Role for CCN1 in lysophosphatidic acid response in PC-3 human prostate cancer cells.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive phospholipids that act as mitogens in various cancers. Both LPA and S1P activate G-protein coupled receptors (GPCRs). We examined the role of CCN1/CYR61, an inducible matricellular protein, in LPA-induced signal transduction in PC-3 human prostate cancer cells. We found that both LPA and S1P induced expression of CCN1 and CCN2 within 2-4 h. CCN1 was induced by 18:1-LPA, but not by 18:0-, 18:2-, or 18:3-LPAs. A free fatty acid receptor-4 agonist inhibited LPA-induced CCN1 induction. CCN1 appeared in the ECM within 2 h after LPA addition. LPA caused biphasic activation of Erk MAPK, with an initial peak at 10-20 min followed by a later phase after 6 h. LPA increased adhesion of PC-3 cells to culture substrates (standard culture plates, fibronectin, or extracellular matrix) at 2 h, an intermediate event between early and late LPA signals. Knockdown of CCN1 suppressed LPA-induced adhesion to ECM or fibronectin. ECM from CCN1 knockdown cells was a poor substrate for adhesion, as compared to ECM from control cells. These results suggest that CCN1 contributes to LPA responses in the tumor microenvironment. The LPA-CCN1 axis holds promise for the development of novel therapeutic strategies in cancer.

Characterization of Lysophospholipase D Activity in Mammalian Cell Membranes.

Lysophosphatidic acid (LPA) is a lipid mediator that binds to G-protein-coupled receptors, eliciting a wide variety of responses in mammalian cells. Lyso-phospholipids generated via phospholipase A2 (PLA2) can be converted to LPA by a lysophospholipase D (lyso-PLD). Secreted lyso-PLDs have been studied in more detail than membrane-localized lyso-PLDs. This study utilized in vitro enzyme assays with fluorescent substrates to examine LPA generation in membranes from multiple mammalian cell lines (PC12, rat pheochromocytoma; A7r5, rat vascular smooth muscle; Rat-1, rat fibroblast; PC-3, human prostate carcinoma; and SKOV-3 and OVCAR-3, human ovarian carcinoma). The results show that membranes contain a lyso-PLD activity that generates LPA from a fluorescent alkyl-lyso-phosphatidylcholine, as well as from naturally occurring acyl-linked lysophospholipids. Membrane lyso-PLD and PLD activities were distinguished by multiple criteria, including lack of effect of PLD2 over-expression on lyso-PLD activity and differential sensitivities to vanadate (PLD inhibitor) and iodate (lyso-PLD inhibitor). Based on several lines of evidence, including siRNA knockdown, membrane lyso-PLD is distinct from autotaxin, a secreted lyso-PLD. PC-3 cells express GDE4 and GDE7, recently described lyso-PLDs that localize to membranes. These findings demonstrate that membrane-associated lyso-D activity, expressed by multiple mammalian cell lines, can contribute to LPA production.

Global Proteomics Analysis of Lysophosphatidic Acid Signaling in PC-3 Human Prostate Cancer Cells: Role of CCN1.

Cysteine-rich angiogenic factor 61 (CCN1/Cyr61) is a matricellular protein that is induced and secreted in response to growth factors. Our previous work showed that 18:1-lysophosphatidic acid (LPA), which activates the G protein-coupled receptor LPAR1, induces CCN1 between 2-4 h in PC-3 human prostate cancer cells in a manner than enhances cell-substrate adhesion. While the time course of induction suggests that CCN1 contributes to intermediate events in LPA action, the roles of CCN1 in LPA-mediated signal transduction have not been fully elucidated. This study utilized a comprehensive global proteomics approach to identify proteins up- or down-regulated in response to treatment of PC-3 cells with LPA for three hours, during the time of peak CCN1 levels. In addition, the effects of siRNA-mediated CCN1 knockdown on LPA responses were analyzed. The results show that, in addition to CCN1, LPA increased the levels of multiple proteins. Proteins up-regulated by LPA included metastasis-associated in colon cancer protein 1 (MACC1) and thrombospondin-1 (TSP1/THBS1); both MACC1 and TSP1 regulated cancer cell adhesion and motility. LPA down-regulated thioredoxin interacting protein (TXNIP). CCN1 knockdown suppressed the LPA-induced up-regulation of 30 proteins; these included MACC1 and TSP1, as confirmed by immunoblotting. Gene ontology and STRING analyses revealed multiple pathways impacted by LPA and CCN1. These results indicate that CCN1 contributes to LPA signaling cascades that occur during the intermediate phase after the initial stimulus. The study provides a rationale for the development of interventions to disrupt the LPA-CCN1 axis.

