Cancer metastasis: Mechanisms of inhibition by melatonin

2.1 Melatonin: mechanisms of action

Melatonin (N-acetyl-5-methoxytryptamine), first identified as a neurohormone in the bovine pineal grand,7 is produced in a great variety of tissue types including the brain, retina, lens, cochlea, Harderian gland, airway, skin, gastrointestinal tract, liver, kidney, thyroid, pancreas, thymus, spleen, lymphocytes, and in the reproductive tract.8-10 In the pineal gland, its synthesis and secretion is controlled by the prevailing light:dark cycle as detected by the eyes where light represses and darkness augments its production.11, 12 Melatonin has multiple means to carry out its effects. Melatonin acts via membrane G protein-coupled receptors (MT1 and MT2) that are expressed in numerous areas of the central nervous system and in peripheral tissues to modulate diverse biological activities.13-16 One of the best-described membrane receptor-mediated actions involve an influence on phasing of circadian rhythms and sleep promotion.17, 18 Additionally, other actions of melatonin are attributed to its ability to bind to nuclear and cytoplasmic interacting partners.19 For example, melatonin is a natural ligand for the retinoid-related orphan nuclear hormone receptor family (RZR/ROR).20 Melatonin's immunomodulatory effects and some of its circadian actions are mediated through these nuclear interactions.21, 22 Furthermore, melatonin interacts with calmodulin,23, 24 an intracellular protein implicated in cytoskeletal rearrangement25 and estrogen signal pathways.26 The cytosolic binding partner of melatonin is the enzyme quinone reductase 2; this enzyme protects against oxidative stress by preventing electron-transfer reactions of quinones.27, 28 Finally, melatonin and its metabolites are potent scavengers of both reactive oxygen (ROS) and reactive nitrogen species.29-32

Mitochondria are the major site of free radicals and related toxic species generation. Melatonin has been consistently shown to not lessen the formation of ROS/RNS at the mitochondrial level, thereby protecting against oxidative or nitrosative damage to electron transport chain proteins; it also limits lipid peroxidation in the inner membrane, thus favoring electron flux and ATP production.33, 34 It was also recently suggested that the mitochondria of all cells may be the site of melatonin synthesis.35

Various lines of evidence have suggested pleiotropic roles of melatonin in the maintenance of health.36-38 A reduction in circulating melatonin levels, polymorphisms of melatonin receptor genes, and circadian disruption are associated with a myriad of physiological and pathological disorders including aging, metabolic syndrome, type 2 diabetes, immune diseases, hypertension, several mood and cognitive disorders, and cancer.39-46 Melatonin, under both in vitro and in vivo conditions, inhibits the growth of numerous cancer types.47-50 Due to its broad spectrum of actions, there are multiple mechanisms underlying melatonin's ability to counteract tumor growth. These include, but are not limited to its varied antioxidant effects, modulation of the cell cycle, induction of apoptosis, inhibition of telomerase activity, ability to antagonize metastasis, prevention of circadian disruption, anti-angiogenesis, epigenetic effects, inhibition of growth factor uptake, stimulation of cell differentiation, and activation of the immune system.51, 52 As a result, melatonin has emerged as an appealing therapeutic target for anticancer therapies with consistent effects on solid tumors over a wide range of doses and cancer types.53 In this review, we discuss recent advancements in understanding the molecular mechanisms by which melatonin inhibits tumor metastases (summarized in Table 1), including experimental evidence and clinical observations, with a focus on the impact of both cancer and non-neoplastic cells within the tumor microenvironment. It is hoped that the knowledge compiled herein will serve as a solid foundation to encourage the use of melatonin to control metastatic diseases.

2.2 Modulation of cell–cell and cell–matrix interaction

Metastasis is the development of secondary tumors distant to the primary site. A key characteristic of metastatic cells is the substantial flexibility in their adhesive interactions with adjacent cells or with ECM components. During cancer cell invasion, the breakdown of cell–cell adhesion allows tumor cells to dissociate from the primary tumor mass, and alterations in cell–matrix interaction potentiate the ability of the cells to invade the surrounding stroma.54 The cell architecture of epithelial cells is characterized by a remarkable polarization of the plasma membrane, maintained by a variety of compositionally and functionally distinct surface structures. These cell-to-cell adhesion complexes include tight junctions (TJs), adherens junctions (AJs), gap junctions (GJs), desmosomes, and hemidesmosomes. Among these, melatonin exhibits an inhibitory role on the invasive properties of cancer through regulating the expression of cell adhesion molecules associated with TJ and AJ.

