and invasion of CRC cells, are shown in Figure 1.
In summary, the knowledge on the impact of selected Nts/NPs/Ntt on increased cell proliferation, migration and invasion of colon tumor cells is based on animal models, as well as in vitro studies using adequate assays and human (e.g., HT-29, H508, SNU-C4, DLD-1, LIM2405, SW480, SW620, Colo320, LoVo, HCT116, HCT8 and Colo205) or mouse (e.g., CT26) colon/colorectal adenocarcinoma cell lines with different metastatic potential. Most of the studies use HT-29 cells and their variants (WiDr, CI.19A), in which the presence of Nt/Np/Ntt receptors was confirmed. Most of the in vitro studies indicate a potential role of many neuroactive molecules in the metastatic process of CRC in vivo. Basic mechanisms of cell signal transduction involving Nts/NPs/Ntt were described, playing a major role in the regulation of genes important for CRC progression (including LM) (e.g., c-Myc, cyclin D1, COX-2, IL-8, MMPs, LEF1/TCF4)).

Colorectal Cancer Cell-Cell and Cell-Extracellular Matrix Loss of Adhesion
In this process, the participation of the NP/NP-Rs components upregulating MMPs production is especially important. The mammalian MMPs, particularly MMP1, degrade EMC and facilitate CRC cell invasion and metastasis. Their levels correlate with the clinical progression of CRC, hematogenous metastases and poor prognosis [102,185]. Production of MMPs (MMP1, MMP2 and MMP3) mediating cell migration was proven in LoVo cells after G-17Gly stimulation [125]. Interestingly, in tissue microarrays (TMA) of CRC LM, MMP1 and MMP2 were identified as consistently under-expressed, compared with primary CRC (pCRC). According to the authors,

Colorectal Cancer Cell-Cell and Cell-Extracellular Matrix Loss of Adhesion
In this process, the participation of the NP/NP-Rs components upregulating MMPs production is especially important. The mammalian MMPs, particularly MMP1, degrade EMC and facilitate CRC cell invasion and metastasis. Their levels correlate with the clinical progression of CRC, hematogenous metastases and poor prognosis [102,185]. Production of MMPs (MMP1, MMP2 and MMP3) mediating cell migration was proven in LoVo cells after G-17Gly stimulation [125]. Interestingly, in tissue microarrays (TMA) of CRC LM, MMP1 and MMP2 were identified as consistently under-expressed, compared with primary CRC (pCRC). According to the authors, MMP1 levels in early CRC stages (II and III) were associated with an increased likelihood of distant metastasis, whereas rectal cancer in stage III recurrence was rather associated with MMP2 [186]. In turn, recent studies on serum MMP2 levels showed upregulation of this marker in CRC patients, as well as its correlation with clinical data (including lymph node and liver metastases) [187].
Increase in MMPs production (mainly MMP7 and MMP1) also upregulated expression of the Nts member ACh [100,102].
Furthermore, a study of CRHR2 signaling showed induction of changes in cell-cell junctions in two CRC cell lines (HT-29, SW620), affecting their ability to maintain cell-cell contact via the Src/ERK pathway. In HT-29 cells, cell adhesion remodelling, modification of cytoskeleton structures, as well as stimulation of migration and invasion ensued. All these phenomena can promote the metastatic potential of human CRC, resembling the EMT process [188]. In turn, other studies show lower expression of CRHR2 in CRC tissues and cell lines compared to control, suggesting contrasting effects of CRHR2/Unc2 signaling on tumor growth and EMT, with decreased expression of EMT-inducers and elevated levels of EMT-suppressors. In other words, downregulation of CRHR2 in CRC could be responsible for cell spread and be a factor of high metastasis risk [96,98].
Another NP-R with a potential role in this stage of LM is NTSR3 (Sortilin) and its soluble form (sNTSR3/Sortilin) [189]. The presence of Sortilin was proven at cell membranes of numerous cancer cells (including CRC cells) [190]. Specific binding of sNTSR3 and its internalization occurs in HT-29 cells, with a potential mechanism in this stage of metastasis based on activation of the FAK/Src-dependent PI3K pathway, accompanied with an increase in intracellular Ca +2 and a decrease of integrin mRNAs [189,191,192]. Hence, sNTSR3 action resulted in modification of desmosome structure, suggesting that these changes might lead to separation and spread of cells in early stages of carcinogenesis, easing metastasis [189,192].
Among neurotrophin receptors, it was shown that downregulation of TrKB increased anoikis sensitivity of CRC cells in vitro, with TrKB-induced anoikis suppression in CRC cells dependent on the PKB (AKT) signaling pathway. In other words, overexpression of TrKB (as an anti-anoikis molecule) could protect CRC cells from anoikis in the circulatory and lymphatic system [182].

