Cysteinyl Leukotriene Pathway and Cancer

Cancer remains a leading cause of death worldwide, despite many advances being made in recent decades. Changes in the tumor microenvironment, including dysregulated immunity, may contribute to carcinogenesis and cancer progression. The cysteinyl leukotriene (CysLT) pathway is involved in several signal pathways, having various functions in different tissues. We summarized major findings of studies about the roles of the CysLT pathway in cancer. Many in vitro studies suggested the roles of CysLTs in cell survival/proliferation via CysLT1 receptor (CysLT1R). CysLT1R antagonism decreased cell vitality and induced cell death in several types of cancer cells, such as colorectal, urological, breast, lung and neurological malignancies. CysLTs were also associated with multidrug resistance of cancer, and CysLT1R antagonism might reverse chemoresistance. Some animal studies demonstrated the beneficial effects of CysLT1R antagonist in inhibiting tumorigenesis and progression of some cancer types, particularly colorectal cancer and lung cancer. The expression of CysLT1R was shown in various cancer tissues, particularly colorectal cancer and urological malignancies, and higher expression was associated with a poorer prognosis. The chemo-preventive effects of CysLT1R antagonists were demonstrated in two large retrospective cohort studies. In summary, the roles of the CysLT pathway in cancer have been delineated, whereas further studies are still warranted.


Unmet Need of Cancer Treatment
Cancer is a major public health issue/concern worldwide and is the leading cause of death in many countries, including Taiwan [1,2]. Many types of procedures and medications are now available for cancer treatment, including surgery, radiotherapy, chemotherapeutic agents and targeted therapy. The development of immunotherapy seems to open a new era of cancer treatment. Check point inhibitor, one of the different types of immunotherapeutic agents, has been demonstrated to treat certain types of cancer alone or in combination with chemotherapeutic agents [3]. Although many anti-cancer treatments are available, drug resistance usually develops sooner or later, and some patients had poor or even no response to the treatment at the beginning. As a result, high cancer mortality remains, suggesting the urgent need of a large improvement in cancer treatment.
The tumor microenvironment is composed of the extracellular matrix and basement membrane, endothelial cells, cancer-associated fibroblasts, neuroendocrine cells, adipose cells, pericytes and tumor-infiltrating immune cells [4]. A tumor and its microenvironment interact constantly and influence each other. Such interactions begin at the very early phase The tumor microenvironment is composed of the extracellular matrix and basement membrane, endothelial cells, cancer-associated fibroblasts, neuroendocrine cells, adipose cells, pericytes and tumor-infiltrating immune cells [4]. A tumor and its microenvironment interact constantly and influence each other. Such interactions begin at the very early phase of tumor formation, and continue during primary growth, local invasion, intravasation and establishment at the metastatic site. The role of the tumor microenvironment has been demonstrated to modulate the aggressiveness, motility, dissemination and colonization of cancer cells in distal organs in the past two decades [5]. Inflammation, including the leukotriene pathway, has been proposed as one of the mechanisms in tumor initiation, progression and metastasis [6]. Cancer-associated immune dysregulation is also a key contributor for tumor progression and metastasis.
By understanding the role of tumor microenvironment and cancer immunology in cancer progression, as well as the underlying detailed molecular mechanisms, we will have a better chance of developing novel diagnostic modalities and therapeutic agents for cancer diagnosis and treatment.

Leukotrienes
Leukotrienes were initially identified in the late 1970s, as a family of inflammatory lipid mediators synthesized from arachidonic acid (AA) in different cells, including mast cells, eosinophils, neutrophils, basophils and macrophages [7].
Due to lacking the peptide side chain of cysteinyl leukotrienes, LTB 4 was not classified as cysteinyl leukotriene [13]. As a classical chemoattractant, LTB 4 could trigger aggregation and adherence of leukocytes to the endothelium. Furthermore, it also regulates the immune responses associated with host-defense against infections. LTB 4 is involved in many inflammatory diseases, including dermatitis, arthritis, nephritis and chronic obstructive pulmonary disease [10].

Cysteinyl Leukotrienes
The cysteinyl leukotrienes (CysLTs) include LTC 4 , LTD 4 and LTE 4 ( Figure 1). Among them, LTE 4 is the most stable, which can be measured in the urine. The urinary LTE 4 level could therefore be used as a marker of 'whole body' leukotriene synthesis [14]. LTC 4 is an 18-kDa membrane protein. It is a noncovalent homodimer. Mg 2+ could augment the enzyme activity of LTC 4 ; on the other hand, Co 2+ and the function of FLAP inhibitor MK886 could inhibit it [15]. LTD 4 increased cytosolic calcium and activated the MAPK pathway in THP-1 cells, a human monocytic leukemia cell line [16,17]. Activation of PKC inhibited LTC 4 synthase activity and attenuates production of CysLTs in an eosinophilic sub-strain of human myeloid leukemia cell line HL-60 [18]. CysLTs are well-known for their role in inflammation, and have been reported to have pro-angiogenic activities [19][20][21].