A hallmark alteration of the cell junction in the early step of metastasis involves the loss by cancer cells of E-cadherin, a prototypical member of the type-1 classical cadherins found at AJs.55 When forming AJs with adjacent epithelial cells, E-cadherin facilitates the congregation of epithelial sheets and retains the quiescence of the cells within these sheets. Reduced expression of E-cadherin was recognized as a prerequisite of invasion and metastasis, whereas augmentation of its expression was shown to impede the development of cancer phenotypes. In addition to its ability to alter the intercellular contacts, loss of E-cadherin contributes to metastatic dissemination by the activation of multiple signaling pathways and induction of numerous transcription factors via its intracellular binding partner, β-catenin.56, 57 Melatonin shifts one human breast cancer cell line, MCF-7, to a lower invasive status by increasing E-cadherin expression.58 Recently, the detailed mechanism by which melatonin modulates E-cadherin induction was unraveled in an investigation into gastric cancer dissemination.59 Melatonin-mediated upregulation of E-cadherin was demonstrated to involve with interference with the interaction between C/EBPβ and NF-κB through the action of calpain induced by endoplasmic reticulum stress.

Another cell junction involved in melatonin-regulated inhibition of cancer invasion and metastasis is TJ. As the most apical structure of epithelial cell junctions, TJs are well known as a control for the paracellular diffusion of ions and various molecules, posing a crucial role in maintaining cell integrity. Alterations in the expression and/or distribution of TJ proteins can lead to perturbations in cohesion of the TJ structure, which in turn allows cancer cells to become invasive and then ultimately result in metastasis.60 Melatonin represses the migration of a human lung adenocarcinoma cell line, A549, which is accompanied by the upregulation of occludin, a transmembrane protein found in the TJ.61 Occludin is required for the leading-edge localization of polarity proteins, aPKC-Par3 and PATJ, and promotes directional migration of epithelial cells by regulating membrane-localized activation of PI3K.62

The expression of genes encoding the adhesion molecules involved in cell-to-ECM interaction is also demonstrably altered in invading cancer cells in response to melatonin. A notable example is the integrin receptors, a group of heterodimeric cell-surface glycoproteins that function in linking molecules of the extracellular space to the intracellular actin cytoskeleton.63 These integral membrane proteins contain two subunits, termed the α- and β-subunits. There are at least eighteen α-subunits and eight β-subunits found in vertebrates, resulting in numerous combinations of different integrin heterodimers, which selectively bind to ECM proteins to activate many intracellular signal pathways and control a range of biological processes. The anti-invasive effect of melatonin on glioma cells under hypoxic conditions was accompanied by a reduced expression of α_vβ₃ integrin.64 Expression of α_v integrin was shown to be correlated with and functionally involved in the maintenance of a highly migratory phenotype in prostate cancer and laryngeal and hypopharyngeal squamous cell carcinoma.65, 66 Paradoxically, in vitro invasive capacity of breast cancer cells was reportedly suppressed by melatonin via the upregulation of β₁ integrin.58 These findings indicate that melatonin collectively contributes to a concerted cross talk between cell–cell and cell–matrix adhesion, thwarting the attempt of malignant cells to extricate themselves from the primary site and migrate into surrounding stroma.

2.3 ECM remodeling by matrix metalloproteinases

Successful metastasis requires not only a local microenvironment to bolster the primary cancer cells to proliferate but also a premetastatic niche to allow disseminated cells to colonize and thrive at a distant location.67 The key constituent of such niche is the ECM, a highly dynamic structure that is present in all tissues and continuously undergoes balanced remodeling. The process of ECM remodeling is mediated by specific enzymes that are responsible for ECM degradation, such as matrix metalloproteinases (MMPs). In addition to serving as a physical scaffold for adhesion proteins of cancer cells to anchor, ECM also functions as a ligand reservoir by sequestering numerous growth factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF).68 Breakdown of ECM by matrix metalloproteinases (MMPs) liberates these growth factors, which in turn promote metastatic spread by breaking down the ECM and by stimulating angiogenesis and lymphangiogenesis.69-71

Melatonin exhibits versatile regulatory actions on MMP gene expression and activity in addition to its anti-inflammatory and antioxidant properties.72 An inhibitory effect of melatonin on the induction and catalytic activity of MMP-9 in a gastric adenocarcinoma cell line has been demonstrated.73 Melatonin interferes with MMP-9 activity through directly docking into the active site of MMP-9 and interacting with key residues within the catalytic site including three histidines that form the coordination complex with the catalytic zinc as well as proline 421 and alanine 191. MMP-9 is a zinc-dependent endopeptidase for gelatin and collagen and mainly participates in the angiogenic switch necessary for tumor development.74 Its expression levels are associated with cancer invasion and metastasis.75 The detailed mechanisms through which melatonin represses the expression of MMP-9 have been revealed. In a study of melatonin-regulated renal cancer metastases, melatonin inhibited NF-κB-mediated MMP-9 transcription and cancer cell invasion by targeting the Akt-Erk/JNK pathways.76 In addition, an epigenetic regulation of melatonin on MMP-9 expression has been proposed in an investigation into oral cancer and nasopharyngeal carcinoma cell migration induced by a phorbol ester, TPA.77, 78 By reducing histone acetylation on the promoter of the MMP-9 gene, melatonin attenuates MMP-9 expression to hinder the motility of oral cancer cells. These results indicate a promising role for melatonin to constrain cancer invasion and metastasis by MMP-9-dependent ECM turnover.