Epithelial to Mesenchymal Transition (EMT) in CRC
EMT is an important process in CRC LM formation [21]. It was recently reported that the GPR56 (GPCR family) is significantly upregulated in some of the CCc lines (e.g., LoVo, DLD-1, SW480, HCT116), compared to control lines (NCM460). However, relatively low expression of GPR56 was detected in HT-29 cells. This peptide promotes CRC cell proliferation, migration and invasion, and is critical in CRC metastasis, due to EMT stimulation via activation of the PI3K/AKT signaling [15].
Another member of NP-Rs, acting on EMT, is ANG II Receptor Type 1 (AT1R). Use of specific AT1R and AT2R blockers caused inhibition of migration of human CRC cell lines (DLD-1 and LIM2405). In turn, ATR1 blocker caused an increase in E-cadherin expression and reduced Zinc finger E-box-binding homeobox 1 (ZEB1) and vimentin, while ATR2 inhibition lowered E-cadherin expression, without changing the levels of ZEB1 and vimentin [109].
Neurotrophin receptors, e.g., TrKB, also participate in EMT induction. In clinical samples, the inverse correlation was described between the expression of TrKB and E-cadherin. SW480 cell line studies confirmed the alleviation of malignant potential of these cells by TrKB knockdown. TrKB might play an important role in EMT and progression to metastasis [193].

Angiogenesis in CRC
A stimulating (e.g., epinephrine, NE), inhibiting (e.g., dopamine, SM), as well as dual role (stimulating or inhibiting) (e.g., NPY) of Nts and NPs on tumor angiogenesis has been described, which might suggest the role of these molecules in progression and metastasis of tumors (including CRC) [194].
Two forms of gastrins (amidated G17 and G17-Gly peptides) were also indicated to increase expression of HB-EGF in Human Umbilical Vein ECs (HUVEC) and microvessel-derived ECs, as well as elevate the levels of MMP2, MMP3 and MMP9. Mean vessel density (MVD) in normal mucosa adjacent to CRC correlated with serum gastrin levels and HB-EGF expression in CRC patients [195]. Pro-angiogenic action of progastrin in CRC was also proven. Stimulation of cadherin phosphorylation in ECs, p125-FAK, paxillin and actin remodelling resulted in EC proliferation/migration, the ability of ECs to form capillary-like structures and enhanced permeability of endothelium [196].

Somatostatin is an endogenous inhibitor of both cell proliferation and angiogenesis.
Interesting research using in vitro receptor autoradiography in submucosal and subserosal vessels localized near human CRC showed 3-5-fold overexpression of SM and SP receptors in the host veins within a close area (2 cm wide) surrounding human pCRC, as compared with veins located at a greater distance (5-10 cm) in control tissue. This finding suggests a regulatory mechanism presence on the levels of tumor vascular bed, which might be crucial for the development of CRC metastasis mechanisms [197].
Another pathway active during the Vascular Endothelial Growth Factor (VEGF)-induced tumor angiogenesis is the CRH/CRHR1 signaling, as one of the mechanisms in colitis-associated CRC [97,161].
A potential role of TrKB, positively regulating the expression of VEGF-A and VEGF-C, was also indicated in an in vitro model [180]. Increased secretion of VEGF-A in mouse rectal CMT93 cells is also caused, in a dose-dependent manner, by AT1R [198].
A list of Nts, NPs and Ntt and their receptors involved in the signaling pathways of the main steps of CRC metastasis (including distant metastasis) is presented in Table 1. Table 1. Neurotransmitters (Nts), Neuropeptides (NPs) and Neurotrophins (Ntt) and their receptors playing a role in the progression of colorectal cancer (including CRC liver metastasis) and the major mechanisms of action of the neuroactive molecules in the most important steps of cancer metastasis.