From outside to inside and back again: the lysophosphatidic acid-CCN axis in signal transduction.

CCN1 and CCN2 are matricellular proteins that are transcriptionally induced by various stimuli, including growth factors. CCN proteins act to facilitate signaling events involving extracellular matrix proteins. Lysophosphatidic acid (LPA) is a lipid that activates G protein-coupled receptors (GPCRs), enhancing proliferation, adhesion, and migration in many types of cancer cells. Our group previously reported that LPA induces production of CCN1 protein in human prostate cancer cell lines within 2-4 h. In these cells, the mitogenic activity of LPA is mediated by LPA Receptor 1 (LPAR1), a GPCR. There are multiple examples of the induction of CCN proteins by LPA, and by the related lipid mediator sphingosine-1-phosphate (S1P), in various cellular models. The signaling pathways responsible for LPA/S1P-induced CCN1/2 typically involve activation of the small GTP-binding protein Rho and the transcription factor YAP. Inducible CCNs can potentially play roles in downstream signal transduction events required for LPA and S1P-induced responses. Specifically, CCNs secreted into the extracellular space can facilitate the activation of additional receptors and signal transduction pathways, contributing to the biphasic delayed responses typically seen in response to growth factors acting via GPCRs. In some model systems, CCN1 and CCN2 play key roles in LPA/S1P-induced cell migration and proliferation. In this way, an extracellular signal (LPA or S1P) can activate GPCR-mediated intracellular signaling to induce the production of extracellular modulators (CCN1 and CCN2) that in turn initiate another round of intracellular signaling.

Lysophosphatidic Acid Signaling in Cancer Cells: What Makes LPA So Special?

Lysophosphatidic acid (LPA) refers to a family of simple phospholipids that act as ligands for G protein-coupled receptors. While LPA exerts effects throughout the body in normal physiological circumstances, its pathological role in cancer is of great interest from a therapeutic viewpoint. The numerous LPA receptors (LPARs) are coupled to a variety of G proteins, and more than one LPAR is typically expressed on any given cell. While the individual receptors signal through conventional GPCR pathways, LPA is particularly efficacious in stimulating cancer cell proliferation and migration. This review addresses the mechanistic aspects underlying these pro-tumorigenic effects. We provide examples of LPA signaling responses in various types of cancers, with an emphasis on those where roles have been identified for specific LPARs. While providing an overview of LPAR signaling, these examples also reveal gaps in our knowledge regarding the mechanisms of LPA action at the receptor level. The current understanding of the LPAR structure and the roles of LPAR interactions with other receptors are discussed. Overall, LPARs provide insight into the potential molecular mechanisms that underlie the ability of individual GPCRs (or combinations of GPCRs) to elicit a unique spectrum of responses from their agonist ligands. Further knowledge of these mechanisms will inform drug discovery, since GPCRs are promising therapeutic targets for cancer.

Report of the 2018-2019 Research and Graduate Affairs Committee.

