Targeting 20-HETE producing enzymes in cancer – rationale, pharmacology, and clinical potential

Figures & data

Figure 1 Synthesis of 20-HETE.

Note: ω/ω-1 hydroxylases of CYP4A and CYP4F introduce a hydroxyl group into positions 20 or 19 of arachidonic acid to form 19-HETE and 20-HETE, respectively.
Abbreviations: 20-HETE, 20-hydroxyeicosatetraenoic acid; CYP, cytochrome P450; 19-HETE, 19-hydroxyeicosatetraenoic acid.

Figure 2 An overview of eicosanoid synthesis pathways.

Note: Schematic overview of three major enzymes of arachidonic acid metabolism and their metabolites.
Abbreviations: CYP, cytochrome P450; LOX, lipoxygenase; EET, epoxyeicosatrienoic acid; PGI₂, prostacyclin synthase; PGE₂, prostaglandin E2; PGF2, prostaglandin F2; PGD₂, prostaglandin D2; TXA₂, thromboxane A2; LTA₄, leukotriene A4; LTB₄, leukotriene B4; PGH2, prostaglandin H2; COX1/2, cyclooxygenase 1/2; HETE, hydroxyeicosatetraenoic acid.

Figure 3 LC-MS-MS detection of HETE standards.

Note: Representative chromatogram generated from LC-MS-MS data, illustrating the separation of various HETEs with different retention times and transitions.
Abbreviations: LC-MS-MS, liquid chromatography-mass spectrometry-mass spectrometry; HETE, hydroxyeicosatetraenoic acid; PG, prostaglandin; DiHETE, diastereomeric 5,6-dihydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid.

Figure 4 Proposed mechanism of action of 20-HETE.

Notes: Mitogens and ligands of G-protein-coupled receptors activate PLA2 and induce release of arachidonic acid, which is converted by CYP4A/F to 20-HETE. Then, 20-HETE, acting presumably via Src, promotes tyrosine phosphorylation of EGFR and consequently activates the Ras-Raf-MEK-ERK cascade, which leads to the proliferation of cancer cells. One of cytoplasmic targets of ERK is I-kappaB kinase-alpha, which phosphorylates I-kappaB-alpha, resulting in the degradation of I-kappaB-alpha by the proteosome and the translocation of NFκB to the nucleus, where it positively regulates the transcription of a number of genes involved in immune and inflammatory responses including vEGF. In addition, 20-HETE also activates cAMP-dependent PKA-phosphorylase kinase-GP (glycogen phosphorylase pathway) to induce hyperglycemia. Besides, 20-HETE stimulates NADPH oxidase-derived superoxide production, which activates L-type Ca²⁺ channels via a PKC-dependent mechanism. Inhibitory interactions are shown by red lines with bars. Stimulatory interactions are shown by green lines with arrows. Activation of the principal signaling cascade is shown by +.
Abbreviations: 20-HETE, 20-hydroxyeicosatetraenoic acid; GPCR, G protein-coupled receptor; EGFR, endothelial growth factor; PLA2, phospholipase A2; NADPH, nicotinamide adenine dinucleotide phosphate; SOS, salt overly sensitive; AA, arachidonic acid; CYP, cytochrome P450; PKC, protein kinase C; PKA, protein kinase A; PhK, phosphorylase kinase; GP, glycogen phosphorylase pathway; cAMP, cyclic adenosine monophos phate; MEK, MAP kinase kinase; ERK, extracellular signal-regulated kinases; IκBα, I-kappaB-alpha; NFκB, nuclear factor-kappaB; VEGF, vascular endothelial growth factor; HET0016, N-Hydroxy-N′-(4-butyl-2-methylphenyl) formamidine; WIT002, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid.

Figure 5 Expression of CYP4A and CYP4F genes at the mRNA level is elevated in human lung cancer tissue samples.