Role of Circulating Tumor Cells (CTC) in Liver Metastasis
CTC aggregates (up to 20 cells) from primary tumor sites might be the precursors of tumor metastasis (including LM) [199]. However, research on sensitive and specific CTC markers still poses numerous difficulties. Numbers of these cells in the blood of metastatic patients is insufficient for effective detection (<1 to <50 in 7.5 mL blood of a metastatic cancer patient), which motivates the search for more modern techniques of their detection, including other panels of cellular markers (apart from epithelial), typical for processes such as EMT, mesenchymal-epithelial transition (MET), with SCs traits, or in an immune evasive state [200,201]. NPs, being the subject of this review, have not yet been indicated as CTC markers.
Recent research shows that regulatory mechanisms of CTC-mediated tumor metastasis involve Tumor-Associated Macrophages (TAMs) by regulating the JAK2/STAT3/miR-506-3p/FoxQ1 axis. The study used different TAMs for evaluation of the associations of their sub-localization with EMT phenotype and ratio of mesenchymal CTC in CRC [202].

Tissue Expression of NP System Components in CRC and Liver Metastasis
CRC is a heterogeneous tumor, containing, apart from epithelial tumor cells, other cellular populations, e.g., cancer-associated fibroblasts (CAFs) [203], myofibroblasts [204], TAMs [202], B and T cells [205], tumor ECs (TECs) [206] and CRC CSCs [207], which might be a source of NPs and their receptors, playing a key role in CRC metastasis (including LM).
A particular prognostic value in CRC is attributed to altered (especially elevated) expression of NPs and their receptors in tumor tissues, compared to control [95,121,135,198,210,212,214,[216][217][218][219].
Peptides secreted in autocrine/paracrine signaling serve an important role in numerous signaling pathways, responsible for an increase in the concentration of cytosolic Ca 2+ and proliferation stimulation [126,211], intensification of CRC migration/invasion [125], enhanced ECs activity in models of angiogenesis [196] or direct pro-carcinogenic effect [220]. Most of the studies of tissue expression, particularly of NP receptors, also suggest their usefulness in the context of CRC therapeutics.
Results of studies on the expression of renin-angiotensin system (RAS) components, conducted on murine models of CRC liver metastases, differ from those obtained from human CRC tissues [224,225]. In mouse metastatic liver, a lower expression of ATG and AT1R was observed, with elevated levels of angiotensin I converting enzyme (ACE) and ANG precursors 1-7 (MasR), compared to the surrounding liver. The treatment with captopril (inhibitor of ACE) in CRC metastases resulted in a decrease in LM volume and downregulation of ATG and AT1R expression, with increased ACE expression in the final stages of tumor growth [224]. Moreover, Wen et al. showed an immunomodulatory role of the RAS via liver Kupffer cells (KCs). Application of captopril increased the number of KCs in the LM in vivo and invasion in vitro, as well as appeared to alter the function of early, anti-tumor KCs during tumor progression [225]. In turn, Shimizu et al. showed that ANG II increases the expression of TGF-β1 in KCs. In other words, collagen build-up in metastasis area via the AT1a pathway was associated with resident KCs induction [226].
Studies on the role and mechanisms of action of Nt/NP/Ntt receptors in colon carcinogenesis and metastasis started almost three decades ago. They mostly concern NTSR [154,156,227], NmB-R [141,164] and TrK [180][181][182][183]. In the 1990s, expression of NTSR was detected in more than 40% of 19 CRC cell lines, with a lack of its presence in the normal colonic epithelium [228]. Furthermore, membrane localization of two structurally different NTSRs, namely NTSR1 (GPCR family) and NTSR3, as well as their internalization after NT stimulation, was described in HT-29 cells [154]. Higher expression of the NTSR1 gene in vivo was observed in colonic adenocarcinoma compared to adenomas. According to the authors, NTSR1 expression might be responsible not only for early stages of CRC development but also for its progression and aggressive forms, as successive NTSR1 tissue expression increase was described from colitis, through dysplasia, to CRC itself [227,229].
When it comes to the research on TrK tissue expression in CRC, the results are consistent for correlation of expression of these receptors with lymph node and peritoneal metastases, with less consistency when it comes to correlation with LM. In one study, only TrKC expression correlated with LM [182], while in another, this correlation was described for overexpression of TrKB [181]. In another, co-expression of both ligand-receptors, namely BDNF/TrKB, correlated with liver and peritoneal metastases [183]. Higher expression of ligands (BDNF) or TrKs correlated with clinical stage [180,182] and worse prognosis in CRC patients [181,183,193].
In the context of Sst1 to Sst5 expression in CRC and LM, the results vary. Loss of Ssst2 mRNA expression was described in Dukes' stage D CRC and hepatic metastasis patients [222]. Other authors considered elevated Sst2 mRNA expression for a bad prognostic factor, as these patients exhibited shorter DFS [230]. Evangelou et al. described a negative correlation between the level of Sst2 and Sst5 protein expression in CRC with invasion and LM. In turn, the level of Sst2 expression was higher in lower-grade and rectum-located tumors, with patients with positive expression of both proteins (Sst2 or sst5) surviving longer [223].