CysLT 1 Receptor (CysLT 1 R)
CysLT 1 R distributes in various human tissues, including the respiratory system, peripheral blood leukocytes, gastrointestinal system and brain [7]. Smooth muscle cells, epithelial cells, interstitial lung macrophages and basophils accumulating in the airways of asthma patients could express CysLT 1 R [7,23,24]. The expression of CysLT 1 R could also be found in peripheral blood leukocytes, including monocytes, macrophages, eosinophils, pregranulocytic CD34 + cells, neutrophils and some B lymphocytes [7]. Tumors of the colon and brain also express CysLT 1 R [22].
CysLT 1 R is involved in several signal pathways. LTD 4 is able to induce elevation of intracellular free Ca 2+ concentration and phosphatidylinositol metabolism [25]. Via non-voltage gated channels, LTD 4 could lead to constriction of the small bronchioles [26]. Activating CysLT 1 R induces phosphorylation of mitogen-activated protein kinase (MAPK) through a G i/o -protein in mesangial cells, airway smooth muscle cells and human mast cells [22].
STAT-1 was demonstrated to be involved in the signal transduction mechanism of the CysLT 1 R; phosphorylation of STAT-1, through protein kinase C (PKC) and ERK1/2 activation, causes expression of ICAM-1 and increased eosinophil adhesion [24]. In studies of human asthma smooth muscle cells, LTD 4 stimulation of G i/o -coupled CysLT 1 Rs leads to the transactivation of the epidermal growth factor receptors (EGFRs) through the intervention of PI3K and ROS, followed by the classical Ras-ERK1/2 signaling pathway. It could then control G1 progression and cell proliferation [23]. CysLT 1 R also plays an important role in Alzheimer's disease. In the study of primary cultured neurons, LTD 4 was demonstrated to cause the production of Aβ by enhancement of βor γ-secretase resulting from activating the CysLT 1 R-mediated NF-κB signaling pathway [27].

CysLT 2 Receptor (CysLT 2 R)
The mRNA of CysLT 2 R mRNA expression is high in the heart, interstitial macrophages of lung, brain, spleen, lymph nodes and peripheral blood [28]. The expression level of CysLT 2 R mRNA is particularly high in the hypothalamus, thalamus, putamen, pituitary gland and medulla of brain. In the peripheral blood, eosinophils particularly express very high level of CysLT 2 R [28]. On the cell surface of human umbilical vein endothelial cells, CysLT 2 R is highly expressed and might be involved in leukotriene-dependent vascular reactions [29].
CysLT 2 R induced microglia M1 polarization through activating the NF-κB pathway, and might promote inflammation and neuronal damage [30]. Aspirin-exacerbated respiratory disease is associated with idiosyncratic CysLT-and mast cells-driven reactions to aspirin. In aspirin-exacerbated respiratory disease, CysLT 2 R signaling on platelets could use RAGE/HMGB1 as a link to the downstream type 2 respiratory immunopathology and IL-33-dependent mast cell activation typical of aspirin-exacerbated respiratory disease [31].

CysLT 3 Receptor (CysLT 3 R)
CysLT 3 R has high-affinity for LTE 4 [32]. CysLT 3 R was demonstrated to be expressed on murine airway epithelial cells and to mediate goblet cell mucin release in response to exogenous LTE 4 [33]. CysLT 3 R could also regulate the numbers of goblet cells in the nasal mucosa. The aeroallergen and LTE 4 -elicited CysLT 3 R-dependent type 2 lung inflammation could be attenuated by blocking IL-25. Therefore, CysLT 3 R may have the potential to be a therapeutic target for inflammatory lung disease [34].

Cysteinyl Leukotriene Pathway Antagonists
In daily clinical practice, medications targeting the CysLT pathway are implicated in the treatment of asthma and allergic rhinitis. In patients with asthma, a chronic inflammatory lung disease characterized by airway hyperreactivity, CysLTs increased vascular permeability and smooth-muscle contraction, which are the causes of patients' symptoms [35]. The pharmacological mechanism of montelukast, zafirlukast and pranlukast in treating asthma is antagonism of CysLT 1 Rs (Table 1). The other drug, zileuton, can be used for treating asthma by inhibiting 5-LO. However, the responses to these pharmacologic blockades remain heterogenous [35].

In Vitro Studies about the Roles of Cysteinyl Leukotriene Pathway in Cancer
Major findings of in vitro studies about the roles of the CysLT pathway in cancer are summarized in Table A1 in Appendix A.

Hematologic Malignancies
Studies about physiological roles of CysLTs in cancer cells have been initiated since 1980s, mainly in leukemia cells [36,37]. Increased LTC 4 production was noted in chronic myeloid leukemia (CML) [37,38]. Some early studies using leukemia cell lines further enriched our knowledge about the role of CysLTs in cell biology [16][17][18].
In line with clinical practice, tyrosine kinase inhibitors, such as imatinib, dasatinib and nilotinib, inhibited cell growth of CML cells, and montelukast further reduced cell proliferation in a dose-dependent manner as key proteins of the leukotriene pathway were expressed in these CML cells [39]. A subsequent study revealed that montelukast, through a CysLT 1 R-dependent pathway, induced apoptosis of CML cells by inducing Bax overexpression, cytochrome C release, PARP-1 cleavage and caspase-3 activation, which could be additive to the pro-apoptotic effect of imatinib; montelukast also altered Wnt/β-catenin signaling, inducing phosphorylation of β-catenin and downregulating the downstream target c-myc [40].
CysLT 1 R expression was demonstrated in human primary mediastinal B-cell lymphoma cell lines (Med-B1, Karpas-1106P); CysLTs induced a calcium signal in Med-B1 cells, which could be blocked by zafirlukast [42]. Some of the human Hodgkin lymphoma cell lines, including L1236 and KMH2 cells, expressed functional CysLT 1 R, responding with a robust calcium signal upon LTD 4 challenge, which could be blocked by zafirlukast [43].

