Background
Lung carcinoma is the most common cancer worldwide, with 1.8 million newly diagnosed patients per year, and has a higher mortality than that of the next top three cancers combined (158,080 vs 115,760 deaths/year) [
1,
2]. While some treatments, such as radiation and immunotherapy, have given hope to lung cancer patients over the past few decades [
3], its 5-year survival rate remains very poor [
4]. That said, anti-angiogenesis treatments and the eradication and functional inhibition of tumour-associated endothelial cells (ECs), have emerged as important cancer treatments [
5,
6]. However, the outcomes of current anti-angiogenic therapies that primarily target vascular endothelial growth factor (VEGF) pathways depend on cancer types and stages, and often lead to the development of resistance, hypertension, proteinuria, or even death [
7]. Thus, more effective and safer anti-angiogenic therapies require further investigation.
Arachidonic acid (AA) released from membrane phospholipids is converted into various bioactive lipid mediators, such as epoxyeicosatrienoic acids (EETs), prostaglandins (PGs), and hydroxyeicosatetraenoic acids (HETEs), by either cytochrome P450 (CYP) epoxygenases, cyclooxygenase (COX), or lipoxygenase (LOX) pathways [
8,
9]. Among them, EETs are particularly powerful pro-angiogenic eicosanoids and are positively linked with cancer progression [
10,
11]. Cyp2c44, the functional homologue of human Cyp2c9, is one of the main CYP epoxygenases for EET biosynthesis in endothelial cells [
12,
13]. Disruption of the
Cyp2c44 gene, or downregulation of its expression, reduces endothelial proliferation and tubular morphogenesis in vitro and inhibits primary tumour growth in vivo [
12,
13]. Taken together, the Cyp2c44-EETs axis may be a vital target for cancer treatment, including lung cancer.
Peroxisome proliferator-activated nuclear receptor alpha (PPARα) is a ligand-activated nuclear receptor that modulates the transcription of specific target genes implicated in lipid metabolism and energy homeostasis [
14,
15]. The PPARα-mediated transcriptional regulation of the
Cyp2c44 gene has been clearly established in previous studies [
12,
16]. Once activated, PPARα translocates into the nucleus, and then binds to the PPAR response element (PPRE) in the promotor of the
Cyp2c44 gene and reduces its expression, thereby indicating why PPARα agonists inhibit angiogenic activity and tumour vascularization [
12,
13]. Unfortunately, application of traditional PPARα agonists were restricted due its insufficient efficacy and hepatotoxicity [
17].
As previously reported, AVE8134 is a specific and high-affinity ligand for PPARα, and shares with Wyeth-14,643 its PPARα selectivity and ability to improve plasma lipid profiles in rodents [
18,
19]. More importantly, AVE8134 has been used in humans and has shown to be well tolerated at doses between 10 and 20 mg/kg body weight per day in contrast with Wyeth [
18,
19]. We assume that, as with Wyeth, AVE8134 downregulates Cyp2c44 expression in the host endothelium, causing a decrease in the production of pro-angiogenic eicosanoid EETs and the inhibition of tumour vascularization, growth, and metastasis. We are proposing to repurpose AVE8134 as a safe agent for the treatment of human cancers.
Discussion
In this study, we proposed that AVE8134 acts as a novel anti-angiogenic drug that could be effective in the treatment of cancers. This is based on the fact that AVE8134 is a high affinity ligand for PPARα and is well tolerated in humans. Unfortunately, AVE8134 was not the strongest anti-tumorigenic and anti-angiogenic drug when compared with two other PPARα ligands and even failed to control lung metastasis. Metabolomics analysis found that PPARα ligands not only decreased EET biosynthesis by downregulating Cyp2c44 expression but also increased the production of a pro-angiogenic factor, 11-HETE, which in return counteracted their benefits on tumour suppression. That is, the effect of PPARα ligands on tumour inhibition depended on the variation in EETs and 11-HETE (ΔEETs-Δ11-HETE). Moreover, we found that the COX inhibitor indomethacin optimized the therapeutic effects of AVE8134 on tumour growth and angiogenesis via decreasing the formation of 11-HETE. Taken together, these results suggest that AVE8134 is an attractive drug for treating cancer if combined with indomethacin.
