Potential interaction between lysophosphatidic acid and tumor-associated macrophages in ovarian carcinoma

Ovarian carcinoma is the deadliest type of gynecological cancer. The unique tumor microenvironment enables specific and efficient metastasis, weakens immunological monitoring, and mediates drug resistance. Tumor associated macrophages (TAMs) are a crucial part of the TME and are involved in various aspects of tumor behavior. Lysophosphatidic acid (LPA) is elevated in the blood of ovarian carcinoma patients, as well as in the tumor tissues and ascites, which make it a useful biomarker and a potential therapeutic target. Recent studies have shown that LPA transforms monocytes into macrophages and regulates the formation of macrophages through the AKT/mTOR pathway, and PPAR γ is a major regulator of LPA-derived macrophages. In addition, TAMs synthesize and secrete LPA and express LPA receptor (LPAR) on the surface. With these data in mind, we hypothesize that LPA can convert monocytes directly into TAMs in the microenvironment of ovarian cancer. LPA may mediate TAM formation by activating the PI3K/AKT/mTOR signaling pathway through LPAR on the cell surface, which may also affect the function of PPAR γ, leading to increased LPA production by TAMs. Thus, LPA and TAMs form a vicious circle that affects the malignant behavior of ovarian cancer.


Introduction
Ovarian carcinoma is the most common cause of mortality from gynecological tumors and the 5th leading cause of cancer death in women [1]. The five-year survival rate is only approximately 46.5% [2]. Several characteristics of ovarian cancer are related to its lethality, including the exfoliation of tumor cells, metastasis and diffusion through peritoneal fluid, and tumor promotion and immunosuppression by the tumor microenvironment (TME) [3]. As an important component of the TME, tumor associated macrophages (TAMs) make a crucial part in ovarian cancer progression, chemotherapeutic resistance, immunosuppression and prognosis. At present, there have been some reports on immunotherapy targeting TAMs [4][5][6][7].

Roles of TAMs in ovarian cancer
The main characteristic of ovarian cancer is early metastasis through peritoneal fluid. Ascites contain a large population of TAMs [8][9][10], forming a unique microenvironment [11]. Macrophages can inhibit apoptosis, promote tumor invasion and proliferation, suppress antitumor immune cells and foster tumor angiogenesis [12,13]. TAMs in the ovarian cancer are generated from monocytes and resident macrophages. Research has shown that ovarian cancer TAMs are similar to monocyte-derived macrophages [14], which adopt the M2 phenotype. TAMs promote tumor progression, chemotherapeutic resistance and immunosuppression [11,12,[15][16][17]. CD163 and CD206/MRC1, which are strongly expressed on TAMs, are receptors for immunosuppressive molecules and predict the early recurrence of ovarian cancer [18][19][20]. CD163 and CD206 mRNA expression is also associated with IL-10 levels in ascites, which indicate a shorter relapse free survival (RFS) in patients with ovarian cancer [21]. The prognosis and survival of ovarian cancer patients are related to the presence of TAMs. Several adverse clinical markers are highly expressed by ovarian cancer TAMs, including CD163, Procollagen C-endopeptidase enhancer 2 (PCOLCE2), IL-6 and IL-10 [22]. TAMs are the primary secretors of most collagens and the main source of most protease inhibitors and make an important part in the synthesis of extracellular matrix (ECM) proteins [11]. Macrophages play a crucial role in ECM remodeling and the invasion of ovarian cancer [14,20,23]. TAMs can synthesize chemokine ligand 5 (CCL5), chemokine ligand 8 (CXCL8), and selectively synthesize CCL18, CXCL2 and CXCL3, all of which can attract monocytes/macrophages and promote tumor progression [24].

Roles of LPA in ovarian cancer
Lysophosphatidic acid (LPA) was initially identified as a ovarian cancer growth factor and was known as an ovarian cancer activator [25,26] and a potential marker of ovarian cancer [27]. LPA can promote ascites formation and tumorigenesis [28]. The raised levels of LPA in blood, tissues and ascites make it a useful biomarker and a potential therapeutic target in ovarian carcinoma [29]. LPA can promote tumor survival and proliferation, cisplatin resistance and increase the production of urokinase plasminogen activator (uPA), additional LPA generation and vascular endothelial growth factor (VEGF) in ovarian cancer. LPA can promote the production of protease and neovascularization mediators, and reduce the apoptosis of tumor cells [30], but has no obvious effect on normal ovarian cells [31]; these roles of LPA are similar to those of TAMs. The tissues and cells in the ovarian carcinoma TME maybe the main source of the increased LPA. The cells involved in LPA production include immune cells, platelets, mesothelial cells and adipocytes [29]. The leading role of LPA in ovarian carcinoma is in cell invasion and migration, and these effects are mainly induced by LPA receptors (LPARs). LPARs are a group of G proteincoupled receptors (GPCRs) for LPA that include LPAR1, LPAR2, LPAR3, LPAR4, LPAR5 and LPAR6 [32][33][34][35][36][37][38][39].. Recent studies showed that LPA is related to the formation of ovarian carcinoma stem cells and enhances their malignant behavior; these effects are mediated by LPAR1 [40,41], which interacts with CD14 [42], a monocyte differentiation antigen which is highly expressed on the cell membrane surface of monocytes/macrophages.