Serum Levels of NP System Components in CRC and Liver Metastasis
Studies of serum concentrations of NP system components in CRC and/or CRC metastases (lymph nodes, liver), were mainly conducted in the context of their prognostic role or application in therapy. An increase in concentrations of Nts (e.g., serotonin) [231] and NPs were mainly observed in pCRC patients compared to control. This observation concerned the following NPs (alphabetically): ET-1 (and big ET-1) [117,208,209,232,233], galanin [67], gastrin/G-17 [234,235], GLP1/GLP2 [236], NT [49,237,238], PYY [236] and SP [239]. Serum VIP concentrations were also higher in metastatic CRC versus control [158].
ET-1 levels were also higher in LM patients compared to those without metastases [208], as well as patients with different types of LM (metachronous/synchronous) compared with control [232]. The systemic plasma levels of big ET-1 were higher in patients with CRC with Dukes' D staging versus localized disease [209]. The levels of big ET-1 of >4.2 pg/mL, age of patient > 70 years and Dukes' stage C, were indicated as factors of bad prognosis and independent prognostic values for OS [233]. However, other authors negate the prognostic value of serum ET-1 concentrations in CRC [232,240].
In the context of the role of gastrin/G-17 in CRC progression, the study results are also inconsistent. Some observed elevated concentrations of these peptides in CRC versus control [235,241], while others did not detect such differences [234]. However, significantly higher serum gastrin levels were demonstrated in patients with lymph node metastasis than patients without metastasis [234]. While gastrin concentrations of European CRC patients were higher in the tumor (>50 pg/mL) than in control, they were not indicated as a CRC risk factor and did not differentiate Helicobacter pylori (Hp) (+) and Hp (−) patients [241]. In precancerous lesions, some authors did not observe any correlation between hypergastrinemia and adenoma development [242], while others have shown that hypergastrinemia is a risk factor for colonic adenomas [243].

