Each year, ~1.4 million new cases of colorectal cancer (CRC) are diagnosed, with almost 700,000 cancer-related deaths, making it the third most common cancer worldwide and the fourth most common cause of cancer death (Torre et al., 2015). Surgical intervention is the cornerstone of treatment but adjuvant chemotherapy and targeted therapies play significant roles in improving survival. Treatment decision is guided by clinical factors (e.g., co-morbid conditions, performance status) and pathological factors (e.g., KRAS mutation status, microsatellite stability status). 5-fluorouracil-based regimens such as FOLFOX or FOLFIRI form the backbone of cytotoxic chemotherapy in the treatment of metastatic disease but are limited by chemoresistance and toxic effects on non-neoplastic healthy tissue (Longley and Johnston, 2005; Fuchs et al., 2007).

Targeted therapies include the anti-angiogenic drug bevacizumab (Avastin®), a humanized monoclonal antibody that binds directly to vascular endothelial growth factor A (VEGF-A; Ferrara et al., 2004). Blocking VEGF inhibits tumor angiogenesis, a pathophysiological process upon which solid tumors depend for growth, survival, and metastasis (Folkman, 1971). Other targeted therapies include anti-epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab, the major advantage of which is the availability of a validated biomarker that reliably predicts patient response—patients with activating KRAS mutations have poorer progression-free and overall survival (Martinelli et al., 2009; Yen et al., 2010). Immune checkpoint inhibition is emerging as a promising treatment strategy in microsatellite instability-high (MSI-H) colorectal tumors, a phenotype resulting from defective DNA mismatch repair and accounting for up to 15% of all CRCs (Boland and Goel, 2010; Le et al., 2015). Pembrolizumab, an anti-programmed cell death protein 1 (anti-PD-1) antibody potentiates T-cell immune responses, circumventing tumor immune evasion, and significantly prolonging progression-free survival in patients with MSI-H tumors (McDermott and Atkins, 2013; Le et al., 2015). Numerous other immunotherapies are currently being evaluated in clinical trials in combination with chemotherapy regimens and/or targeted therapies.

While CRC survival rates are improving, there is a need for more effective therapies as the survival benefit associated with targeted therapies is only ~4–5 months (McCormack and Keam, 2008; Bokemeyer et al., 2012). Many novel strategies are currently under investigation, one of which is to target cysteinyl leukotriene (CysLT) signaling. The focus of this mini-review is to evaluate the link between CysLTs and the hallmarks of cancer.

Cysteinyl leukotrienes (CysLTs) are a subfamily of eicosanoids, lipophilic signaling molecules that regulate both acute and chronic inflammation (Henderson, 1994). These potent bioactive lipids are rapidly generated de novo from cell membrane-associated arachidonic acid (AA), an essential polyunsaturated fatty acid in response to cell activation (Clark et al., 1991). Once mobilized to the cytosol, AA is metabolized by 5-lipoxygenase (5-LOX) in conjunction with 5-lipoxygenase-activating protein (FLAP) to yield leukotriene A₄ (LTA₄), which undergoes further metabolism to form leukotrienes B₄ (LTB₄) and C₄ (LTC₄) (Kanaoka and Boyce, 2004). Subsequently LTC₄ is exported from the cell via multi-drug resistance-associated proteins 1 and 4, and metabolized to LTD₄ and LTE₄ (Lam et al., 1989). LTC₄, LTD₄, and LTE₄, referred to collectively as CysLTs, are structurally similar but exhibit functional diversity (Laidlaw and Boyce, 2012).

The biological actions of CysLTs are mediated via ligation of the widely distributed G-protein-coupled receptors, CysLT₁ and CysLT₂ (Figure 1; Peters-Golden et al., 2006). The role of newly-identified CysLT receptors GPR17 and GPR99 remains to be established. GPR17 is an orphan P2Y-like receptor with dual specificity for uracil nucleotides and CysLTs, while GPR99 has been proposed as a potential LTE₄-selective CysLT receptor (Ciana et al., 2006; Kanaoka et al., 2013). Cross-regulation occurs between CysLT receptors—CysLT₂ negatively regulates CysLT₁ signaling via receptor heterodimerization and GPR17 has been reported as a ligand-independent negative regulator of CysLT₁ (Lynch et al., 1999; Jiang et al., 2007; Maekawa et al., 2009).

CysLTs play recognized roles in promoting the inflammatory response, bronchoconstriction and vascular permeability (Davidson et al., 1987; Lee et al., 2004). CysLTs have also recently emerged as important regulators of intestinal homeostasis, with endogenous CysLT production mediating the survival and proliferation of intestinal epithelial cells (Paruchuri et al., 2006). Dysregulated CysLT signaling has been implicated in colorectal adenocarcinomas with increased CysLT₁ and decreased CysLT₂ levels in patient tumor samples compared with surrounding normal tissue (Magnusson et al., 2007). Interestingly, unlike the majority of G-protein coupled receptors, CysLT₁ and CysLT₂ are located both at the plasma membrane and the nuclear membrane (Magnusson et al., 2010). This subcellular receptor localization is critically important for CRC patient survival - patients with high nuclear CysLT₁ expression have a poorer prognosis than patients with high cytoplasmic expression (Magnusson et al., 2010). In contrast, patients with high nuclear CysLT₂ expression have a better overall survival expectancy, indicating the existence of an inverse relationship between nuclear CysLT₁ and CysLT₂ expression, and suggesting that CysLT₂ has a protective role in CRC (Magnusson et al., 2010).