Notes: Real time-PCR of lung TissueScan cancer survey panel using primers for CYP4A/F genes. Matched control normal tissues are included for comparison between normal and disease samples. The amount of PCR product is correlated with the amount of original RNA transcript and the data are expressed in arbitrary units. *Indicates tumor samples demonstrating striking up-regulation in CYP4A/F enzymes.
Abbreviations: CYP, cytochrome P450; mRNA, messenger ribonucleic acid; PCR, polymerase chain reaction; RNA, ribonucleic acid.

Table 1 Primers used to detect human genes encoding CYP4A/F by real-time PCR

Enzyme	Forward	Reverse
CYP4A11	5′-ATGAAGTGTGCCTTCAGCCA-3′	5′-AAGGCATTCCTCACACGGG-3′
CYP4A22	5′-AATGGGAAGAGCTCCTTGGC-3′	5′-AAGGCATTCCTCATACAGC-3′
CYP4F2	5′-AAGCACCCAGAATACCAGGA-3′	5′-TCATGCACATGGTCAGGAAG-3′
CYP4F3	5′-CTGTCGGCAGGAGGTACAAG-3′	5′-CCTCAGGCTCTCCTTAATGC-3′

Note: The 20-HETE-producing CYP450 isoform-specific primer sequences to detect the expression of CYP4A and CYP4F genes at the mRNA level in human cancer tissues are outlined.

Abbreviations: CYP, cytochrome P450; PCR, polymerase chain reaction; 20-HETE, 20-hydroxyeicosatetraenoic acid; mRNA, messenger ribonucleic acid.

Figure 6 Inhibitors, agonists, and antagonists of 20-HETE.

Note: Depicted are the chemical structures of inhibitors of 20-HETE producing ω-hydroxylases as well as 20-HETE analogs that exhibit agonist or antagonist activity.
Abbreviations: 20-HETE, 20-hydroxyeicosatetraenoic acid; 5,14-20-HEDGE, N-[20-hydroxyeicosa-5(Z), 14(Z)-dienoyl]glycine; 5,14–20, -HEDE, -20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid. CYP450, Cytochrome P450; WIT002-20-hydroxyeicosa-6(Z),15(Z)-dienoic acid; HET0016, N-Hydroxy-N′-(4-butyl-2-methylphenyl) formamidine.

Table 2 Expression of CYP4A and CYP4F in human cancer samples and the effects of inhibitors of 20-HETE synthesis and signaling upon cancer cells of different origin

Human cancer type	Estimated new cases in the United States in 2012	Estimated deaths in the United States in 2012	Increased expression of CYP4A or CYP4F enzymes in cancer tissues	Antiproliferative effect of HET0016	Antiproliferative effect of WIT002	References
Breast cancer	226,870 (female) 2190 (male)	39,510 (female) 410 (male)	4F2, 4A11, CYP4Z1	Yes	Not studied	63,45
Colon and rectal cancer	103,170 (colon) 40,290 (rectal)	51,690 (colon and rectal combined)	4F2, 4A22, 4F3	Not studied	Not studied	63
Ovarian cancer	22,280	15,500	4A11, 4A22, 4F2	Not studied	Not studied	63
Kidney cancer	64,770	13,570		Yes	Yes	66
Lung cancer	226,160	160,340	4F2	Yes	Yes	63,80
Prostate cancer	241,740	28,170	Both normal and cancer tissues have high levels of expression	Yes	Not studied	63,82
Thyroid cancer	56,460	1780	4A11, 4A22, 4F2,	Not studied	Not studied	63
Liver cancer	28,720	20,550	Both normal and cancer tissues have high levels of expression	Not studied	Not studied	63
Brain tumor	22,910	13,700		Yes	Yes	65,77,79

Abbreviations: CYP, cytochrome P450; 20-HETE, 20-hydroxyeicosatetraenoic acid, HET0016, N-Hydroxy-N′-(4-butyl-2-methylphenyl) formamidine; WIT002, 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid