The Role of Eicosanoids in Gynecological Malignancies.

Eicosanoids, bio-active lipid molecules, elicit a wide range of biological effects that plays a role in physiological and pathophysiological conditions ( Harizi et al., 2008 ; Buczynski et al., 2009 ; Chhonker et al., 2018 ). Eicosanoid driven processes have been shown to have increasing importance in the development, progression and metastasis of gynecological malignancies. Gynecological malignancies are cancers that initiate in the reproductive organs of women that include vulvar, vaginal, cervical, uterine, and ovarian cancers. The eicosanoid pathway in cancer has been reviewed in detail ( Panigrahy et al., 2010 ; Wang and DuBois, 2010 ; Gomes et al., 2018 ; Umamaheswaran et al., 2018 ); briefly, arachidonic acid (AA) is liberated from membrane phospholipids by phospholipase A2 (PLA2) and metabolized by one of three pathways cyclooxygenase (COX), lipoxygenase (LOX) and P450 epoxygenase which then produce a wide range of prostanoids, leukotrienes, epoxyeicosatrienoic acids (EETs), and hydroxyeicosatetraenoic acids (HETEs) ( Wang and DuBois, 2010 ; Gomes et al., 2018 ). The most prominent eicosanoids to be identified to play a role in cancer are those involved in the COX and LOX pathways ( Jones et al., 2019 ); however, products of the cytochrome P450 epoxygenase pathway are shown to play an emerging role in angiogenesis, inflammation and cancer [as reviewed by ( Panigrahy et al., 2010 )]. In gynecological malignancies, eicosanoids can act directly on the cancer cells, indirectly in the tumor microenvironment and, in many cases, at the confluence of infectious diseases such as HIV and HPV, inflammation and carcinogenesis ( De Nola et al., 2019 ). This review will highlight the role of eicosanoids in the development and progression of gynecological malignancies. The cyclooxygenase (COX) pathway converts arachidonic acid to PGH 2 which is then further converted to 5 different eicosanoids, prostaglandin E 2 (PGE 2 ), prostaglandin F 2α (PGF 2α ), prostaglandin D 2 (PGD 2 ), Prostaglandin I 2 (PGI 2 ) (prostacyclin), and thromboxane (TXA 2 ) by specific PG synthases which are then exported to signal through their cognate receptors ( Wang and DuBois, 2010 ; Reader et al., 2011 ) ( Figure 1 ). The COX pathway has been the main focus of research especially concerning PGE 2 . PGE 2 binds to four different G protein-coupled receptors EP1-4 which signal to different downstream signaling pathways ( Reader et al., 2011 ). In the lipoxygenase pathway both HETEs and leukotrienes are produced by multiple subfamilies of LOX enzymes: 5-, 8-, 12-, 15-LOX. 5- (ALOX5) and 12-LOX (ALOX12) have been reported to have pro-carcinogenic roles, whereas 15-LOX-2 (ALOX15B) may have an anticancer effect ( Guo et al., 2011 ). The role of 15-LOX-1 (ALOX15) is controversial (as reviewed in Guo et al., 2011 ). The two major isoforms of 12-LOX are platelet (p12-LOX) and leukocyte (l12-LOX) LOX with pLOX defined as the main 12-LOX in humans. 12-LOX catalyzes the stereospecific oxygenation of AA to form 12(S)-hydroperoxyeicosatetraenoic acid (HPETE), which is converted to 12(S)-hydroxyeicosatetraenoic acid (12-HETE) ( Wang and DuBois, 2010 ; Guo et al., 2011 ) ( Figure 2 ). It has been hypothesized that HETEs can bind to GPCRs, with 3 identified thus far, and at higher concentrations they can also activate nuclear transcription factors peroxisome proliferator-activated receptors (PPARs) ( Panigrahy et al., 2010 ; Choi and Bothwell, 2012 ; Powell and Rokach, 2015 ; Garcia et al., 2017 ; Hoxha and Zappacosta, 2020 ). In addition to HETEs, 5-LOX, and 5-LOX activating protein (FLAP) convert AA to a series of leukotrienes including LTA 4 , LTB 4 , LTC 4 , LTD 4 , and LTE 4 that when exported can signal through cognate receptors including leukotriene B4 receptors (BLT) and cysteinyl leukotriene receptors (CysLTR) ( Wang and DuBois, 2010 ). Arachidonic acid (AA) is liberated from the plasma membrane via phospholipase A2 (PLA2). AA can be recycled by the Lands cycle, which is a reacylation/deacylation cycle, that serves to keep the concentration of free AA at a low level. AA is converted by cyclooxygenase 1 or 2 (COX-1/COX-2) to PGH 2 which is then converted to PGE 2 , PGF 2α , PGD 2 , PGI 2 , or TXA 2 by prostaglandin specific synthases. PGE 2 is exported out of the cell by multidrug resistance-associated protein four (MRP4) where it can bind to its receptors, the E series of prostaglandin receptors on the plasma membrane, EP1-4. Each of the G-protein-coupled- receptors signal through a different intracellular pathway: EP1 leads to elevation of intracellular calcium through Gαq, EP3, which exists in multiple isoforms, can lead to different responses with the majority acting to inhibit cAMP through Gαi as well as an increase in IP3/intracellular calcium; EP2 and EP4 cause stimulation of cyclic AMP (cAMP) production and protein kinase A (PKA) by sequential activation of Gαs and adenylate cyclase (AC); EP4 can also activate phosphoinositide-3-kinase (PI3K) through Gαi. PGE 2 is imported back into the cell through prostaglandin transporter (PGT) where it can either be re-exported or inactivated by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to 15-keto-PGE 2 . 