In vitro data corroborate these findings as malignant intestinal cell lines Caco-2 and SW480 demonstrate higher CysLT₁ but lower CysLT₂ expression levels compared to non-cancerous Int 407 intestinal epithelial cells (Magnusson et al., 2007). CysLT₂ signaling also results in terminal differentiation of Caco-2 cells and growth inhibition (Magnusson et al., 2007). All-trans retinoic acid (ATRA), an anti-cancer agent promoting cell differentiation, acts in part by upregulating CysLT₂ mRNA expression and LTC₄ synthase (Bengtsson et al., 2013).

Interestingly, CysLT₁ expression increases CRC tumor burden in a gender-specific manner in vivo—In the Apc^Min/+ mouse model of familial adenomatous polyposis/sporadic CRC, female mice lacking the CysLT₁ gene (Cysltr1 ^−/− Apc^Min/+) develop significantly less polyps, less systemic inflammation, and increased regulatory T-cell tumor infiltration, a negative prognostic factor in CRC, compared to Cysltr1 ^+/+ Apc^Min/+ mice.

Analysis of The Cancer Genome Atlas (TCGA) RNA-sequencing dataset reveal that GPR17 and GPR99 are expressed in CRC patient samples, with GPR99 more highly expressed relative to GPR17 based on FPKM relative expression values. Kaplan-Meier survival analysis was conducted using a log-rank test with the “survival” package in R and grouping 41 tumor samples based on median expression. We found no significant correlation of either gene with overall survival. While both receptors have been identified in the colon, further investigation in a larger cohort of CRC patients is required to confirm these findings.

CysLTs have multiple roles in many hallmarks of cancer (Figure 2).

Overall, targeting CysLT signaling is a promising option for anti-cancer therapy due to its effects on multiple oncogenic pathways described above (Figure 2). Commercially available anti-asthmatic drugs which target the CysLT₁, have demonstrated notable in vivo potential as anti-cancer agents. Montelukast, a CysLT₁-selective antagonist significantly reduces tumor volume in a HCT-116 CRC-specific murine xenograft model, via a combination of anti-proliferative and pro-apoptotic effects (Savari et al., 2013). CysLT₁ antagonism also exhibits an anti-angiogenic effect, with reduced blood vessel formation and VEGF expression levels in colorectal tumors (Savari et al., 2013). Furthermore, pre-treatment of HCT-116 cells with montelukast prior to inoculation completely inhibited tumor initiation in BALB/c nu/nu mice. Montelukast also has antagonistic actions at GPR17—it is unknown if this receptor is important for the drug's anti-neoplastic effect (Ciana et al., 2006). Pranlukast significantly attenuates chemotherapy-triggered morbidity in mice via a MGST2-LTC₄ pathway, and so may have therapeutic potential in combination with chemotherapy to reduce toxic side effects or to reduce the risk of metastases (Dvash et al., 2015). Furthermore, CysLT₁ antagonists potently inhibit the growth of HCT-116 colon cancer cells, in addition to prostate, urothelial, and neuroblastoma cancer cell lines (Matsuyama et al., 2007, 2009; Sveinbjörnsson et al., 2008; Savari et al., 2013).

No studies to date have investigated the effects of specific CysLT₂ agonists or antagonists on tumor progression specifically. In the case of CRC, selective CysLT₂ agonism would appear to be the desired approach, but this needs potent selectivity in order to avoid negative effects of CysLT₁ agonism on the intestine. Due to cross regulation between CysLT receptors, such a targeting strategy would need to be approached with caution (Laidlaw and Boyce, 2012).

Targeting 5-LOX or FLAP to inhibit endogenous production of CysLTs is an alternative strategy that would circumvent the increasing number of CysLT isoreceptors. Zileuton, a 5-LOX inhibitor, prevents colonic polyp formation in an APC^Δ468 mouse model of polyposis and significantly decreases tumor burden in LoVo and HT29 colon cancer murine xenograft models (Melstrom et al., 2008; Gounaris et al., 2015). MK-866, a FLAP inhibitor, reduces proliferation of Caco-2 and HT29 colon cancer cell lines (Ford-Hutchinson, 1991; Cianchi et al., 2006).

These studies highlight the importance of CysLT signaling in intestinal biology, and its potential role in the tumorigenesis of colorectal adenocarcinoma. CysLT signaling is linked with many hallmarks of cancer—cell proliferation and survival, cell migration, angiogenesis, genomic instability, glycolytic metabolic switch, and tumor-promoting inflammation. Data largely centers on CysLT₁ and CysLT₂ with further investigation necessary to establish the importance of GPR17 and GPR99 in this context.

Targeting CysLT signaling is a promising pharmacological strategy, as evidenced by the numerous CysLT antagonists which have anti-tumor efficacy in in vitro and in vivo CRC models. In particular, blockade of LTC₄ has recently emerged as a promising new therapeutic opportunity in limiting toxicity to current chemotherapy drugs. A notable advantage of many CysLT receptor antagonists is their favorable safety profile. CysLT signaling modulators may have potential to synergize with other targeted therapies to benefit survival rates. In summary, CysLT signaling is gaining increasing recognition as a mediator of colorectal tumorigenesis and pharmacological manipulation of this signaling pathway represents an exciting opportunity that merits further translational research work.

LB conducted the literature review and wrote the paper. CB revised and edited the paper. AM revised and edited the paper. BM provided analysis of RNA sequencing dataset. WG provided analysis of RNA sequencing dataset. JO revised and edited the paper. BK revised and edited the paper.

This work is supported by Irish Cancer Society Research Scholarship CRS13BUT.