Colorectal Cancer
The role of the CysLT pathway has been extensively studied in intestinal epithelium and associated tumors. The expression of CysLT 1 R was demonstrated in intestine 407 (Int 407), a non-transformed human embryonic intestinal epithelial cell line and two human colon cancer cell lines, Caco-2 and SW-480 [44].
Several studies have demonstrated the role of the CysLT pathway in cell survival of colorectal cancer cells. Overexpression of CysLT 1 R increased cell viability in Caco-2 cells [44]. A study using Int 407 found that LTD 4 was capable of preventing apoptosis induced by NS-398, a COX-2 inhibitor [45]. Further study revealed that LTD 4 not only reversed the apoptosis induced by COX-2 inhibition but also reduced the apoptotic potential by lowering the basal level of caspase 8 activation and truncated Bid generation [46]. LTD 4 enhanced proliferation via a distinct Ras-independent, PKC -dependent activation of Erk-1/2 and a parallel Ras-dependent signaling pathway [47]. A study using Int 407 and Caco-2 cells demonstrated that LTD 4 induced upregulation of COX-2 and Bcl-2 through a pertussis toxin sensitive G-protein and MAPK pathway [48]. In both Int 407 and Caco-2 cells, LTD 4 stimulation induced activation and nuclear translocation of cytosolic phospholipase A 2 α, an important regulator of colon tumor growth, via a calcium-dependent mechanism involving activation of PKC and the MAPK pathway [49]. LTD 4 increased the level of free β-catenin in Int 407 cells; the increased free β-catenin translocated to the nucleus where it activated TCF/LEF transcription factors; the increased free β-catenin also translocated to the mitochondria where it associated to the antiapoptotic protein Bcl-2 [50]. Further studies showed that LTD 4 increased mitochondrial metabolic activity and gene transcription and increased reactive oxygen species levels and subsequent activations of the p65 subunit of NF-κB, presumably through β-catenin accumulation in the mitochondria [51]. Collectively, these findings lend credence to the idea that the CysLT pathway potentially provides intrinsic oncogenic signals involving cell survival and anti-apoptosis.
Furthermore, LTD 4 increased the motility of Int 407 cells via a PI3K/Rac signaling pathway [52]. LTD 4 increased β-catenin level in colon cancer cells [53]. LTD 4 induced nuclear translocation of β-catenin, upregulated β-catenin target genes and increased the proliferation and migration in HCT116 cells, but not in HT29 cells; the effect could be prevented by pretreatment with ZM198,615, a CysLT 1 R antagonist [53].
CysLT 1 Rs were found in the plasma membrane and outer nuclear membrane in both Int 407 and Caco-2 cells [54]. The colorectal carcinoma cell line, Caco-2 cells, appeared to have greater intracellular formation of CysLTs and more CysLT 1 Rs in both plasma membrane and outer nuclear membrane than the non-tumor cell line, Int 407 cells [54]. Another study found that the basal level of CysLT 1 R was higher in several colon cancer cell lines (HT-29, SW-480, Caco-2 and HCT-116) compared to Int 407 cells [55]. LTD 4 significantly increased CysLT 1 R expression in Int 407 cells, but not in colon cancer cell lines; LTD 4 induced upregulation of CysLT 2 R in several colon cancer cell lines [55]. LTD 4 could also induce nuclear translocation of CysLT 1 Rs from the plasma membrane to the nucleus in Int 407 cells, but not in Caco-2 cells [54].
A study using several intestinal epithelial cell lines found the autocrine pattern of endogenously produced CysLTs which mediated the survival and proliferation of intestinal epithelial cells via CysLT 1 R signaling [56]. MK571, a CysLT 1 R antagonist, induced apoptosis and dose-dependent proliferation inhibition in two non-tumor cell lines (Int 407 and IEC-6 cells), but only led to proliferation reduction without apoptosis in the tumor intestinal cell lines (Caco-2, SW480, HCT-116 and HT-29 cells); the presence of nuclear CysLT 1 Rs in intestinal cancer cells, which are inaccessible to the receptor antagonist, might provide a clue to the finding [56]. In a study with HT-29 and SW-480, montelukast prevented LTD 4 -induced colony formation and disrupted colonospheres as well as downregulation of cancer stem cell markers (ALDH1 and DCLK1), suggesting the beneficial effect in minimizing cancer stem cells of CysLT 1 R inhibition [57]. In addition, 1,4-dihydroxy quininib reduced clonal formation and gene silencing of CysLT 1 R significantly reduced expression of angiogenic marker calpain-2, which further confirmed the importance of CysLT 1 R in cancer progression and angiogenesis [58].
The expression of low-affinity CysLT 2 R, compared with CysLT 1 R, was higher in a nontumor cell line (Int 407) but was lower in two colon cancer cell lines (Caco-2 and SW480) [59]. Similar to CysLT 1 R, CysLT 2 R was found to be located both at the plasma membrane and the nuclear membrane [59]. Although CysLT 2 R signaling had no effect on cell proliferation or apoptosis of Caco-2 cells, LTC 4 increased the activity of alkaline phosphatase and aminopeptidase N, suggesting the role of CysLT 2 R in cellular differentiation [59]. A subsequent study showed that IFN-α could upregulate CysLT 2 R in Caco-2 cells [60]. LTC 4 induced expression of mucin-2, and the effect could be blocked by AP 100984 (a specific CysLT 2 R antagonist) but not by montelukast (a specific CysLT 1 R antagonist) [60]. CysLT 2 R signaling was able to suppress cell migration induced by epidermal growth factor (EGF) signaling in Int 407 cells [60]. All-trans retinoic acid (ATRA) treatment increased CysLT 2 R expression without affecting CysLT 1 R level, and upregulated LTC 4 synthase in SW480 cells; the effect was not observed in HCT-116 cells, which was ATRA-resistant [61]. ATRA did not affect cell proliferation or induce apoptosis of SW480 cells [61]. ATRA induced MUC-2 expression and alkaline phosphatase activity in SW480 cells, and AP 100984 reduced the effect [61].