2.4 Cytoskeleton reorganization

As discussed above, the dynamic interplay between cell–cell and cell–matrix adhesion contributes to the plasticity of cancer cells that allows them to respond to external cues, from either ECM components or ECM-associated molecules. These signaling events subsequently orchestrate the changes in the organization of cytoskeletal proteins, which are essential to drive directional cell migration and invasion. The cytoskeletal elements, composed of actin microfilaments, microtubules and intermediate filaments, are extensively integrated and play a key role in controlling cell shape and movement. Actin microfilaments and microtubules are dynamic polymer structures that are capable of organizing cytoplasmic organelles and intracellular compartments, determining cell polarity and generating both expansion and contractile forces.79 During cell migration, these subcellular organelles affect the stabilization of cell–cell and cell–matrix adhesion through their interactions with cadherins and integrins, respectively, and generate protrusive forces at the front and retraction forces at the rear.80 Their reorganization is central to the cross talk between E-cadherin and integrins, which is regulated by the phosphorylation of two key kinases: myosin light-chain kinase (MLCK) and Rho-associated protein kinase (ROCK), a downstream effector of Rho GTPase.81 These two kinases are pivotal in regulating the collective migration of cells by controlling the cytoskeletal rearrangement associated with the two adhesion types.

Increased expression of E-cadherin and β₁ integrin is involved in the slowing of breast cancer cell migration by melatonin.58 A follow-up study further demonstrated that melatonin inhibits cancer cell invasion and metastasis via ROCK-regulated microfilament and microtubule organization that converge in a migration/anchorage switch in the migrating leader cells.82 The ROCK family consists of two isoforms, ROCK1 and ROCK2, sharing 65% overall homology and 92% homology in the kinase domain. Both kinases play primary roles in the organization of the cytoskeleton and are implicated in a wide range of fundamental functions in cancer progression.83 In addition to interfering with the kinase activity, melatonin is effective in restraining cancer migration through the downregulation of ROCK-184, 85 and MLCK.61 These findings highlight a role of melatonin in cytoskeleton rearrangement by targeting several key signaling molecules during cancer metastasis.

2.5 Epithelial–mesenchymal transition

The morphogenesis process of epithelial–mesenchymal transition (EMT), in which epithelial cells shift toward the mesenchymal state and become migratory, was first recognized as a principal process in embryonic development and organogenesis (Figure 2).86 Recently, mounting evidence has indicated that manipulation of EMT also affects cancer progression and metastasis.87, 88 The molecular and cellular changes in EMT are characterized by a loss of epithelial cell–cell junctions and apical–basal polarity, rearrangement of cytoskeleton, and gain of migratory phenotypes.89 This switch in cell architecture and behavior is mediated by a series of transcriptional and signaling events elicited from various extracellular stimuli.90 Signaling pathways, such as NF-κB, Wnt, Notch, Hedgehog, AP-1, and growth factor signaling, induce or modulate the EMT process.91-93 Many of these signal cascades converge at the level of EMT-associated transcription factors, of which Snail, Slug, Twist, and Zeb are commonly unregulated in metastatic cells that are undergoing EMT;87 they function to regulate a hallmark of EMT and cancer metastasis, the downregulation of E-cadherin.94-98

Melatonin impedes the EMT process and cancer cell dissemination through interference with NF-κB signaling.59 The effects of NF-κB on melatonin-mediated inhibition of EMT are partly attributed to its direct and indirect regulation of EMT-associated transcription factors, Snail, Slug, Twist, and Zeb,99 which negatively control the expression of E-cadherin. In addition to suppressing an epithelial phenotype, NF-κB also contributes to the induction of vimentin,100-102 a cytoskeletal protein vital for cell migration, and promotes and maintains a mesenchymal state. Decreased expression of vimentin was observed when the migration and invasion rate of mammospheres formed by breast cancer cells was repressed after melatonin treatment (NF-κB inhibition).103 Also upregulated by NF-κB to display metastatic behaviors is another mesenchymal marker, MMP-9.104-106 An inverse correlation between melatonin receptor 1A (MTNR1A also known as MT1) and MMP-9 expression in renal cancer and normal kidney tissues has been noted.76 By targeting NF-κB and Akt-MAPK pathways, melatonin inhibited MMP-9 transactivation and renal cancer metastasis. These data collectively link NF-κB to melatonin's actions on curbing the EMT and cancer metastasis.