Genetic and Epigenetic Changes of Selected NPs and CRC Liver Metastasis
Profiles of many genes responsible for CRC development, and most probably associated CRC LM, were described. However, there is a lack of knowledge on LM-specific mutations that could be applied in everyday medical practice [245][246][247][248][249][250][251]. According to some authors, combinations of oncogenic changes are more important than specific events that determine the metastatic genotype of tumor cell early in carcinogenesis [245,248]. Although some of the papers comparing the genetic changes in pCRC and metastatic tumors, or advanced stages of CRC versus small tumors with less metastatic potential did not show significant changes in the Nt/NP/Ntt genes [246,247,249,251], there are some reports of such changes [245,250,252].
In the study of Koehler et al., among 23 up-and down-regulated transcripts in the high-stage and low-stage CRC group, upregulation of Ntt-3 precursor (BDNF, NGF2) was described [245]. Lim et al. defined 3 classes of gene mutations in CRC, assessing the presence or absence of mutations during LM development. Around 60% of changes were classified as Class 1 (shared between primary tumor and LM), which suggests the clonal origin of the primary tumor and LM. This research indicated 11 mutation-associated splicing events in the LM transcriptomes, including the splite-site GPR56 mutation, which can result in a premature stop codon for all functional domains of GPR56 (tumor-suppressor) and be responsible for CRC development. Although the analysis shows several interesting changes at the exome and transcriptome levels, the authors did not indicate significant LM-specific mutations [250]. Interestingly, among the differentially expressed mRNA (DEMs) in CRC, was a GRP, a significant increase of which could serve as an independent DFS prognostic gene [252].
The most common epigenetic alterations in CRC include aberrant methylation of DNA [178,179,[253][254][255][256][257]. Hyper-or hypo-methylation might serve as an epigenetic biomarker for early detection, prognosis and response to chemotherapy in CRC [244,258,259]. The only, as of now, epigenetic marker approved by the US Food and Drug Administration (FDA) for CRC screening, is the methylation of the septin 9 (SEPT9) gene [259]. A search was also conducted for similar epigenetic biomarkers among the NP and Ntt genes. Therefore, e.g., expression of NT and NmN expressed in fetal colon, is reexpressed in~25% of colon cancers. Differential expression of both these genes was also described in vitro-positive in human colon cancer KM12C cell line and negative in KM20, which was associated with epigenetic changes. Gene silencing in KM20 cells concerns methylation of the CpG sites in a distal consensus AP-1 site in the NT and NmN promoter. In turn, NT and NmN gene expression in KM12C was associated with demethylation of the CpG sites [253]. Methylation of the NTSR1 promoter in some human CRC cells (KM12c, Caco2 and DLD1) and NTSR2 in all six CRC cell lines (KM12c, Caco2, DLD1, HT-29, HCT116 and SW480) was also noted [157]. The NTSR1 gene is also often methylated in CRC in vivo, with the higher level of methylation occurring in laterally growing, large, non-invasive tumors, which is associated with better prognosis [260]. Other studies suggest that methylation-associated silencing of NTSR1 is inversely correlated with invasiveness of CRC, and that a low level of methylation activates NTSR1 and is responsible for malignant potential of CRC [257].
Studies of the SP precursor, TAC1, showed that high methylation levels of these markers in serum at 6-month follow-up, and SEPT9 at 1-year follow-up, were independent predictors for tumor recurrence and unfavorable CSS [261]. Furthermore, methylation inhibited the expression of the NTRK3 gene, observed in 60% of colon adenomas, and 67% of colon adenocarcinomas. Loss of these genes' expression was associated with neoplastic transformation in vivo and in vitro [179].
Genetic/epigenetic changes also lead to activation of another potential proto-oncogene, TrKC. A decrease in TrKC expression was observed in a major portion of human CRCs. It was demonstrated that TrKC silencing by promoter methylation can limit tumor cell death. Additionally, the existence of two different tumor-associated TrKC mutants was proven in sporadic CRC. One of them is responsible for a gain-of-oncogenic function, while the other causes a loss of pro-apoptotic function, negating the tumor-suppressive effect of TrKC [178].
Other CRC-associated hypermethylation events, which significantly differentiated CRC and non-neoplastic tissue, as well as adenomas versus control, affected the GLP1R [254]. Studying the methylation levels of NPY, proenkephalin (PENK) and Wnt inhibitor factor 1 (WIF1), a potential diagnostic value of the combination of these three markers was proven in CRC [255]. In a study by Mitchell et al., the NPY gene was among the 7 others methylated in >50% of CRC samples, compared with a low level of methylation in non-neoplastic colorectal tissue [256].
Identification of genes prognostic for CRC with the use of bioinformatic methods showed that low expression of 4 out of 10 of them, including GCG, is associated with an unfavorable prognosis [262]. Similarly, recent studies confirmed that GCG is among the top five downregulated genes in CRC [263,264]. However, this gene is not significantly associated with distant metastases (including LM).
Other studied epigenetic alterations concern ET-2 and ET-3. It was proven that epigenetic inactivation through hypermethylation of EDN2 and EDN3 is common in both rat and human colon cancers [110].
The candidate methylation targets in pCRC included SM (88%) and the TAC1 (47%) genes. The degree of methylation was, in both cases, associated with a decrease in mRNA production. The intensity of methylation in the case of TAC1 was higher in Dukes A/B than C/D. Hence, it cannot be associated with tumor progression (including LM). Significantly higher methylation of the SM was observed in low-level microsatellite stability (MSI-L) than non-MSI-L CRC [265]. In later publications on the SM gene, methylation status analyses showed a significantly higher level of promoter hypermethylation in CRC than in healthy young individuals, which can be linked to decreased SM production in CRC and uncontrolled cellular proliferation [221]. A significant correlation was found between serum methylation levels of SM (mSM) and CSS, which allowed to indicate mSM as an independent predictor for poor CSS. Patients with high serum mSM were noted to have a higher risk for cancer-specific death (HR = 1.96, 95% CI: 1.06-3.62) [266]. The molecular mechanisms of SM gene promoter methylation in CRC development and progression (including distant metastasis) are unknown. Common and significant DNA methylation was also observed in three other genes, among them, sst2, in CRC tissues compared to adjacent normal tissues [258].
It was also reported that hypomethylation of genes, e.g., ACE, might play a prognostic role in CRC. This process is important in enhancing of cell proliferation, colony formation, inhibition of apoptosis and is related to CRC prognosis [244].
Furthermore, regarding the CRHR2 gene, a recent study indicates higher levels of hypermethylation of the gene in patients with z colitis-associated CRC versus non-tumorous mucosa, which could be used in screening, prognostics, as well as evaluation of therapy outcomes in patients with UC-associated CRC [267]. These studies could also determine that hypermethylation of CRHR2 may be responsible (similarly as in the case of SM) for lowered tissue expression of this protein [96], but also cell spreading, being a high metastasis risk factor in CRC [98].