Pancreatic Cancer and Hepatoma
A comprehensive study revealed CysLT 1 R expression in several pancreatic cancer cell lines (PA-TU-8988T, SUIT-2 and PANC1 cells, but not in MIAPaCa-2 cells); LTD 4 promoted the proliferation of pancreatic cancer cells, whereas treatment with montelukast caused cell cycle arrest at G0/G1 phase without inducing apoptosis [62].
In a recent study using human hepatoma cell lines, CysLT antagonists (pranlukast and montelukast) inhibited ADAM9 activity and upregulated level of membrane-bound MHC class I-related chain A (mMICA), which might facilitate natural killer cell-mediated cytotoxicity, suggesting the potential of using leukotriene receptor antagonists along with regorafenib in the treatment of hepatoma [63].

Breast Cancer
LTB 4 and LTD 4 inhibited MCF-7 breast cancer cell growth, and a leukotriene antagonist (LY171883) and a 5-LO inhibitor (MK886) further lifted the inhibitory effect, suggesting that LT-receptors mediated the growth-inhibitory effect of LTB 4 or LTD 4 [72]. CysLTR antagonists, montelukast and zafirlukast reduced cell viability of MDAMB-231, a triplenegative breast cancer cell line, in a dose-dependent manner; zafirlukast mainly exerted its action on cell cycle, while montelukast mainly induced apoptosis [73].
Similar to the findings in colon cancer, activating CysLT 2 R signaling with LTC 4 (preferentially binds to CysLT 2 R, rather than CysLT 1 R) did not affect cell proliferation or apoptosis of breast cancer cells but reduced cell migration [74].

Lung Cancer
Inhibition of 5-LO resulted in interruption of 5-LO-mediated growth factor signaling, resulting in significant growth reduction and enhanced apoptosis in a number of lung cancer cell lines [75]. Furthermore, 5-LO inhibitors (AA861 or ETH 615-139) and zafirlukast (a CysLT 1 R antagonist) blocked the release of organic osmolytes (taurine, meAIB) and the concomitant cell volume restoration following hypoosmotic swelling of A549 cells; inhibition of 5-LO or CysLT 1 R did not affect caspase-3 activity during hypoxia [76].
Montelukast and zafirlukast inhibited proliferation and induced apoptosis of glioblastoma cells (A172 and U-87 MG cells) in a concentration-dependent manner; both medications decreased Bcl-2 expression without affecting Bax level [79]. Montelukast induced more apoptosis than zafirlukast in A172 cells, but not in U-87 MG cells; zafirlukast caused cell cycle arrest at G0/G1 phase by upregulating the expression of p53 and p21 and showed a greater antiproliferative effect than montelukast [79]. Montelukast and zafirlukast, but not zileuton, significantly inhibited migration and invasion of glioblastoma cells, as well as inhibiting the expression and activities of MMP-2 and MMP-9 in glioblastoma cells and primary human astrocytes, suggesting the important role of CysLT 1 R in migration and invasion of glioblastoma [80].

Drug Resistance and Cysteinyl Leukotriene Pathway
The LTD 4 receptor has been highly associated with multidrug resistance protein (MRP), a membrane transporter of multiple anticancer drugs. MK571, a CysLT 1 R antagonist, modulated MRP-associated multidrug resistance in HL60/AR and GLC4/ADR cells, MRP-overexpressing multidrug resistant human leukemia and human small cell lung cancer cell lines, respectively, in a dose-dependent manner [83]. Similarly, increased expression of multidrug-resistance-associated protein 1 (MRP1) was observed in multidrug resistant phenotype of prostate cancer, and adding non-toxic doses of MK571, zafirlukast or buthionine sulfoximine significantly increased the sensitivity of the MDR models to cytotoxic drugs [84].
ONO-1078, a peptide LTD 4 receptor antagonist, inhibited the transporting activity of MRP and increased vincristine uptake, resulting in increased sensitivity to vincristine of multidrug-resistant CV60 and its parental drug-sensitive KB-3-1 cell line [85]. Similarly, ONO-1078 enhanced the sensitivity of lung cancer NCI-H520 cells to several chemotherapeutic agents, including vincristine, doxorubicin and etoposide, through inhibiting the function of MRP [86].
To investigate the role of CysLT 1 R in chemoresistance, a study established 5-FUresistant colon cancer cell lines by culturing with increasing concentration of 5-FU over a period of 6-8 months [87]. The 5-FU-resistant colon cancer cell lines expressed increased CysLT 1 R, which regulated 5-FU resistance via β-catenin activation and promoted 5-FUresistance-derived stemness [87]. Montelukast restricted the motility of 5-FU-resistant colon cancer cells, sensitized them to 5-FU and decreased 5-FU-resistance-derived stemness, suggesting that inhibition of CysLT 1 R signaling with montelukast might reverse drug resistance of colon cancer [87].

