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Extracellular Phosphatidic Acid: A Novel Regulator of Lung Cancer Cell Migration – Role of the PI3K/AKT Pathway

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Ana Gómez-Larrauri, Patricia Gangoiti, Laura Camacho, Natalia Presa, César Martin and Antonio Gómez-Muñoz

Submitted: 01 October 2025 Reviewed: 27 November 2025 Published: 24 February 2026

DOI: 10.5772/intechopen.1014164

Integrating Metabolomics with Proteomics, Transcriptomics, and Genomics for Medical Advancements IntechOpen
Integrating Metabolomics with Proteomics, Transcriptomics, and Ge... Edited by Abdul-Hamid Emwas

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Integrating Metabolomics with Proteomics, Transcriptomics, and Genomics for Medical Advancements [Working Title]

Dr. Abdul-Hamid Emwas and Prof. Helena U. Zacharias

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Abstract

Lung cancer is one of the most lethal types of cancer, being responsible for the majority of cancer-associated deaths worldwide. The main therapeutic strategy for the treatment of lung cancer is chemotherapy, but the currently available drugs are not sufficiently effective to overcome the disease. In the present chapter, we show that extracellular phosphatidic acid (PA), a bioactive phospholipid involved in inflammatory responses and cell proliferation, induces rapid phosphorylation of AKT and mammalian target of rapamycin (mTOR), and that pretreatment of the cells with LY294002, 10-DEBC, or rapamycin, which specifically inhibit phosphatidylinositol 3-kinase (PI3K), AKT, or mTOR, respectively, blocks PA-stimulated lung cancer cell migration. Additionally, AM966 and Ki16425, which inhibit lysophosphatidic acid receptor 1 (LPAR1), completely block PA-stimulated phosphorylation of mTOR and lung cancer cell migration, suggesting that stimulation of chemotaxis by PA involves its interaction with LPA receptors. Moreover, exogenous PA induced phosphorylation of focal adhesion kinase (FAK), which is upstream of Rac1 activation, and inhibition of FAK or Rac1 also blocked PA-stimulated lung cancer cell migration. It can be concluded that the PA-LPA1-PI3K-AKT-mTOR pathway and the FAK/Rac1 axis are highly relevant factors for regulation of lung cancer cell migration, and that inhibition of PA generation may be of crucial importance for reducing lung cancer dissemination.

Keywords

  • focal adhesion kinase
  • phosphatidic acid
  • phosphatidylinositol 3-kinase
  • AKT
  • lung cancer cell migration
  • mammalian target of rapamycin (mTOR)

1. Introduction

Lung cancer is responsible for the majority of cancer-associated deaths worldwide. It affects millions of people every year, costing billions of dollars to health care systems [1]. GLOBOCAN 2020 provided an estimate of 2.2 million new lung cancer cases (11.4%) and almost 1.8 million lung cancer deaths (18.0%) worldwide in 2020 [2, 3]. The American Cancer Society’s estimates for lung cancer in the US for 2025 are 226,650 new cases of lung cancer (110,680 in men and 115,970 in women approximately) and about 124,730 deaths from lung cancer (64,190 in men and 60,540 in women) (https://www.cancer.org/cancer/types/lung-cancer/about/key-statistics.html).

There are two major lung cancer subtypes: non-small cell lung cancer (NSCLC) and small cell lung cancer. NSCLC is the most common type, accounting for over 85% of all cases [4]. NSCLC can be further classified into three types: large cell carcinoma, squamous cell carcinoma, and adenocarcinoma, with the latter being the most common type of NSCLC and representing about 40% of all NSCLC cases [5]. Some rare subtypes of lung cancer vary in frequency by race, ethnicity, or age [6]. Although the incidence of rare lung cancer diseases is less than 5% of all NSCLC, it can affect over 90,000 people annually worldwide, a number higher than that of other malignancies, including some types of leukemia, or bone and male genital cancers [6].

Chemotherapy remains the main therapeutic strategy for the treatment of lung cancer, but the currently available drugs are not sufficiently effective for curing the disease, especially in patients with advanced NSCLC. Therefore, new therapeutic strategies to reduce lung cancer malignancy are urgently needed. In this connection, targeting specific receptors or intracellular components of cell signaling pathways involved in cell proliferation and motility may offer promising approaches [7-9]. A key signaling cascade that is implicated in lung cancer growth and dissemination is the AKT/mammalian target of rapamycin (mTOR) pathway, which is triggered by stimulation of phosphatidylinositol 3-kinase (PI3K) (reviewed in [10]). The binding of agonists, such as growth factors or cytokines, to their cell surface receptors, including G protein-coupled receptors, often leads to PI3K activation and subsequent phosphorylation (stimulation) of AKT to promote invasion and metastasis in different types of cancer [5]. Besides conventional growth factors, which are usually of peptide (protein) nature, some bioactive lipids, including phosphatidic and lysophosphatidic acids (PA and LPA, respectively), have been shown to regulate key biological functions that are associated with tumor promotion. These include regulation of cell growth, differentiation, survival, or cell migration in different cell types [11–17]. In particular, LPA exerts most of its biological effects through binding to six different G protein-coupled receptors, designated LPAR1-6 [18]. By contrast, no specific receptor for PA has, to date, been identified. Also, although some initial studies suggested that the biological actions of PA were caused by its metabolism to LPA, recent evidence indicates that PA itself can promote cell activation independently of conversion to LPA. Specifically, PA is implicated in tumor growth and development through activation of a variety of signaling cascades and is a key regulator of mTOR, GTPase-activating proteins, guanine nucleotide exchange factors, or protein kinase C-α, which are proteins involved in tumorigenesis and tumor dissemination [17, 19]. PA can be synthesized de novo in the endoplasmic reticulum or can be generated intracellularly by the actions of LPA acyl transferases, diacylglycerol kinase, or phospholipase D activities [17, 20]. Also importantly, PA can be secreted by cells into the extracellular environment and is found in plasma at low micromolar concentrations [11]. Moreover, PA is present in extracellular vesicles, which are secreted by a great variety of cell types, including cancer cells [21–23]. This would enable PA to interact with the plasma membrane of cells to elicit at least some of its biological effects.

By using the Boyden chamber or transwell assay, we demonstrate here that extracellular PA promotes migration of lung cancer (adenocarcinoma) cells and that, at least partially, the mechanism whereby PA exerts this action involves activation of the PI3K/AKT/mTOR pathway, as well as activation of FAK and Rac1. The latter findings were determined by using specific inhibitors of these kinases and Rac1 in combination with Western-blotting techniques, as indicated in Section 2.

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2. Experimental procedures

2.1 Materials

Human adenocarcinoma A549 cells were obtained from ATCC (American Type Culture Collection) (Manassas, VA, USA). The culture medium, Dulbecco’s Modified Eagle’s Medium (DMEM), was purchased from Lonza (Basel, Switzerland). Fetal bovine serum (FBS) was obtained from Gibco. Fatty acid-free bovine serum albumin (BSA), crystal violet, the FAK inhibitors Y-15 (1,2,4,5-benzenetetraamine) and PF573228 rapamycin, the Rac1 inhibitor, and the LPA1 inhibitor Ki16425 were from Sigma-Aldrich (St. Louis, MO, USA). The LPA1 inhibitor AM966 was purchased from Cayman Chemicals (Ann Arbor, MI, USA). LY290042 and 10-DEBC hydrochloride were from Tocris Bioscience (Bristol, UK). Phosphatidic acid (isolated from egg yolk lecithin), 1,2-dipalmitoyl-sn-glycero-3-phosphate (16:0 PA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (16:0-18:1 PA), and 1,2-dioleoyl-sn-glycero-3-phosphoethanol were obtained from Avanti Polar Lipids (Birmingham, AL, USA). Secondary antibodies (goat anti-rabbit IgG conjugated to horseradish peroxidase) and primary antibodies to phospho-AKT (Ser 473), total AKT, phospho-mTOR, total mTOR, phospho-p70S6K, total p70S6K, phospho-S6, total S6, phospho-FAK, total FAK, phospho-ERK1/2 (Thr-202/Tyr-204), and total ERK1/2 were obtained from cell signaling (Danvers, MA, USA).