Angiogenesis is not only an important mechanism by which tumours obtain sufficient nutritional support and remove metabolic waste, but it is also required for the growth of numerous solid tumours and their metastases [
32,
33]. Tumours remain in a dormant state until they become vascularized and these immature vessels increase the chance of tumour cells entering circulation and immigrating to distant organs [
32,
34]. Angiogenesis is a complex multi-step process, involving endothelial cell proliferation, migration, sprouting, and transforming into tube-like structures [
35]. A variety of endogenous factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factors (bFGF), epidermal growth factor (EGF) and EETs, promote angiogenesis and contribute to the development of tumours. This makes these factors ideal targets for cancer treatments [
36,
37]. CYP epoxygenases and their metabolites, EETs, are upregulated in human tumours and have been identified as powerful pro-angiogenetic mediators [
23,
38]. Overexpression of CYP epoxygenases or inhibiting EET hydrolysis by soluble epoxide hydrolase inhibitors (sEHi) demonstrated their capacity to promote tumour growth and metastasis in many preclinical studies [
23,
34]. CYP epoxygenase inhibitors will hopefully enter into clinical trials for cancer treatments [
23,
39]. PPARα agonists exhibited a reduction in tumour growth and vascularization by suppressing Cyp2c44 expression, which connects them with clinical tumor treatment [
12,
13,
16].
PPARs are members of the steroid receptor superfamily and there are three subtypes: PPAR-α, −δ, and -γ. These receptors are important ligand-activated transcription factors involved in the regulation of cell proliferation and energy metabolism [
15,
40]. Interestingly, increasing evidence has showed that PPAR activation exhibited multiple functions in tumour progression [
41]. Compared with the unified conclusion that PPARγ inhibited the growth of various tumours [
42], the effects of PPARα on tumour progression were diverse and depended on the tissue type or PPARα ligands [
13]. In some studies, PPARα deficiency inhibited tumorigenesis through increasing the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) [
43]. Moreover, PPARα activation enhances breast cancer cell proliferation by upregulating cyclin E levels [
44]. Conversely, PPARα activation with Wyeth-14,643 or fenofibrate was also reported to inhibit endothelial cell growth and non-small cell lung cancer (NSCLC) progression via binding to the PPER area in the promoter of mouse
Cyp2c44 [
12,
13,
16]. Besides the paradoxical roles of PPARα, another nonnegligible issue impeding its use in clinical trials is that PPARα agonists typically increase the incidence of liver hepatomegaly and tumours through induction of cell proliferation and oxidative stress [
17]. This study compared the effects of three different PPARα agonists on tumour progression and liver hepatomegaly and found that the novel PPARα ligand AVE8134 was an ideal choice for tumour treatment given its effectiveness and safety. Inconsistent with previous studies, this study suggests that PPARα ligands not only reduce EET production via downregulating Cyp2c44 expression but also increase 11-HETE biosynthesis. Increased 11-HETE was shown to be a pro-angiogenic and -tumorigenic factor, which partially cancelled out the benefits from decreased EETs in tumour-bearing mice. Thus, the combined utilization of drugs that inhibit 11-HETE formation may solve this issue and enhance the anti-tumour effect of AVE8134.
11-HETE, a bioactive metabolite derived from AA, is mainly generated from COX enzymes, while LOX and CYP enzymes, and non-enzymatic catalytic pathways may also contributed to its production [
30,
31]. Previous research has reported that the COX2 specific inhibitor, celecoxib, reduced 11-HETE production in lung cancer A549 cells [
45]. AA can bind to the COX active site in a specific catalytic arrangement that leads to 11-HETE production, which is inhibited by aspirin treatment [
29,
46]. Moreover, the non-specific COX inhibitor indomethacin was observed to reduce the formation of 11-HETE in bovine coronary artery endothelial cells [
29,
30]. These results suggest that the formation of 11-HETE relies on COX enzymes. Although increased 11-HETE is described as a biomarker ranging from coronary events to cancers, its biological function remains unclear [
47]. This study found that 11-HETE stimulated endothelial proliferation, migration, and angiogenesis, as well as the subsequent growth and metastasis of tumours.
Although the control region of the
COX2 gene possesses one response element for PPAR (PPRE), there is no evidence that indicates that PPARα activation affects its expression [
48]. However, a previous study has reported that PPARγ activation participates in the transcriptional activation of the
COX2 gene [
49]. That said, PPARα may change the catalytic arrangement at the COX active site. This may be important given the fact that COX-2 is an inducible isoform of COXs and its overexpression is linked to various cancers [
50]. Both COX1 and COX2 inhibitors have been reported to inhibit tumour progression and this inhibition underlies their anti-inflammation and anti-angiogenesis effects [
51,
52]. The COX inhibitor indomethacin has been used previously as a non-steroidal anti-inflammatory drug for the treatment of various inflammatory diseases, such as arthritis, fever, and various headache syndromes [
53]. In subsequent preclinical and clinical studies, researchers found that indomethacin exhibits anti-tumour activity [
52], although the underlying mechanisms are unclear. This study found that indomethacin synergistically strengthened the anti-tumour effects of AVE8134 by inhibiting the production of 11-HETE. Although indomethacin alone inhibited the activation of endothelial cells and slightly suppressed the growth of TC-1 lung tumours, its joint effects with AVE8134 seem more powerful. Thus, combining the novel PPARα ligand AVE8134 with the COX inhibitor indomethacin provides a new and effective strategy for the treatment of cancer.
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