Animal Studies about the Roles of Cysteinyl Leukotriene Pathway in Cancer
Major findings of animal studies about the roles of the CysLT pathway in cancer are summarized in Table A2 in Appendix A.

The Role of Vascular Permeability Mediated by Cysteinyl Leukotrienes
CysLTs may modulate vascular permeability, and therefore may affect drug delivery or tumor metastasis. An animal study using a rat C6 glioma model showed that LTE 4 selectively opened the blood-tumor barrier and increased the tumor uptake of intravenously injected methotrexate [88].
In contrast, CysLTR antagonists may inhibit tumor metastasis by inhibiting capillary permeability. An animal study using transplantable rat colon adenocarcinoma RCN9 cells implanted via the cisterna magna of male Fisher rats showed that pranlukast, but not montelukast, could inhibit tumor cell migration through brain capillary [89]. Using Lewis lung carcinoma metastasis model in mice, the effect of both pranlukast and montelukast on inhibiting tumor cell migration through peripheral capillary was demonstrated [89].
A study using male C57BL/6 mice, including wild type, Cysltr1 −/− and Cysltr2 −/− , implanted with Matrigel plugs or subcutaneously injected with Lewis lung carcinoma cells demonstrated the important role of CysLT 2 R in regulating tumor angiogenesis, metastasis and endothelial cell dysregulation [90]. CysLT 2 R-regulated angiogenesis was shown in isolated mouse endothelial cells and in Matrigel implants [90]. The growth and metastases of implanted Lewis lung carcinoma cells were significantly reduced in CysLT 2 R-null mice, compared with wild-type or CysLT 1 R-null mice. In wild-type mice subcutaneously implanted with Lewis lung carcinoma cells, the expression of CysLT 2 R, but not CysLT 1 R, was increased in tumor vasculature, while BayCysLT 2 (a selective CysLT 2 R antagonist), but not MK571 (a CysLT 1 R antagonist), reduced tumor growth, angiogenesis and lung metastasis of tumor cells [90].

Colorectal Cancer
The role of CysLTs in colon cancer have been investigated in several studies using a xenograft model of nude mouse subcutaneously injected with human colon cancer cells. LTD 4 promoted cancer-initiating cells in initiating tumor growth by allowing immunomodulation of the tumor microenvironment [91]. The important role of CysLT 1 R in colon tumorigenesis was shown in a study using a colitis-associated colon cancer mice model induced by azoxymethane/dextran sulfate sodium, showing higher relative body weight, reduction in inflammation and polyps with lower-grade dysplasia and decreased nuclear expression of β-catenin and COX-2 in mice with global disruption of CYSLTR1 gene expression [92]. In studies using a xenograft model, CysLT 1 R antagonists (montelukast and ZM198,615) inhibited proliferation and induced apoptosis of tumor cells, resulting in reduced size of the inoculated tumor [57,93]. Montelukast also significantly decreased amounts of cancer-stem cells, as well as macrophage infiltration and decreased expression of ALDH1A1, DCLK1 and BCL2 in the tumor tissue [57]. As CysLTs might involve in angiogenesis, 1,4-dihydroxy quininib significantly reduced tumor volume and the expression of angiogenic marker calpain-2 [58].

Pancreatic Cancer
The chemo-preventive effect and therapeutic potential of montelukast on pancreatic cancer was studied with a Syrian golden hamster model, using N-nitrosobis (2-oxopropyl) amine (BOP) to induce pancreatic ductal carcinomas, which were shown to be similar to those in humans morphologically and molecularly; montelukast suppressed pancreatic carcinogenesis by suppressing cell proliferation via the LTD 4 -CysLT 1 R axis [62].

Lung Cancer
The effects of leukotriene pathway inhibitors (zafirlukast, MK886 and Zileuton) on preventing lung cancer were investigated with a mice model of lung tumors induced by vinyl carbamate injection; leukotriene pathway inhibitors (zafirlukast, MK886 and zileuton) prevented lung tumor formation and slowed the growth and progression of adenomas to carcinoma [94]. A later study, using an orthotopic immunocompetent mouse model of lung cancer with Lewis lung carcinoma cells injected into lungs of syngeneic mice, found increased production of leukotrienes (LTB 4 , LTC 4 , LTD 4 and LTE 4 ), in dependent on cytosolic phospholipase A 2 , in the tumor microenvironment [95]. Our study using mice with Lewis lung carcinoma cells injected subcutaneously (to monitor the tumor growth on a daily basis) showed that montelukast significantly hampered the tumor growth, and the tumor tissue of the montelukast group showed markedly decreased Ki-67 expression and markedly increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells [77].

Neurological Malignancies
An animal study using experimental brain tumor of intracerebral C6 glioma in a Sprague-Dawley rat model found that pretreatment with LTC 4 significantly extends survival in rats treated with cisplatin [96].

Clinical Studies about the Roles of Cysteinyl Leukotriene Pathway in Cancer
Major findings of clinical studies about the roles of the CysLT pathway in cancer are summarized in Table A3 in Appendix A.