2.2 Cell culture

Human A549 lung cancer cells were cultured in RPMI 1640 culture medium supplemented with heat-inactivated FBS (10%), gentamicin (50 mg/L), and L-glutamine (2 mM). The culture medium was replaced every 3 to 4 days with freshly prepared medium, maintaining a cell density of about 6 × 104 cells/cm2 in T75 flasks. Experiments were performed in RPMI 1640 in the absence of FBS but supplemented with BSA (0.1%), except for cell migration experiments, where BSA was maintained at 0.2%. In cases where inhibitors were used, they were added to cell cultures 90 min before the addition of the agonist. PA, which was sonicated in nanopure water, was added to the cells as required.

2.3 Cell viability assay

Human A549 lung cancer cells were cultured in 96-well plates (104 cells/well) in a volume of 100 µl RPMI 1640 culture medium supplemented with 10% FBS, which had been previously inactivated by heating at 56 ºC for 30 min with gentle swirling every 5 to 10 min to avoid protein clotting, and the cells were left to grow overnight. After this time, the culture medium was discarded and replaced with freshly prepared medium supplemented with BSA (0.1%) in the absence of FBS. Agonists or inhibitors were then added as required. The cells were washed twice with PBS and stained with 50 μl of a solution of crystal violet (0.5% of the dye in 20% methanol) with gentle shaking at room temperature for 20 min. The cells were then washed twice with water and allowed to dry for about 1 hour. Methanol (200 µl) was then added to each well to elute the dye, for which the plates were kept shaking for 20 min. Absorbance were read at a wavelength of 570 nm in a Biotek Power Wave XS spectrophotometer.

2.4 Measurement of cell migration

Cell migration was assessed using the Boyden chamber assay, also known as the transwell assay, with 24-well culture plates (from Corning Costar) containing 12 chemotaxis chambers equipped with 8.0-µM pore diameter filters. Prior to seeding the cells, the filters were coated with 6 µg of human fibronectin in a volume of 30 µl to ensure optimal attachment of the cells. Cells (5 x 104 in 100 μl) were placed on top of the fibronectin-coated filters in serum-free RPMI 1640 culture medium supplemented with 0.2% BSA. The cells were allowed to adhere for 90 min. The chambers were then transferred into the lower wells of the plates, where agonists had been previously added in a volume of 300 µl of the same culture medium supplemented with 0.2% BSA and no FBS. When inhibitors were used, they were added to both the chambers and the lower wells. The cells were further incubated for 90 min before placing the chambers in the lower wells. When required, the chambers were removed from the lower wells, and non-migrated cells that did not pass through the filters and remained attached to the upper side of the filters were eliminated using cotton swabs. The cells that migrated through the filters remained attached to the lower side of the filters and were fixed by placing the chambers in wells containing 300 µl of para-formaldehyde (5% in phosphate-buffered saline (PBS)) for 30 min. After this period, the para-formaldehyde solution was discarded, and the filters carrying the cells were washed twice with PBS. Staining of the cells was performed by placing the chambers in wells containing 300 µl of 0.1% crystal violet in PBS for 20 min. Subsequently, the upper side of the filters was cleaned again with cotton swabs to remove any residual cells that had not passed through the filters. Counting of the migrated cells was conducted using a Nikon Eclipse 90i light microscope equipped with NIS-Elements 3.0 software. Alternatively, an inverted Nikon Eclipse Ts2 light microscope was used without the need to unmount the filters. Cell counting was performed by randomly selecting five microscope fields per well at 100x magnification.

2.5 Western blotting analysis

Human A549 lung cancer cells (2 × 105 cells/well) were placed in 6-well plates in 1 ml RPMI 1640 medium supplemented with 10% heat-inactivated FBS and allowed to grow for 24 h. Agonists and/or inhibitors were added to the cells in serum-free medium, as required. Cells were then carefully washed three times with cold PBS and collected in lysis buffer containing 100 mM Tris-HCl pH 8, 0.685 M NaCl, 1% (v/v) IGEPAL, 2.5 mM EDTA, 10% (v/v) glycerol, and 1 μg/ml of a commercial cocktail of protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Cell lysis was performed by sonicating and vortexing the samples, and the concentration of protein in the samples was determined with a commercial kit from BioRad. Protein separation and subsequent identification of selected bands were achieved by SDS-PAGE electrophoresis, followed by transfer of the proteins onto nitrocellulose or polyvinylidene difluoride (PVDF) membranes, and incubation with appropriate antibodies as previously established [24].