The 2018-2019 Research and Graduate Affairs Committee (RGAC) was charged with critically evaluating the leadership development support necessary for pharmacy researchers, including postdoctoral trainees, to develop the skills needed to build and sustain successful research programs and analyzing how well those needs are being met by existing programs both within AACP and at other organizations. The RGAC identified a set of skills that could reasonably be expected to provide the necessary foundation to successfully lead a research team and mapped these skills to the six domains of graduate education in the pharmaceutical sciences established by the 2016-2017 RGAC (Table 1). In addition, the RGAC identified competency in team science and the bench-to-bedside-to-beyond translational spectrum as being critical elements of research leadership. The universality of these skills and their value prompted the RGAC to make two related recommendations to AACP: [Table: see text] Recommendation 1: AACP should promote the development and use of strategies to ensure intentional and ongoing professional development, such as Individual Development Plans. Recommendation 2: AACP should explore collaborative research leadership development opportunities between faculty at research-intensive institutions and faculty at non-research-intensive institutions. The RGAC also examined programs available at AACP and other national organizations that could help pharmacy faculty develop foundational skills for research leadership (Table 2). The RGAC administered two surveys, one to administrators responsible for research at colleges and schools of pharmacy and one to faculty members at pharmacy schools, to gather information about training needs, programming and support available for research leadership development. Administrators and faculty agreed that research is important for career advancement for faculty, and almost all administrators reported their schools provide funds, release time and mentoring for participation in research career development. However, a lack of faculty awareness regarding programs and available support may be a barrier to participation. The RGAC therefore makes two recommendations and one suggestion related to AACP programming: [Table: see text] Recommendation 3: AACP should expand research leadership development opportunities building from existing programs such as ALFP and AACP Catalyst, with consideration placed on developing programs that promote collaborative research. Recommendation 4: AACP should collaborate with other professional organizations to expand research leadership development opportunities across the academy. Suggestion 1: Colleges and schools of pharmacy should take a proactive role in promoting and facilitating research leadership development for faculty. The RGAC separately examined the research leadership development needs of postdoctoral trainees, recognizing the distinct needs of trainees along the PhD or PhD/PharmD, PharmD/fellowship, and PharmD/residency paths. A review of organizational resources and opportunities for post-doctoral trainees available from national organizations, including AACP, was undertaken (Table 5). The RGAC sees an opportunity for AACP to foster research development of those trainees whose career track will likely be in clinical practice and makes one recommendation and one suggestion related to postdoctoral trainees: Recommendation 5: AACP should support and/or develop programs and activities for pharmacy residents seeking to transition into faculty positions to acquire the skills necessary to develop and lead research programs. Suggestion 2: Colleges and schools of pharmacy should include postdoctoral trainees with academic interests in research leadership development opportunities available to junior faculty. In addition, the RGAC proposed one policy statement that was adopted July 2019 by the AACP House of Delegates: Policy Statement: AACP recognizes the positive role that research leadership development can play in the success of early and mid-career faculty.

The role of CCN4/WISP-1 in the cancerous phenotype.

CCN proteins are secreted into the extracellular environment where they interact with both components of the extracellular matrix and with cell surface receptors to regulate cellular function. Through these interactions, CCNs act as extracellular ligands to activate intracellular signal transduction pathways. CCN4/WISP-1, like other CCNs, plays multiple physiologic roles in development and also participates in pathogenesis. CCN4 is of particular interest with respect to cancer, showing promise as a biomarker or prognostic factor as well as a potential therapeutic target. This review focuses on recent work addressing the role of CCN4 in cancer. While CCN4 has been identified as an oncogene in a number of cancers, where it enhances cell migration and promoting epithelial-mesenchymal transition, there are other cancers where CCN4 appears to play an inhibitory role. The mechanisms underlying these differences in cellular response have not yet been delineated, but are an active area of investigation. The expression and activities of CCN4 splice variants are likewise an emerging area for study. CCN4 acts as an autocrine factor that regulates the cancer cells from which it is secreted. However, CCN4 is also a paracrine factor that is secreted by stromal fibroblasts, and can affect the function of vascular endothelial cells. In summary, current evidence is abundant in regard to establishing potential roles for CCN4 in oncogenesis, but much remains to be learned about the functions of this fascinating protein as both an autocrine and paracrine regulator in the tumor microenvironment.

Free fatty acid receptor (FFAR) agonists inhibit proliferation of human ovarian cancer cells.

Many cellular actions of omega-3 fatty acids are mediated by two G protein-coupled receptors, FFA1 and FFA4, free fatty acid receptor (FFAR) family members that are activated by these dietary constituents. FFAR agonists inhibit proliferation of human prostate and breast cancer cells. Since omega-3 fatty acids can inhibit ovarian cancer cell growth, the current study tested the potential role of FFARs in the response. OVCAR3 and SKOV3 human ovarian cancer cell lines express mRNA for FFA1; FFA4 mRNA was detected at low levels in SKOV3 but not OVCAR3. Lysophosphatidic acid (LPA) and epidermal growth factor (EGF) stimulated proliferation of both cell lines; these responses were inhibited by eicosopentaneoic acid (EPA) and by GW9508, a synthetic FFAR agonist. The LPA antagonist Ki16425 also inhibited LPA- and EGF-induced proliferation; FFAR agonists had no further effect when added with Ki16425. The results suggest that FFARs are potential targets for ovarian cancer therapy.

Free Fatty Acid Receptors and Cancer: From Nutrition to Pharmacology.