Hematological Malignancies
A study analyzing blood samples from 17 CML patients in the chronic phase and 15 healthy medication-free volunteers found that mature CML CD16 (+) neutrophils had aberrantly increased expression of LTC 4 synthase, which might be responsible for stimulating the proliferation of human bone marrow-derived myeloid progenitor cells [97]. A subsequent study showed that urinary excretion of LTE 4 was significantly higher in CML patients than healthy controls, supporting the increased LTC 4 synthase activity in CML patients; they further found that neutrophilic LTC 4 synthase expression and activity were markedly elevated and were normalized with imatinib mesylate treatment [98]. A study using multiplex single cell polymerase chain reaction analyzed the expression of the mediators of the leukotriene pathway in bone marrow BCR-ABL + CD34 + CD38 − cells at diagnosis found the majority of cells expressed CysLT 1 R and CysLT 2 R, but not ALOX5; treatment with zileuton or montelukast failed to suppress cell growth, suggesting that targeting the CysLT pathway may not be a promising strategy to eradicate leukemia stem cells in CML patients [99].
The lymph node biopsy specimens from 57 non-Hodgkin lymphoma patients were assessed using immunohistochemical staining, and it was found that primary mediastinal B-cell lymphoma was the only type showing CysLT 1 R expression in tumor cells, while other types of lymphoma included in the study showed no CysLT 1 R expression [42]. The same study group further assessed biopsy specimens from 29 Hodgkin lymphoma patients with immunohistochemical studies and assessed the micro-dissected Hodgkin Reed-Sternberg cells with microarray analysis, showing that CysLT 1 R was expressed by primary Hodgkin Reed-Sternberg cells, which were surrounded by CysLT-producing eosinophils, macrophages and mast cells; the findings might suggest that CysLTs were important mediators in the pathogenesis of Hodgkin lymphoma, contributing to the aberrant cytokine network [43].

Colorectal Cancer
Several studies have analyzed tissue samples from colorectal cancer patients with immunohistochemical staining, in situ hybridization and tissue microarrays, showing higher CysLT 1 R and 5-LO expression, particular in the nuclei, in the cancer tissues than in normal colon tissues [54]. Nuclear accumulation of CysLT 1 R was strongly correlated with stronger staining of proliferative marker Ki-67 [54]. Higher expression of CysLT 1 R, particularly in the nucleus, was associated with a poorer prognosis [44,100], whereas higher nuclear expression of CysLT 2 R was associated with an earlier stage and better prognosis [59,100]. Aggressive tumors generally expressed less CysLT 2 R and IFN-α receptor and more EGFR, with a negative correlation between CysLT 2 R and EGFR expression, suggesting a potential protective role of CysLT 2 R against tumor progression [60]. In addition, 1,4-dihydroxy quininib significantly decreased the secretion of both angiogenic factor TIE-2 and adhesion molecule VCAM-1 in human ex vivo colorectal cancer tumor explants [58].

Esophageal Cancer and Gastric Cancer
A study assessed esophageal biopsy specimens with specific enzyme immunoassays found higher expression levels of LTB 4 and CysLTs in esophageal adenocarcinoma tissue compared to Barrett's metaplasia and control tissues [101]. However, another study analyzing esophageal biopsy samples with immunohistochemistry and quantitative reverse transcription-polymerase chain reaction showed significantly lower expression levels of CysLT 1 R and CysLT 2 R in esophageal cancer tissues than in control squamous epithelium [102].
Assessing with immunohistochemistry, gastric cancer tissue showed significantly increased immunoreactive score of CysLT 1 R than tumor-free gastric mucosa; the intestinal type had more CysLT 1 R and CysLT 2 R expression than the diffuse type [103].

Pancreatic Cancer and Hepatoma
In a study enrolling 92 hepatocellular carcinoma patients and 20 healthy control subjects, remarkably higher circulating LTD 4 level was noted in patients of hepatocellular carcinoma, particular in those with chronic hepatitis B infection or metastasis [104].
Immunohistochemical analyses of 108 pancreatic ductal adenocarcinoma tissues revealed that high CysLT 1 R expression was associated with worse overall survival [62].

Urological Malignancies
Strong CysLT 1 R expression was noted in several urological cancer samples, including renal cell carcinoma [64,68], bladder cancer (transitional cell carcinoma) [64,65], prostate cancer and prostatic intraepithelial neoplasia [64,67] and testicular cancer (including seminoma, embryonal carcinoma, yolk sac tumors, choriocarcinoma and teratoma) [64,66], than in the corresponding normal tissue, and more extensive and intense expression was noted in cancer with a higher grade or an advanced stage. Very weak CysLT 1 R expression was observed in relatively normal tissues (normal kidney tissue, normal bladder tissue, benign prostatic hyperplasia, normal prostate tissue and normal testis tissue) [64].

Breast Cancer
A study investigating 144 breast cancer specimens with tissue microarray and immunohistochemistry found that breast cancers with higher CysLT 1 R and lower CysLT 2 R expression levels were associated with higher histological grade and worse overall survival [74].

Other Malignancies
CysLT 1 R staining was evident in neuroblastoma surgical specimens and the adjacent vasculature, suggesting a potential treatment target [78].
A study assessing data of 80 primary uveal melanoma samples in The Cancer Genome Atlas (TCGA) showed that higher expression of CYSLTR1 and CYSLTR2 genes were significantly associated with poorer disease-free survival and overall survival [81]. However, the study group also examined the tissue from 52 patients of primary uveal melanoma using tissue microarray and only found poorer overall survival in those with higher CysLT 1 R expression, whereas CysLT 2 R expression was not associated with survival [81].