2.6 Statistical analysis

Results are expressed relative to the control value in each experiment and are given as the mean ± SD of the number of experiments indicated. Basal cell migration (control condition) after 24 hours of incubation was about 20 ± 3 cells (mean ± SD of cells that migrated through the chamber filter in six different experiments), which is consistent with our previously reported work (see reference number 20). Statistical analyses were performed using Student’s t-tests. Values of p < 0.05 were considered statistically significant.

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3. Results

3.1 Extracellular phosphatidic acid promotes lung cancer cell migration

PA is a bioactive phospholipid with mitogenic and proinflammatory properties that is involved in tumor promotion. However, although exogenous PA can promote myoblast proliferation [24], it does not stimulate lung cancer cell growth. Nonetheless, cancer cells are able to migrate and invade different tissues to establish new malignant foci that culminate in metastasis. In this chapter, we show that PA stimulates lung cancer cell migration after 24 hours of incubation, whereas similar concentrations of phosphatidylethanol (PEtOH), an abnormal phospholipid that is also synthesized by the action of phospholipase D but in the presence of ethanol, did not significantly alter cell motility (Figure 1).

Figure 1.

Stimulation of lung cancer cell migration by PA. Lack of effect of phosphatidylethanol. A. A549 cells were incubated with vehicle or treated with 20 µM PA, or 20 µM PEtOH for 24 h, as indicated. Optimal concentrations of PA for stimulation of lung cancer cell migration (10–20 µM) were previously established [25]. Data are expressed relative to the control value and are given as means ± SD of three independent experiments performed in duplicate (**p < 0.01). B. Cells were treated with 10 or 20 µM PEtOH for 24 h, and cell viability was determined as detailed in Materials and Methods. Data are expressed relative to the control value and are given as the means ± SD of three different experiments performed in triplicate.

3.2 Exogenous phosphatidic acid induces AKT phosphorylation: Implication of the PI3K/AKT pathway in the stimulation of lung cancer cell migration by PA

We reported recently that part of the mechanism by which PA promotes lung cancer cell migration involves the activation of mitogen-activated protein kinases (MAPKs) [25]. However, a major pathway that is particularly relevant in the spreading of lung cancer is the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. In fact, it was previously reported that in NSCLC, anomalous activation of the mitogen-activated protein kinase (MEK)/extracellularly regulated kinases 1 and 2 (ERK1-2) and PI3K/AKT pathways leads to lung cancer cell dissemination [26]. Therefore, to investigate whether an association exists between PI3K/AKT and PA-regulated lung cancer cell migration, the human A549 lung cancer (adenocarcinoma) cells, a kind of NSCLC, were used. Figure 2A shows that PA rapidly (within minutes) stimulates the phosphorylation of AKT, which lies downstream of PI3K activation. The implication of the PI3K/AKT pathway in PA-stimulated lung cancer cell migration was studied by preincubating the cells with LY290042 or 10-DEBC, which specifically inhibit PI3K or AKT, respectively, prior to challenging the cells with exogenous PA. Figure 2B, C shows that PA-stimulated cell migration was significantly reduced by these inhibitors at concentrations at which they did not affect cell viability (Figure 2D, E).

Figure 2.

PA induces phosphorylation of AKT in lung cancer cells. Implication of PI3K/AKT in PA-stimulated lung cancer cell migration. A. Treatment of A549 lung cancer cells with PA (10 µM) led to AKT phosphorylation. The phosphorylated form of AKT (P-AKT) was identified by Western blotting using a specific antiphospho-AKT (Ser473) antibody. Controls for equal loading of protein were performed using specific antibodies to total AKT and GAPDH, as indicated. B, C. Cells were pretreated for 90 min with or without LY294002 (10 µM) or 10DECB (1 µM), which specifically inhibit PI3K or AKT, respectively, prior to addition of PA, and cell migration was determined as indicated in the materials and methods section. Data are expressed relative to the control value and are given as the means ± SD of three independent experiments performed in duplicate. (*p < 0.05, control versus PA-treated cells, ##p < 0.01, PA-treated cells versus PA-treated cells in the presence of inhibitor, LY = LY294002, or DECB = 10DECB). D, E. LY294002 (10-20 µM) or 10DECB (1-5 µM) did not significantly affect cell viability, which was monitored by staining the cells with crystal violet (see materials and methods). Data are expressed relative to the values without inhibitor (0 µM) and are given as the means ± SD of three independent experiments performed in triplicate.