Targeting NP/NP-R System in CRC Liver Metastasis Molecular Therapy
Based on the knowledge of NPs/NP-Rs, attempts were made to introduce several drugs for patients with advanced stages of CRC, such as the label phase I/II study with G17-DT (Gastrimmune), with no observed tumor regression [268]. Falciani et al. applied the tetra-branched NT armed with 5-fluorouridine (5-FdU) and showed a 50% reduction in tumor growth as compared to animals treated with placebo [269]. In in vitro conditions, superior drug internalization of DOPC-NT 4 Lys(C 18 ) 2 liposomes with NT8-13 fragments and containing Doxorubicin (peptide-functionalized liposomes) was proven over the "pure" DOPC liposomes [270]. When it comes to NTSR1, the use of radioligand therapy with NTSR1-targeting agent, 177 Lu-3BP-227, inhibited tumor growth and decreased its volume by 55-88% [271]. Effectiveness of VIP hybrid antagonist neurotensin(6-11)VIP(7-28) was also proven, both in vitro and in an animal model (rat), in inhibition of tumor growth and decrease of its volume or staging [272]. Based on the knowledge about BBS/GRP, using an antagonist of these peptides, RC-3940-II, the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice decreased by 25% to 67%. A better effect was obtained in combined therapy with RC-3940-II, 5-Fluorouracil (5-FU) and irinotecan [273].
If LM arises during the progression of CRC, a standard procedure dictates liver resection [14,16]. Proper and rapid regeneration of the liver after surgical LM removal has importance equal to the initial outcome of surgery [274]. Apart from hepatectomy, a combination of chemotherapy and targeted drugs, e.g., anti-VEGF and anti-EGFR monoclonal antibodies, are also applied [14]. A search continues for other forms of LM therapy, e.g., thermal ablation with hepatectomy, which could lower patient mortality and help to avoid two-stage hepatectomy [275].
In the context of this review, there are also attempts to apply the knowledge about the NP/NP-R system in the development of LM therapy. As an example, inhibition of the RAS system was tested as a therapeutic approach [225,274]. A mouse model of partial hepatectomy showed that early liver regeneration (LR) is promoted by captopril (ACE inhibitor) via an increased number of HSCs and MMP9 levels. The results of this study could be valuable in ensuring enough liver regeneration and prevention of tumor recurrence after hepatectomy [274]. Research by Wen et al., conducted on an orthotopic murine model of LM, showed varying effects of RAS component action on the number of KCs in the liver, possibly leading to in LM progression. The use of captopril and ANG-(1-7) increased the numbers of KCs in the liver (but not in the metastatic tumor), with the former reducing LM growth [225].
Of patients with different models of liver metastatic CRC, therapeutic approaches using neuropeptides (or their agonists/antagonists) were tested. However, their number was limited, including (chronologically): Sst2 [276], Gal, serotonin and octreotide [277], RAS [225,278,279] and NPY [280] (Table 3). Most of the used therapies inhibited tumor growth and liver metastasis formation. Among the treatments based on the knowledge that many NPs can act as growth factors or oncogenes, there are currently no drugs qualified for clinical trials in the context of CRC. However, some NPs, e.g., NPY methylated (mNPY) ctDNA, might serve a role of universal and easily applicable biomarkers in CRC patients treated with regorafenib. A high baseline level of mNPY ctDNA correlated with shorter patient survival, with the results potentially helping with treatment monitoring [280].
To prevent LM, a search is underway for inhibitors of its different stages, including CRC cell proliferation with NP-R participation using, for example, miRNAs. It was proven in in vitro and in vivo conditions that miR-148b inhibits cell proliferation and carcinogenesis via downregulation of the CCK2R gene on a translational level [281]. In studies on HT-29-bearing mice, significant inhibition of tumor growth by Nts dopamine was also observed, but only in combination with 5-FU. While dopamine did not exhibit a direct effect on tumor growth, it inhibited angiogenesis by decreasing TEC proliferation via VEGF-R2, MAPK and FAK kinase phosphorylation [282]. Polydopamine nanoparticles are also a promising immunotherapy candidate. Applied in tumor-bearing mice, they delayed tumor progression due to a sufficient amount of cytotoxic T lymphocytes (CTLs) and M1-type TAMs as well as the deficient number of immunosuppression-related cells in the tumor tissues [283]. Gao et al. demonstrated that CCKR-targeted immunotoxin (rCCK8PE38) also has potential as a new immunotherapy agent, decreasing tumor size in nude mice with HCT-8 tumor xenografts, as well as acting cytotoxically in two CRC cell lines [284].