The effects of fatty acids on cancer cells have been studied for decades. The roles of dietary long-chain n-3 polyunsaturated fatty acids, and of microbiome-generated short-chain butyric acid, have been of particular interest over the years. However, the roles of free fatty acid receptors (FFARs) in mediating effects of fatty acids in tumor cells have only recently been examined. In reviewing the literature, the data obtained to date indicate that the long-chain FFARs (FFA1 and FFA4) play different roles than the short-chain FFARs (FFA2 and FFA3). Moreover, FFA1 and FFA4 can in some cases mediate opposing actions in the same cell type. Another conclusion is that different types of cancer cells respond differently to FFAR activation. Currently, the best-studied models are prostate, breast, and colon cancer. FFA1 and FFA4 agonists can inhibit proliferation and migration of prostate and breast cancer cells, but enhance growth of colon cancer cells. In contrast, FFA2 activation can in some cases inhibit proliferation of colon cancer cells. Although the available data are sometimes contradictory, there are several examples in which FFAR agonists inhibit proliferation of cancer cells. This is a unique response to GPCR activation that will benefit from a mechanistic explanation as the field progresses. The development of more selective FFAR agonists and antagonists, combined with gene knockout approaches, will be important for unraveling FFAR-mediated inhibitory effects. These inhibitory actions, mediated by druggable GPCRs, hold promise for cancer prevention and/or therapy.

Differential Expression of Ccn4 and Other Genes Between Metastatic and Non-metastatic EL4 Mouse Lymphoma Cells.

BACKGROUND: Previous work characterized variants of the EL4 murine lymphoma cell line. Some are non-metastatic, and others metastatic, in syngenic mice. In addition, metastatic EL4 cells were stably transfected with phospholipase D2 (PLD2), which further enhanced metastasis. MATERIALS AND METHODS: Microarray analyses of mRNA expression was performed for non-metastatic, metastatic, and PLD2-expressing metastatic EL4 cells. RESULTS: Many differences were observed between non-metastatic and metastatic cell lines. One of the most striking new findings was up-regulation of mRNA for the matricellular protein WNT1-inducible signaling pathway protein 1 (CCN4) in metastatic cells; increased protein expression was verified by immunoblotting and immunocytochemistry. Other differentially expressed genes included those for reproductive homeobox 5 (Rhox5; increased in metastatic) and cystatin 7 (Cst7; decreased in metastatic). Differences between PLD2-expressing and parental cell lines were limited but included the signaling proteins Ras guanyl releasing protein 1 (RGS18; increased with PLD2) and suppressor of cytokine signaling 2 (SOCS2; decreased with PLD2). CONCLUSION: The results provide insights into signaling pathways potentially involved in conferring metastatic ability on lymphoma cells.

Positive and Negative Cross-Talk between Lysophosphatidic Acid Receptor 1, Free Fatty Acid Receptor 4, and Epidermal Growth Factor Receptor in Human Prostate Cancer Cells.

Lysophosphatidic acid (LPA) is a lipid mediator that mediates cellular effects via G protein-coupled receptors (GPCRs). Epidermal growth factor (EGF) is a peptide that acts via a receptor tyrosine kinase. LPA and EGF both induce proliferation of prostate cancer cells and can transactivate each other's receptors. The LPA receptor LPA1 is particularly important for LPA response in human prostate cancer cells. Previous work in our laboratory has demonstrated that free fatty acid 4 (FFA4), a GPCR activated by ω-3 fatty acids, inhibits responses to both LPA and EGF in these cells. One potential mechanism for the inhibition involves negative interactions between FFA4 and LPA1, thereby suppressing responses to EGF that require LPA1 In the current study, we examined the role of LPA1 in mediating EGF and FFA4 agonist responses in two human prostate cancer cell lines, DU145 and PC-3. The results show that an LPA1-selective antagonist inhibits proliferation and migration to both LPA and EGF. Knockdown of LPA1 expression, using silencing RNA, blocks responses to LPA and significantly inhibits responses to EGF. The partial response to EGF that is observed after LPA1 knockdown is not inhibited by FFA4 agonists. Finally, the role of arrestin-3, a GPCR-binding protein that mediates many actions of activated GPCRs, was tested. Knockdown of arrestin-3 completely inhibits responses to both LPA and EGF in prostate cancer cells. Taken together, these results suggest that LPA1 plays a critical role in EGF responses and that FFA4 agonists inhibit proliferation by suppressing positive cross-talk between LPA1 and the EGF receptor.