3.3 Extracellular phosphatidic acid induces phosphorylation of mTOR: Implications of mTOR/S6 and LPA1 in the stimulation of lung cancer cell migration by PA

A relevant downstream effector of PI3K/AKT is mTOR, which is a kinase involved in the regulation of key pathophysiological processes, including cell proliferation and migration. Figure 3A shows that treatment of the A549 lung cancer cells with PA causes rapid and time-dependent phosphorylation of mTOR and its downstream kinase effector p70S6K, which phosphorylates ribosomal protein S6, a relevant regulatory element of translation. The implication of the mTOR pathway in PA-stimulated lung cancer cell migration was evaluated using rapamycin, which is a specific and well-established inhibitor of mTOR. As shown in Figure 3B, C, non-toxic concentrations of rapamycin completely inhibited this process, suggesting that mTOR is essential for the stimulation of lung cancer cell migration by PA. Of interest, our lab recently reported that the stimulation of lung cancer cell migration by PA involved prior interaction of the phospholipid with LPA receptor 1 (LPA1), followed by MAPK activation [25]. We now show that AM966, which specifically inhibits the LPA1 receptor, and Ki16425, an inhibitor of LPA1 and LPA3 receptors, block phosphorylation of the mTOR effector protein S6 (Figure 3D), suggesting that the mTOR/S6 pathway is downstream of LPA1 activation by PA. Noteworthy, in agreement with our previous work, both LPA1 inhibitors completely blocked PA-stimulated lung cancer cell migration [25]. Furthermore, both Ki16425 and AM966 inhibitors block the phosphorylation of ERK1-2, thereby confirming that the mitogen-activated protein kinase (MEK)/ERK1-2 pathway is also implicated in PA-stimulated lung cancer cell migration (Figure 3D). Also, it should be noted that the phosphate moiety of PA seems to be crucial for the phospholipid to interact with the LPA receptor, as incorporation of ethanol into the molecule to form phosphatidylethanol rendered the phospholipid inactive (Figure 1B).

Figure 3.

PA promotes phosphorylation of mTOR, p70S6K, and S6 in lung cancer cells. Implication of mTOR and the LPA1 receptor in PA-stimulated cell migration. A. Treatment of A549 cells with PA (10 µM) caused phosphorylation of mTOR, p70S6K, and S6. Phosphorylated proteins were identified by Western blotting analysis using specific antibodies. Controls for equal loading of protein were performed using specific antibodies to total mTOR, p70S6, S6, and GAPDH, as indicated. B. Cells were pretreated for 90 min with or without rapamycin (1 µM), which specifically inhibits mTOR, prior to addition of PA, and cell migration determined as detailed in the materials and methods section. Data are expressed as in Figure 2 (*p < 0.05, control versus PA-treated cells, ##p < 0.01, PA-treated cells versus PA-treated cells in the presence of rapamycin). C. Rapamycin at concentrations ranging from 0.1 to 1 µM did not significantly alter cell viability. Data are expressed as in Figure 2. D. Inhibition of PA-induced phosphorylation of ERK1-2 and S6 by the LPA1 receptor antagonists AM966 and Ki16425. Cells were pretreated for 90 min with or without the LPA1 receptor antagonists, Ki16425 (10 µM) or AM966 (1 µM), before addition of PA (10 µM). phosphorylated proteins were identified by Western blotting as indicated above.

3.4 Phosphatidic acid induces phosphorylation of focal adhesion kinase (FAK): Implication of FAK in the stimulation of lung cancer cell migration by PA

FAK is an important protein tyrosine kinase involved in cell migration processes [27, 28]. Figure 4A shows that PA causes rapid (within minutes) phosphorylation of FAK, which is positioned upstream of ERK1-2 and PI3K phosphorylation and can control the activities of these kinases [29–32]. To assess the possible implication of FAK in PA-stimulated lung cancer cell migration, A549 cells were challenged with PA in the presence of PF573228 or Y-15, which specifically inhibit FAK [27, 28, 33]. Both FAK inhibitors completely blocked the stimulation of cell migration by PA (Figure 4B, C) at concentrations at which they did not significantly alter cell viability (Figure 4D, E), suggesting that FAK is a relevant factor in this process.