15-keto-PGE 2 can signal through PPARγ. PGE 2 can be converted to PGA 2 through a dehydration reaction. PGD 2 through a series of dehydrogenation reactions creates PGJ 2 , delta-12-prostaglandin J2 (d-12-PGJ 2 ) and 15-deoxy-delta12,14-prostaglandin J2 (15-d-PGJ 2 ). PGI 2 can either signal through PPARδ intracellularly or exported via multidrug resistance protein (MRP) to signal through the IP receptor. TXA 2 is exported out of the cell via MRP to signal through the TP receptor. PGF 2α is exported out of the cell via MRP where it can signal via the FP receptor on the cell surface. Arachidonic acid (AA) is liberated from the plasma membrane via phospholipase A2 (PLA 2 ). AA can be recycled by the Lands cycle, which is a reacylation/deacylation cycle, that serves to keep the concentration of free AA at a low level. AA is converted by different lipoxygenase enzymes to form either hydroperoxyeicosatetraenoic acid (HPETEs) which is then reduced to the corresponding hydroxy compound. 12-HETE can signal through the G-protein-coupled receptor 12-HETE (12-HETER). 8-HETE, 15-HETE and leukotriene LTB 4 can signal through PPAR. 5-LOX and 5-lipoxygenase activating protein (FLAP) convert AA to leukotrienes through a series of reactions using 5-LOX and as well as LTA 4 hydrolase and LTC 4 synthase including LTA 4 , LTB 4 , LTC 4 , LTD 4 , and LTE 4 . LTB 4 is exported via multidrug resistance protein 4 (MRP4) to signal through leukotriene B4 receptor BLT1/2. LTC 4 and LTD 4 is exported via multidrug resistance protein 1 (MRP1) to bind to cysteinyl leukotriene receptor 1 or 2 (CysLTR1/2). The cytochrome P450 (CYP) epoxygenase pathway, a largely neglected pathway in cancer, leads to the creation of regioisomeric epoxyeicosatrienoic acids (EETs) or HETEs ( Panigrahy et al., 2010 ; Panigrahy et al., 2011 ). EETs are then metabolized by soluble epoxide hydrolase (sEH) to form the corresponding fatty acid diols ( Wang et al., 2019 ) ( Figure 3 ). Arachidonic acid is metabolized by the CYP ω-hydroxylases to 7-, 10-, 12-, 13-, 15-, 16-, 17-, 18-, 19-, and 20-HETEs, the principal metabolite being the pro-inflammatory 20-HETE ( Panigrahy et al., 2010 ). Although EETs are primarily metabolized by sEH, a few studies have observed that the 5,6-EET and 8,9-EET are but substrates for COX-1 and COX-2 ( Zhang et al., 1992 ; Carroll et al., 1993 ; Moreland et al., 2007 ; Rand et al., 2017 ). Three EET regioisomers were found to be substrates for COX, and EET substrate preference for both COX-1 and COX-2 were estimated as 8,9-EET > 5,6-EET > 11,12-EET, whereas 14,15-EET was inactive. 8,9-EET is metabolized by COX to form ct-8,9-E-11-HET (8,9,11-EHET) and ct-8,9-E-15-HET (8,9,15-EHET) ( Choi and Bothwell, 2012 ; Rand et al., 2017 ; Rand et al., 2019 ). Receptors for EETs have only recently been identified. A low affinity receptor for 11,12-EET, GPR40, has been identified in vascular cells that upon stimulation can lead to increase in Cx43 and COX-2 expression in endothelial cells via ERK phosphorylation ( Park et al., 2018 ). A high affinity binding protein that may be a receptor was identified in smooth muscle, endothelial and U937 cells that can bind 8,9-EET, 11,12-EET, and 14,15-EET ( Chen et al., 2011 ). Is it not yet known if these receptors play a role in gynecological malignancies. The cytochrome P450 epoxygenase pathway creates a series of regioisomeric epoxyeicosatrienoic acids (EETs) or hydroperoxyeicosatetraenoic (HETEs). Arachidonic acid (AA) is metabolized by the CYP ω-hydroxylases to 7-, 10-, 12-, 13-, 15-, 16-, 17-, 18-, 19-, and 20-HETEs. HETEs can bind to PPAR intracellularly or bind their receptor on the plasma membrane surface. EETs are metabolized by soluble epoxide hydrolase (sEH) to form the corresponding fatty acid diols. 5,6-EET and 8,9-EET are also substrates for COX-1 and COX-2 forming ct -8,9-E-11-HET and ct -8,9-E-15-HET. EETs can also signal through their cognate receptors.