Eicosopentaneoic Acid and Other Free Fatty Acid Receptor Agonists Inhibit Lysophosphatidic Acid- and Epidermal Growth Factor-Induced Proliferation of Human Breast Cancer Cells.

Many key actions of ω-3 (n-3) fatty acids have recently been shown to be mediated by two G protein-coupled receptors (GPCRs) in the free fatty acid receptor (FFAR) family, FFA1 (GPR40) and FFA4 (GPR120). n-3 Fatty acids inhibit proliferation of human breast cancer cells in culture and in animals. In the current study, the roles of FFA1 and FFA4 were investigated. In addition, the role of cross-talk between GPCRs activated by lysophosphatidic acid (LPA), and the tyrosine kinase receptor activated by epidermal growth factor (EGF), was examined. In MCF-7 and MDA-MB-231 human breast cancer cell lines, both LPA and EGF stimulated proliferation, Erk activation, Akt activation, and CCN1 induction. LPA antagonists blocked effects of LPA and EGF on proliferation in MCF-7 and MDA-MB-231, and on cell migration in MCF-7. The n-3 fatty acid eicosopentaneoic acid inhibited LPA- and EGF-induced proliferation in both cell lines. Two synthetic FFAR agonists, GW9508 and TUG-891, likewise inhibited LPA- and EGF-induced proliferation. The data suggest a major role for FFA1, which was expressed by both cell lines. The results indicate that n-3 fatty acids inhibit breast cancer cell proliferation via FFARs, and suggest a mechanism involving negative cross-talk between FFARS, LPA receptors, and EGF receptor.

Omega-3 fatty acids and other FFA4 agonists inhibit growth factor signaling in human prostate cancer cells.

Omega-3 fatty acids (n-3 FAs) are proposed to have many beneficial effects on human health. However, the mechanisms underlying their potential cancer preventative effects are unclear. G protein-coupled receptors (GPCRs) of the free fatty acid receptor (FFAR) family, FFA1/GPR40 and FFA4/GPR120, specifically bind n-3 FAs as agonist ligands. In this study, we examined the effects of n-3 FAs in human prostate cancer cell lines. Initial studies established that the long-chain n-3 FAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid, inhibit proliferation of DU145 cells in response to lysophosphatidic acid (LPA), a mitogenic lipid mediator. When added alone to serum-starved DU145 cells, EPA transiently activates signaling events, including p70S6K phosphorylation. However, when added 15 minutes prior to LPA, EPA suppresses LPA-induced activating phosphorylations of ERK, FAK, and p70S6K, and expression of the matricellular protein CCN1. The rapid onset of the inhibitory action of EPA suggested involvement of a GPCR. Further studies showed that DU145 and PC-3 cells express mRNA and protein for both FFA4 and FFA1. TUG-891 (4-[(4-fluoro-4'-methyl[1,1'-biphenyl]-2-yl)methoxy]-benzenepropanoic acid), a selective agonist for FFA4, exerts inhibitory effects on LPA- and epidermal growth factor-induced proliferation and migration, similar to EPA, in DU145 and PC-3 cells. The effects of TUG-891 and EPA are readily reversible. The FFA1/FFA4 agonist GW9508 (4-[[(3-phenoxyphenyl)methyl]amino]-benzenepropranoic acid) likewise inhibits proliferation at doses that block FFA4. Knockdown of FFA4 expression prevents EPA- and TUG-891-induced inhibition of growth and migration. Together, these results indicate that activation of FFA4 initiates signaling events that can inhibit growth factor-induced signaling, providing a novel mechanism for suppression of cancer cell proliferation.

Effects of lysophosphatidic acid on calpain-mediated proteolysis of focal adhesion kinase in human prostate cancer cells.