The hypothesis
The interplay between LPA and tumor associated macrophages plays a critical role in driving ovarian cancer malignancy and offers a potential target for therapy. We propose that this hypothesis is supported by three points, as showed in the Fig. 1: 1. LPA transforms monocytes directly into TAMs in the ovarian cancer TME. 2. LPA regulates TAM formation by activating the PI3K/AKT/mTOR signaling pathway through LPAR on the cell surface, which may also affect the function of peroxisome proliferators-activated receptor gamma (PPAR γ). 3. TAMs produce more LPA. Together, LPA and TAMs form a vicious circle that affects the malignant behavior of ovarian cancer.

LPA and TAMs play similar roles
LPA regulates a variety of tumor-promoting factors and inflammatory factors in epithelial ovarian cancer, including IL-6, IL-8, VEGF, matrix metallopeptidases (MMPs), CXCL12, cytochrome c oxidase subunit 2 (COX2), uPA, cyclin D1, CXCL1 [43]. Macrophages produce many factors that contribute to tumor growth, including VEGF, IL-1, IL-6, nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α) and macrophage colony-stimulating factor (M-CSF) [44,45]. LPA enhances the expression and secretion of IL-13 in T cells [46]. M2 macrophages can be activated by IL-13 or IL-4 [47]. The macrophage derived phospholipase PLA2G7 can produce extra-cellular LPA, which participate in the progress of ovarian carcinoma, and is related to the early recurrence of ovarian cancer [21,48]. LPA helps cancer cells avoid the immune system by improving the chemotaxis of Th2 cells [49] and inhibiting the activation of CD8+ T cells [50]. M2 macrophages have poor antigen-presenting ability, participate in the Th2 reaction, inhibit Th1 adaptive immunity, and promote tumor progression [51,52]. TAMs express high levels of tumor growth factors and cytokines, such as CCL18, KITLGG, semaphorin-6B, S100 calcium-binding protein B (S100B) and VEGFB. Furthermore, TAMs preferentially express cytokines and growth factors, such as CCL18, that promote tumor progression, growth and recurrence in ovarian cancer [11]. The levels of CCL18 in cancer tissue are related to metastasis and the shorter overall survival of patients with ovarian cancer, which seems to be associated with the increase in the mTOR Complex 2 (mTORC2) signaling pathway [53]. LPARs (LPAR1-6) are GPCRs and the LPA signal is mainly induced by these six GPCRs, which activate extracellular signal-regulated kinases 1/2 (ERK1/2), phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), Ca2+ mobilization, RAC, RAS and Rho, and stimulate ovarian cancer cell survival and migration [54,55]. Therefore, we conjecture that LPA is involved in the functions of TAMs, perhaps in a manner related to the PI3K/AKT/mTOR signaling pathway (Fig. 1).

Relationship between TAMs and LPA
The functional annotation of TAM genes in the ovarian cancer TME most frequently reveals the GPCRs pathway. TAMs participate in the formation of lipids and play a crucial role in the synthesis of LPA. Additionally, macrophages and T cells express LPAR5 and LPAR6 [56]. LPAR6 is the main LPA receptor expressed on ovarian cancer TAMs [11]. In ovarian cancer, LPA is mainly generated by TAMs, and the role of LPA in macrophage polarization was previously reported [57]. Furthermore, it is TAMs, not tumor cells, which produce LPA in fat-free medium. LPA-induced genes in macrophages are related to cell movement and migration in ovarian cancer microenvironment [56]. Accordingly, ovarian cancer TAMs may activate LPAR and the relevant signaling pathway by synthesizing and secreting LPA, thus promoting the invasion and metastasis of ovarian cancer (Fig. 1).
LPA regulates macrophage polarization [11]. Recent research has shown that LPA can convert human monocytes into macrophages [57]. LPA-stimulated macrophages express high levels of CD68 and levels of CD14, CD64, CD68 and CD206 comparable to those expressed by macrophages stimulated with human M-CSF. AKT/mTOR signaling stimulated by LPA makes a significant part in the development of murine macrophages, and PPAR γ is an important transcriptional regulator of LPA-induced macrophage development [57]. Existing data prompt the hypothesis that LPA may activate the PI3K/AKT/mTOR pathway through LPAR to directly induce the polarization of monocytes/macrophages to TAMs in ovarian cancer. This mechanism needs to be better understood in future studies before the application of clinical immunotherapy in ovarian cancer (Fig. 1).

Conclusion
In ovarian carcinoma, elevated levels of LPA in blood, tissue and ascites lead to the conversion of monocytes into ovarian cancer TAMs in the TME. The molecular mechanism may involve LPA binding to LPAR, which activates the PI3K/AKT/mTOR pathway and affects the function of PPAR γ, resulting in a cascade of reactions and changes. Finally, LPA produced by TAMs and TAMs themselves form a vicious circle that affects the metastasis and invasion of ovarian carcinoma. Further study of the interaction between TAMs and LPA in ovarian cancer will bring about a better further understanding of ovarian cancer pathogeny and will provide theoretical evidence for the treatment and early diagnosis of ovarian carcinoma. This vicious circle is a potential target of immunomodulatory therapy for ovarian cancer.  PCOLCE2: Procollagen C-endopeptidase enhancer 2; CCL: Chemokine (C-C motif) ligand; CXCL: Chemokine (C-X-C motif) ligand; uPA: urokinase plasminogen activator; MMP: Matrix metallopeptidase; COX2: Cytochrome c oxidase subunit 2; S100B: S100 calcium-binding protein B; mTORC2: mTOR Complex 2; ERK: Extracellular signal-regulated kinases