Figure 4.

PA promotes FAK phosphorylation in lung cancer cells: Implication of FAK in PA-stimulated cell migration. A. Treatment of A549 cells with PA (10 µM) stimulated FAK phosphorylation. Phosphorylated FAK (P-FAK) was identified by Western blotting using a specific antibody. Controls for equal loading of protein were performed using specific antibodies to total FAK and GAPDH. B, C. Cells were pretreated for 90 min with or without PF573228 (10 µM) or Y15 (10 µM), which specifically inhibit FAK, prior to addition of PA (10 µM), and cell migration was determined as detailed in the materials and methods section. Data are expressed as in Figure 2 or 3 (*p < 0.05 or **p < 0.01, control versus PA-treated cells, ##p < 0.01, PA-treated cells versus PA-treated cells in the presence of the corresponding inhibitor, PF = PF573228). D, E. PF573228 or Y15 (both up to 10 µM) did not significantly alter cell viability. Data are expressed relative to the values without inhibitor (0 µM) and are given as means ± SD of three independent experiments performed in triplicate.

An important downstream target of FAK is the small homomeric G protein Rac1 [34]. Figure 5 shows that PA-stimulated cell migration was substantially decreased by pretreating the cells with a specific inhibitor of Rac1, suggesting that this G protein is also relevant to the regulation of lung cancer cell migration by the phospholipid.

Figure 5.

Implication of Rac1 in PA-stimulated lung cancer cell migration. A. Cells were pretreated for 90 min with or without Rac1 inhibitor (100 µM) prior to the addition of PA (10 µM). Cell migration was measured as detailed in Materials and Methods. Data are expressed as in Figure 2 or 3 (*p < 0.05, control versus PA-treated cells; #p < 0.05, PA-treated cells versus PA-treated cells in the presence of inhibitor). B. The Rac1 inhibitor (up to 200 µM) did not significantly affect cell viability. Data are expressed as in Figure 2 or 3.

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4. Discussion

Although extensive research over several decades has aimed to develop effective treatments for lung cancer, the condition remains a major focus of scientific inquiry, as no current medication or chemotherapy approach has proven capable of curing the disease. Hence, deciphering the molecular mechanisms or signaling pathways governing lung cancer cell growth and dissemination is of vital importance. A significant class of molecules with increasing relevance in cancer biology over the last few years includes bioactive lipids, such as the glycerophospholipid PA. The latter is critical for the recruitment of guanine nucleotide-exchange factors for small GTPases, including Dedicator of Cytokinesis 2 (DOCK2) and SOS (Son of Sevenless), to the plasma membrane, keeping Ras in its active form [16, 35]. Notably, PA regulates Ras, a protein implicated in tumorigenesis that is involved in 20-30% of all human cancers when mutated [36, 37]. PA has also been implicated in cell trafficking and the release of factors that enhance tumorigenesis, including type 1 matrix metalloproteinases, which are enzymes implicated in metastasis [38]. Another important mechanism whereby PA may play a relevant role in cancer cell biology is by promoting the activation of mTOR [39, 40], which is a key regulator of cell proliferation and survival [41], and is also implicated in cell motility and metastasis [42]. Regarding lung cancer, we recently reported that PA induces migration of human lung adenocarcinoma cells through a mechanism involving its interaction with the LPA1 receptor, followed by activation of ERK1-2, p38, and c-Jun N-terminal kinase (JNK), as well as upregulation of the Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 pathway [25]. This finding was particularly interesting, as activation of ERK, JNK, and p38 does not typically occur simultaneously. Nonetheless, concomitant stimulation of these three kinases was critical for promoting ME180 cervical carcinoma cell migration by LPA [43] and for regulating lung cancer cell migration by interleukin-1β (IL-1β) [44]. Interestingly, secretion of IL-1β is a major mechanism whereby PA exerts its pro-inflammatory actions [45].