The Wnt/β-catenin pathway represents another key regulator of the EMT. β-catenin remains a core element of the adherens junction by virtue of its interaction with E-cadherin.107 During the EMT, loss of E-cadherin leads to the dissociation of β-catenin from the junctional complex. As such, β-catenin then translocates to the nucleus and binds to the T-cell factor/lymphocyte enhancer factor (TCF/LEF), a main transcription factor that turns on the expression of Wnt target genes, including Snail and Slug. In the absence of Wnt signaling, a destruction complex, composed of glycogen synthase kinase 3 β (GSK3β), axin, adenomatous polyposis coli (APC), and casein kinase 1, phosphorylates excess cytoplasmic β-catenin and targets it for ubiquitin-dependent degradation. In a xenograft model of breast cancer, the phosphorylation of GSK3β, resulting in inhibition of its activity, is regulated in a circadian manner.108 Melatonin activates GSK3β by interfering with Akt phosphorylation to induce β-catenin turnover and hinders the EMT, revealing a promising role for circadian gating of EMT in metastatic spread via regulation of GSK3β.

Recently, Rsk2, a key signaling node activated by the HER2/MAPK/Erk pathway, was identified as being involved in the melatonin-mediated suppression of EMT and late-staged metastasis in breast cancer cells.109 HER2 is a member of the human epidermal growth factor receptor (HER) family that plays an important role in the progression of certain aggressive types of breast cancer.110 This finding provides insight into a therapeutic use of melatonin in patients with HER2-positive breast cancer.

2.6 Angiogenesis

An extensive vascular network is required in the tumor mass to provide the supply of oxygen and nutrients and the route for malignant cells to escape from the primary site. The process whereby new blood vessels form from pre-existing ones is called angiogenesis. Tumor angiogenesis requires complex interactions between endothelial cells and the microenvironment, allowing the endothelial sprouts to grow, convert into endothelial tubules, and connect with other vessels.111 Several key signaling pathways have been defined that mediate angiogenesis.112 This list includes, but is not limited to, growth factors, integrins, cell-surface receptors, cytokines, chemokines, lipids, and the ECM.

Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic factor thought to be crucial for tumor angiogenesis and metastasis.113 As the VEGF family is present not only in cancer but also in the adjacent non-neoplastic cells, such as endothelial cells, it has emerged as a therapeutic target in anticancer treatment.114 The anti-angiogenic activity of melatonin is related to a decline in VEGF secretion in patients with advanced cancer.115 Various studies using cancer cell lines demonstrated that melatonin destabilized the transcription factor, hypoxia-inducible factor-1α (HIF-1α), which enhances the expression of VEGF.116-119 Several distinct mechanisms underlying melatonin's actions in reducing the levels of HIF-1α and VEGF have been proposed. In a mouse model of renal cancer, the action of melatonin on HIF-1α and VEGF expression was associated with its antioxidant activity and independent of its membrane receptors.115 Such inhibition of HIF-1α-induced transcription is explained by the altered activity of the ubiquitin ligase, von Hippel–Lindau (VHL) protein, which recognizes HIF-1α as a substrate and remains part of the oxygen-sensing mechanism of the cell.120 Another line of evidence, however, related melatonin-mediated inhibition of HIF-1α and VEGF to its interaction with the nuclear receptor RZR/RORγ in human gastric cancer cells.119 In addition, a post-transcriptional regulation through which melatonin modified HIF-1α and HIF-2α concentrations via changes in microRNA species has been described in prostate cancer cells.119

Endothelin-1 (ET-1) is a vasoconstrictor that orchestrates different stages of tumor angiogenesis.121 Through the binding to its cognate receptors, a family of G protein-coupled receptors, ET-1 upregulates MMP-2 and VEGF in endothelial and ovarian cancer cells, respectively.122, 123 It has been reported that melatonin reduced ET-1 expression and secretion in colon cancer cells via the inactivation of FoxO1 and NF-κB,124 revealing an additional role of melatonin in modulating angiogenesis.

In addition to acting on the cancer cells, melatonin directly exhibits anti-angiogenic effects by suppressing the proliferation and migration of endothelial cells.125 A wide spectrum of melatonin receptors, including MT1, MT2, RORα, and RORβ, but not RORγ, was found to be expressed in a line of human primary endothelial cells, the human umbilical vein endothelial cell (HUVEC).126 Through these receptors, high concentrations of melatonin markedly reduced HUVEC proliferation via the Erk/Akt/NF-κB pathway. Inhibition of NF-κB by melatonin in HUVEC also protected the permeability of endothelial monolayer by attenuating MMP-9 expression and activity;127 a marked rise in vascular permeability is a hallmark of angiogenesis. These results demonstrated that melatonin functions as an anti-angiogenic agent through the reduction in VEGF secretion by cancer cells and by directly antagonizing endothelial cell activation.