The Main Headlines of the Review
In vitro and/or animal model studies on the mechanisms of Nts and NPs/Ntt action, confirm the participation of these molecule systems (mainly their overexpression) in main stages in CRC liver metastasis.
In vivo studies confirm the production of numerous Nts/NP/Ntt and their receptors in neural structures (neurons, nerve fibers) or EECs, tumor microenvironment cells (TECs, TAMs, myofibroblasts), as well as in pCRC and metastatic cells. It is worth noting that peptide expression concerns different tumor cell populations, while the presence of their receptors is mostly characteristic for cancer cells. Liver metastasis presence was positively correlated with tissue expression/overexpression of many NP/NP-Rs (e.g., AT1R, BDNF, BDNF/TrKB, ET-1, gastrin, progastrin, SP/NK-1R system, TrKB and TrKC), as well as a lack or lower expression of some of them (e.g., AT2R, CRHR2, GRP/GRPR, Sst3 and Sst5).
Studies on the role of the serum NP/NP-R concentration were mostly conducted in recent years and indicate a diagnostic-prognostic role of these peptides. Higher levels of Nts/NPs were noted in pCRC versus control (serotonin, ET-1, big ET-1, galanin, G17, GLP1/GLP2, NT, PYY and SP), as well as in metastatic CRC than pCRC (gastrin, VIP), metastatic CRC versus control (ET-1) or in CRC compared to other types of GIT cancer (pancreatic, gastric cancers) (NT, VIP).
While there is a lack of description of liver metastasis typical mutations/epigenetic alterations concerning Nts/NPs/Ntt, a continuation of studies is essential in the elucidation of the mechanism of not only the development, but also metastasis, involving aberrant methylated DNA, or the discovery of a more universal epigenetic profile of CRC and LM.

1.
Studies on the role of NP/NP-Rs in the promotion of the invasion-metastasis cascade in CRC, show the complexity of brain-large intestine-tumor interactions, caused by their different forms of release to the extracellular environment (endocrine, autocrine, paracrine and neurocrine). More research is needed to understand the exact mechanisms of neuronal-tumor cells communication.

2.
Many steps of CRC promotion, progression and liver metastasis are connected to the activity of pro-inflammatory (CRHR1, NPY, NT), anti-inflammatory (CGRP, CRHR2, VIP) or dual role (SP) NPs, regulation of the local immunological profile (CRH/CRHRs), dysfunctions of the protective/enterotrophic role of NPs on epithelial cells (NT/NTSR system), structural-functional changes in ENS innervation of the large intestine in CRC (including PNI), or other tumor-promoting factors (bacterial GIT infections, such as H. pylori).

3.
The knowledge on the mechanisms regulating tumor growth and different steps of metastasis, as well as effects of the action of a numerous group of Nts/NPs/Ntt as growth factors, have implications for future therapeutic strategies. For obtaining the best treatment outcomes, it is important to use signaling pathways common for many NPs, as well as the development of broad-spectrum antagonists.
Author Contributions: Both authors worked on the information compilation, analysis and manuscript writing equally. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.