Chemopreventive Effects of Cysteinyl Leukotriene Inhibition
A large nationwide retrospective cohort study using the Taiwan National Health Insurance Research Database investigated the effect of CysLTR antagonists (LTRAs), including montelukast and zafirlukast, in reducing cancer incidence in asthma patients [105]. Comparing 4185 LTRA users with 20,925 LTRA non-users, we found that using LTRA decreased cancer risk in a dose-dependent manner, and the chemo-preventive effect of LTRA was markedly observed in terms of lung, colorectal, liver and breast cancer [105]. Another national retrospective cohort study used data from the Department of Veteran Affairs to investigate the association between leukotriene inhibition and lung cancer incidence among U.S. Veterans with asthma [106]. The researchers found that 23,730 patients with leukotriene pathway inhibiting medication (montelukast, zafirlukast, or zileuton) exposure, compared with 534,736 patients without exposure, had significantly reduced risk of lung cancer [106].

Future Perspectives
In summary, inflammation plays important roles in tumor initiation, progression and metastasis [6], and the role of the CysLT pathway in cancer has been demonstrated in several cancers using different approaches. Increased 5-LO expression and downstream CysLT pathway have been recognized as inflammation to shape the tumor microenvironment and are associated with cancer proliferation, migration and invasion. Serial change in the expression level of CysLT 1 R within the spectrum from normal tissue to low-grade and then advanced malignancies has been documented in several cancers. For example, very weak CysLT 1 R expression was observed in relatively normal tissues or benign lesions of urological organs [64]. In contrast, strong CysLT 1 R expression was noted in several urological cancer samples, with higher expression associated with a higher tumor grade or an advanced stage [64][65][66][67][68]. Similarly, higher CysLT 1 R and 5-LO expression was noted in colon cancer tissues than in normal colon tissues, with higher expression level of CysLT 1 R associated with higher tumor Ki-67 level and poorer prognosis [44,54,100]. The role of CysLT 1 R in colon tumorigenesis was demonstrated in a study using a colitisassociated colon cancer mice model, showing polyps with lower-grade dysplasia in mice with disruption of CYSLTR1 expression [92]. These findings demonstrated the important role of the CysLT pathway in carcinogenesis and tumor progression.
While the findings still appear somewhat heterogenous, further studies are warranted to clarify the role of the CysLT pathway in cancer biology, including cancer cells and their microenvironment, of different cancers. In our opinion, the following aspects require further investigation: (1) the roles of CysLTs and CysLTRs in proliferation, stemness, invasion and migration of cancer cells, as well as their secretomes and interactions with other cells in the tumor microenvironment; (2) the roles of CysLTs and CysLTRs in the cells in tumor microenvironment, particularly tumor-associated immune cells, which establish a cancerpromoting milieu; (3) the effects of CysLT inhibition, particularly CysLTR antagonists, in chemoprevention; and (4) the effects of CysLT inhibition, particularly CysLTR antagonists, as adjunct for the currently available standard anti-cancer treatment modalities. With more understanding about the role of the CysLT pathway in cancer, we will have better chance to provide precision care, including diagnostic and therapeutic approaches, for cancer patients.

Conflicts of Interest:
The authors declare no conflict of interest related to this article.

Author, Year Cancer Cells Major Findings
Magnusson C, 2007 [59] Int 407 and two human colon cancer cell lines (Caco-2 and SW-480) The expression of CysLT 2 R, compared with CysLT 1 R, was higher in Int 407 but lower in two colon cancer cell lines (Caco-2 and SW480). CysLT 2 R was found to be located both at the plasma membrane and the nuclear membrane. CysLT 2 R signaling led to terminal differentiation of Caco-2 cells but had no effect on cell proliferation or apoptosis.
Bengtsson AM, 2013 [61] colon cancer cell lines (Caco-2, SW-480, HCT-116) All-trans retinoic acid (ATRA) treatment increased CysLT 2 R expression without affecting CysLT 1 R level, and upregulated LTC 4 synthase in SW480 cells; the effect was not observed in HCT-116 cells. ATRA did not affect cell proliferation or induce apoptosis of SW480 cells. ATRA induced MUC-2 expression and alkaline phosphatase activity in SW480 cells, and a CysLT 2 R antagonist (AP 100984) reduced the effect.

Author, Year Cancer Cells Major Findings
Matsuyama M, 2007 [67] human prostate cancer cell lines (LNCaP, PC3, DU-145) and normal stromal prostate cell lines Montelukast significantly inhibited the proliferation and induced apoptosis of prostate cancer cell lines, but not normal stromal prostate cell lines.
Funao K, 2008 [68] human renal cell carcinoma cell line (Caki-1) and normal proximal tubular endothelial cells Montelukast significantly inhibited the proliferation and induced apoptosis of Caki-1, but not normal proximal tubular endothelial cells.
Brussel JP, 2004 [84] PC3 and DU-145, human prostate cancer cell lines Prostate cancer cell with multidrug resistant phenotype had increased expression of multidrug-resistance-associated protein 1 (MRP1) and adding MK571, zafirlukast or buthionine sulfoximine significantly increased the sensitivity to cytotoxic drugs.

Author, Year Cancer Cells Major Findings
Nagayama S, 1998 [85] human epidermoid carcinoma cell line (KB-3-1) and its multidrug resistant subclone (CV 60) ONO-1078, a LTD 4 receptor antagonist, inhibited the transporting activity of MRP and increased the sensitivity to vincristin of CV 60 and KB-3-1 cells.
Nakano R, 1998 [86] NCI-H520, a human lung cancer cell line ONO-1078 enhanced the sensitivity of lung cancer NCI-H520 cells to vincristine, doxorubicin and etoposide through inhibiting the function of MRP.
Montelukast restricted the motility of 5-FU-R colon cancer cells, sensitized them to 5-FU, and decreased 5-FU-R-derived stemness. Table A2. Selected animal studies investigating the cysteinyl leukotriene pathway in cancer.