The core of the present study is the demonstration that exogenous PA promotes lung cancer cell migration through stimulation of the PI3K/AKT/mTOR pathway. Similar to the activation of MAPKs and STAT3 [25], PA induced rapid phosphorylation of AKT and its downstream effector mTOR, leading to subsequent phosphorylation of p70S6K and its direct target S6, a protein that has been defined as a potential therapeutic target against cancer [46]. In addition to being phosphorylated (stimulated) by AKT, mTOR can be activated in a PI3K/AKT-independent manner by PA, probably involving PLD activation [47, 48]. In this context, we previously reported that exogenous PA was able to stimulate PLD activity, thereby increasing the intracellular levels of PA [49], which may then contribute to the enhancement of mTOR activation. However, the fact that inhibitors of PI3K completely block S6 phosphorylation, as we show here, indicates that the PI3K/AKT/mTOR pathway is the main operative mechanism through which PA stimulates lung cancer cell migration. As shown in this chapter, PA-induced phosphorylation of protein S6 and ERK1-2 was completely inhibited by specific inhibitors of the LPA1 receptor. These findings, together with our previously published work showing inhibition of PA-stimulated migration of these same cells by the same specific inhibitors of the LPA1 receptor, indicate that PA regulates lung cancer cell migration through binding to LPA1 followed by activation of the PI3K/AKT/mTOR and ERK1-2 pathways. It should also be noted that stimulation of FAK phosphorylation, a kinase that is upstream of PI3K and ERK1-2 activation [50, 51], may also play an important role in PA-stimulated cell migration, as blockade of this kinase or inhibition of its downstream effector Rac1 abolished the stimulation of cell migration by PA.

It should also be noted that LPAR1 is the major receptor expressed in A549 lung cancer cells, with minimal expression of LPAR2 and LPAR3, and a total absence of the other LPA receptors in these cells [52]. So, the observation that the blockade of LPAR1 leads to complete inhibition of PA-stimulated lung cancer cell migration also suggests that the other LPA receptors may not be as relevant as LPAR1 for promoting migration/invasion of the lung cancer cells. The fact that PA can interact with LPAR1 to elicit cell migration should encourage researchers to focus their attention on PA metabolism, in particular on PA formation, to reduce LPAR1-mediated lung cancer cell migration/invasion when LPA levels are low or when the synthesis of LPA is compromised.

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5. Conclusion

The existence of extracellular PA, which is present in plasma, led to the proposal of the hypothesis that PA could trigger receptor-mediated effects. Using the Boyden chamber (or transwell) assay in combination with analytical procedures to detect and identify specific proteins (Western blotting) has made it possible to observe that exogenous PA promotes lung cancer cell migration. The results of the present study demonstrate that a major mechanism by which PA exerts this action implicates the stimulation of the PI3K/AKT/mTOR/S6 and FAK/Rac1 pathways, also involving the activation of the MAP kinases ERK1 and 2. More importantly, the finding that PA can substitute for LPA to activate LPAR1 and subsequent signaling pathways may be highly relevant for designing novel therapeutic strategies to reduce lung cancer dissemination when the levels of LPA are depleted, or when the activity or expression of LPA-producing enzymes is low. Further research is now necessary for the translation of these results from the bench to the bedside to improve patient care.

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Abbreviations

ERK

Extracellular signal-regulated kinase

 FAK

Focal adhesion kinase

 JAK

Janus kinase

 JNK

C-Jun N-terminal kinase

 LPA

Lysophosphatidic acid

 LPAR

LPA receptor

 MAPK

Mitogen-activated protein kinase

 MEK

Mitogen-activated protein kinase

 mTOR

Mammalian target of rapamycin; NSCLC, non-small cell lung cancer

 PA

Phosphatidic acid; PI3K, phosphatidylinositol 3-kinase

 PKC

Protein kinase C; STAT3, signal transducer and activator of transcription 3.
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Acknowledgments

Work in AGM and CM laboratories is supported by the “Departamento de Educación, Viceconsejería de Universidades e Investigación del Gobierno Vasco.” Basque Country, Spain (Grant number IT1720-22).

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Conflict of Interest

The authors declare no conflicts of interest.

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Author Contributions

AGM designed the research study. AGL and PG performed the research. LC and NP provided help with some experiments. AGL and PG analyzed the data. CM provided advice on methodology and assistance with some experiments. AGM and AGL wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

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Written By

Ana Gómez-Larrauri, Patricia Gangoiti, Laura Camacho, Natalia Presa, César Martin and Antonio Gómez-Muñoz

Submitted: 01 October 2025 Reviewed: 27 November 2025 Published: 24 February 2026

© 2026 The Author(s). Licensee IntechOpen. This content is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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