PS, DR, GR, and JR contributed written sections and figures. MC and AF contributed edits. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This manuscript aimed to provide a comprehensive review of the role of eicosanoids in gynecological malignancies. With increasing evidence, it is demonstrated that eicosanoid driven processes are associated with the progression and spread of gynecological malignancies. These bio-active lipids have direct effects on cancer cells as well as indirect effects on the tumor microenvironment. Most of the studies reported in gynecological malignancies focused on the cyclooxygenase pathway especially the role of PGE 2 and PGF 2α and their receptors. The least reported pathway was the P450 cytochrome epoxygenase pathway in which only ovarian cancer had associative studies involving the pathway but with a lack of mechanistic investigation; thus, this pathway is one that should be examined more in the future for all gynecological malignancies. Given the rare nature of vaginal and vulvar cancer, there is not an abundance of research available; however, the studies performed are directed towards the cyclooxygenase pathway and exploring all of the eicosanoid pathways would be of benefit in order to find much needed treatments and information related to tumorigenesis of these malignancies. It was demonstrated that the COX pathway plays a significant role in the pathogenesis of cervical cancer alone and in conjunction with HPV and HIV; however, there was a lack of mechanistic studies in interrogating which receptors are involved in the tumorigenic signaling pathways. In uterine cancer, it would be of benefit to see more studies performed on the potential for PGE 2 receptors EP1 and EP3 in uterine cancer given that to date most of the investigations have been correlative. In ovarian cancer, the data presented for the potential role for the lipoxygenase pathway in ovarian cancer tumorigenesis and modulation of the tumor microenvironment supports the need for additional studies including antagonizing the receptors for both leukotrienes and HETEs. Given the potential anti-neoplastic effects on some of the prostaglandins such as the cyclopentenone prostaglandins as well as the association of PGD 2 with good prognosis in high grade serous ovarian cancer ( Alves et al., 2019 ) as well as the positive effects of blocking PGE 2 EP4 in modulating the tumor microenvironment ( Hennequart et al., 2017 ), additional mechanistic studies exploring modulation of COX signaling downstream of the COX enzymes as a monotherapy and in combination with additional therapeutic interventions is warranted. Overall there is a theme that inhibition of COX pathways leads to potentiation of chemotherapeutic treatment in multiple gynecological malignancies including ovarian ( Li et al., 2012a ; Li et al., 2013 ), uterine ( Hasegawa et al., 2013 ; Reader et al., 2019 ) and vulvar ( Kim et al., 2009a ). Additional studies are warranted to explore the role of and the mechanism behind adding inhibition of prostanoid receptors to chemotherapy and other treatment modalities in order to conserve potential anti-neoplastic eicosanoid activities for development of novel treatments for gynecologic malignancies.

Vaginal cancer represents only 1–2% of gynecologic malignancies and is under-studied due to its rarity. There were only 5,350 US cases and 1,430 deaths from this disease in 2019 ( Siegel et al., 2019 ). Approximately 10–50% of patients with vaginal cancer or its precursor lesion [high grade vaginal intraepithelial neoplasia (VAIN)] have a history of hysterectomy or radiation for cervical cancer. HPV infection and immunosuppression are strong risk factors for development. The majority arise in the upper third of the canal and involve the posterior wall and are squamous in histology. Less common subtypes include clear cell adenocarcinoma, which is characteristically associated with in utero diethylstilbestrol (DES) exposure or malignant degeneration of endometriosis, and embryonal rhabdomyosarcoma, the most common vaginal malignancy in children ( Cardenes et al., 2013 ). COX-2 expression has been found to be a prominent feature in mucosal inflammation of the vagina leading to disease susceptibility and transmission ( Joseph et al., 2012 ), as well as rectovaginal endometriotic implants ( Fagotti et al., 2004 ). Recently, we have observed lower expression of EP4 in vaginal tissues of patients with pelvic organ prolapse and higher expression in women with radiation-induced vaginal stenosis after treatment for gynecologic malignancy compared to healthy controls ( Santayana et al., 2018 ). There also appears to be differential expression between luminal, basal, and intermediate (smooth muscle) layers of the vagina. Additional studies are underway to better elucidate the role of EP4 in both alterations of tissue integrity as well as its relevance in gynecologic malignancy, but this preliminary data raises interest in a possible pharmacologic role for use of EP4 inhibitors to modify a number of biologic processes in vaginal cancer.

The work presented has been supported by Department of Obstetrics, Gynecology, and Reproductive Medicine (JR, GR, DR), the Kaleidoscope of Hope Ovarian Cancer Foundation (JR), The Foundation for Women’s Cancer Research (DR), Maryland Department of Health’s Cigarette Restitution Fund Program (AF, JR), Baltimore Veterans Affairs Administration (AF) the STAR-PREP program of the National Institute of General Medical Sciences at the University of Maryland School of Medicine (MMC).