Author, Year Animal Model of Cancer Major Findings
The role of vascular permeability mediated by cysteinyl leukotrienes Chio CC, 1995 [88] Rat C6 glioma model LTE 4 selectively opened the blood-tumor barrier and increased the tumor uptake of intravenously injected methotrexate.
Nozaki M, 2010 [89] male Fisher rats, transplantable rat colon adenocarcinoma RCN9 cells implanted via the cisterna magna; Lewis lung carcinoma metastasis model in mice Pranlukast, but not montelukast, inhibited tumor cell migration through brain capillary; both pranlukast and montelukast inhibited tumor cell migration through peripheral capillary.
Duah E, 2019 [90] male C57BL/6 mice, including wild type, Cysltr1 −/− and Cysltr2 −/− , implanted with Matrigel plugs or subcutaneously injected with Lewis lung carcinoma (LLC) cells CysLT 2 R regulated angiogenesis in isolated mouse endothelial cells and in Matrigel implants in mice. The growth and metastases of implanted LLC cells were significantly reduced in CysLT 2 R-null mice than in wild-type or CysLT 1 R-null mice. In wild-type mice, the expression of CysLT 2 R, but not CysLT 1 R, was increased in tumor vasculature, and BayCysLT 2 (a selective CysLT 2 R antagonist), but not MK571 (a CysLT 1 R antagonist), reduced tumor growth, angiogenesis and lung metastasis of Lewis lung carcinoma cells in wild-type mice.   Colon cancer tissue had a significantly lower CysLT 2 R expression than paired normal tissue; lower expression of CysLT 2 R in colon cancer was associated with later stage and poorer prognosis.
Magnusson C, 2011 [60] specimens from 78 colon cancer patients The tissue microarray showed that aggressive tumors generally expressed less IFN-α receptor and more EGFR, with a negative correlation between CysLT 2 R and EGFR expression.
Magnusson C, 2010 [100] tissue microarray of 329 colorectal cancer patients Higher nuclear expression of CysLT 1 R was associated with a poorer prognosis, whereas higher nuclear expression of CysLT 2 R was associated with a better prognosis.
Butler CT, 2019 [58] human ex vivo colorectal tumor explants representing Dukes' stage A, B and C colorectal cancer In human ex vivo colorectal cancer tumor explants, 1,4-dihydroxy quininib significantly decreased the secretion of both angiogenic factor TIE-2 and adhesion molecule VCAM-1.

Esophageal cancer and gastric cancer
Shutt JD, 2012 [101] esophageal biopsy specimens from 14 Barrett's metaplasia, 2 high-grade dysplasia, 11 esophageal adenocarcinoma and 11 squamous control Increased expression levels of LTB 4 and CysLTs were found in esophageal adenocarcinoma tissue than in Barrett's metaplasia and control tissues.
Venerito M, 2016 [102] 19 patients with esophageal squamous cell cancer and 9 sex-and age-matched patients with functional dyspepsia Significantly decreased expression levels of CysLT 1 R and CysLT 2 R were noted in esophageal cancer tissues compared with control squamous epithelium. Tumor specimens were obtained from patients with prostate cancer (n = 151), prostatic intraepithelial neoplasia (n = 20), benign prostatic hyperplasia (n = 20), and normal prostate (n = 20) Prostate cancer tissue had a significantly more extensive and intense CysLT 1 R expression, in nuclei and cytoplasm, than other groups, and the expression was stronger in those with higher Gleason score.
Funao K, 2008 [68] Specimens from 58 patients with renal cell carcinoma and 20 patients with normal kidney tissues Significantly more extensive and intense CysLT 1 R expression, in nuclei and cytoplasm, was observed in renal cell carcinoma tissues, compared with normal kidney tissue; the expression of CysLT 1 R was stronger in cancer tissues with higher grade.

Breast cancer
Magnusson C, 2011 [74] specimens from 144 breast cancer patients Breast cancers with higher CysLT 1 R and lower CysLT 2 R expression levels were associated with higher histological grade and worse overall survival. Slater K, 2020 [81] data of 80 primary uveal melanoma samples in The Cancer Genome Atlas (TCGA) and tissue from 52 patients of primary uveal melanoma in the Liverpool Ocular Oncology Centre The data from TCGA showed that higher expression of CYSLTR1 and CYSLTR2 genes were significantly associated with poorer disease-free survival and overall survival. However, analysis with tissue microarray of 52 patients only showed poorer overall survival in those with higher CysLT 1 R expression but CysLT 2 R expression was not associated with survival.

Chemopreventive effects of cysteinyl leukotriene inhibition
Tsai MJ, 2016 [105] 4185 CysLTR antagonist (LTRA) users and 20,925 LTRA non-users from Taiwan National Health Insurance Research Database LTRA use decreased cancer risk in a dose-dependent manner in asthma patients. The chemopreventive effect of LTRA was markedly observed in terms of lung, colorectal, liver and breast cancer.
Sutton SS, 2021 [106] 23,730 patients with leukotriene pathway inhibiting medication exposure and 534,736 patients from the data from the Department of Veteran Affairs Patients with leukotriene pathway inhibiting medication (montelukast, zafirlukast, or zileuton) exposure had a significantly reduced risk of lung cancer than those without exposure.