BACKGROUND: Calcium-mediated proteolysis plays an important role in cell migration. Lysophosphatidic acid (LPA), a lipid mediator present in serum, enhances migration of carcinoma cells. The effects of LPA on calpain-mediated proteolysis were, therefore, examined in PC-3, a human prostate cancer cell line. METHODS: Cultured PC-3 cells were used in studies utilizing pharmacologic interventions, immunoblotting, and confocal immunolocalization. RESULTS: Focal adhesion kinase (FAK), a tyrosine kinase involved in cell adhesion, is rapidly proteolyzed in serum-starved PC-3 cells exposed to the calcium ionophore, ionomycin; Nck, p130CAS, PKCα, and Ras-GAP are also degraded. Thapsigargin, which causes more moderate increases in intracellular calcium, induces partial proteolysis of these proteins. Calpain inhibitors block the proteolytic responses to ionomycin and thapsigargin. Ionomycin does not induce proteolysis in cells maintained in serum, suggesting a protective role for growth factors contained in serum. LPA causes minor FAK proteolysis when added alone, but protects against ionomycin-induced proteolysis in a time-dependent manner. LPA also protects against the cell detachment that eventually follows ionomycin treatment. The response to LPA is blocked by an LPA receptor antagonist. A similar effect of LPA is observed in ionomycin-treated Rat-1 fibroblasts. In PC-3 cells, the protective effects of LPA and serum are correlated with phosphorylation and redistribution of paxillin, suggesting roles for phosphorylation-mediated protein-protein interactions. CONCLUSIONS: The complex effects of LPA on calpain-mediated proteolysis of FAK and other adhesion proteins are likely to play a role in the ability of LPA to promote attachment, migration, and survival of prostate cancer cells.

Control of neurite outgrowth by RhoA inactivation.

cAMP induces neurite outgrowth in the rat pheochromocytoma cell line 12 (PC12). In particular, di-butyric cAMP (db-cAMP) induces a greater number of primary processes with shorter length than the number induced by nerve growth factor (NGF). db-cAMP up- and down-regulates GTP-RhoA levels in PC12 cells in a time-dependent manner. Tat-C3 toxin stimulates neurite outgrowth, whereas lysophosphatidic acid (LPA) and constitutively active (CA)-RhoA reduce neurite outgrowth, suggesting that RhoA inactivation is essential for the neurite outgrowth from PC12 cells stimulated by cAMP. In this study, the mechanism by which RhoA is inactivated in response to cAMP was examined. db-cAMP induces phosphorylation of RhoA and augments the binding of RhoA with Rho guanine nucleotide dissociation inhibitor (GDI). Moreover, RhoA (S188D) mimicking phosphorylated RhoA induces greater neurite outgrowth than RhoA (S188A) mimicking dephosphorylated form does. Additionally, db-cAMP increases GTP-Rap1 levels, and dominant negative (DN)-Rap1 and DN-Rap-dependent RhoGAP (ARAP3) block neurite outgrowth induced by db-cAMP. DN-p190RhoGAP and the Src inhibitor PP2 suppress neurite outgrowth, whereas transfection of c-Src and p190RhoGAP cDNAs synergistically stimulate neurite outgrowth. Taken together, RhoA is inactivated by phosphorylation of itself, by p190RhoGAP which is activated by Src, and by ARAP3 which is activated by Rap1 during neurite outgrowth from PC12 cells in response to db-cAMP.

Differential expression of FAK and Pyk2 in metastatic and non-metastatic EL4 lymphoma cell lines.

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to phorbol 12-myristate 13-acetate (PMA). In sensitive cells, PMA causes Erk MAPK activation and Erk-mediated growth arrest. In resistant cells, PMA induces a low level of Erk activation, without growth arrest. A relatively unexplored aspect of the phenotypes is that resistant cells are more adherent to culture substrate than are sensitive cells. In this study, the roles of the protein tyrosine kinases FAK and Pyk2 in EL4 phenotype were examined, with a particular emphasis on the role of these proteins in metastasis. FAK is expressed only in PMA-resistant (or intermediate phenotype) EL4 cells, correlating with enhanced cell-substrate adherence, while Pyk2 is more highly expressed in non-adherent PMA-sensitive cells. PMA treatment causes modulation of mRNA for FAK (up-regulation) and Pyk2 (down-regulation) in PMA-sensitive but not PMA-resistant EL4 cells. The increase in Pyk2 mRNA is correlated with an increase in Pyk2 protein expression. The roles of FAK in cell phenotype were further explored using transfection and knockdown experiments. The results showed that FAK does not play a major role in modulating PMA-induced Erk activation in EL4 cells. However, the knockdown studies demonstrated that FAK expression is required for proliferation and migration of PMA-resistant cells. In an experimental metastasis model using syngeneic mice, only FAK-expressing (PMA-resistant) EL4 cells form liver tumors. Taken together, these studies suggest that FAK expression promotes metastasis of EL4 lymphoma cells.

Phospholipase D2 Enhances Epidermal Growth Factor-Induced Akt Activation in EL4 Lymphoma Cells.

Phospholipase D2 (PLD2) generates phosphatidic acid through hydrolysis of phosphatidylcholine. PLD2 has been shown to play a role in enhancing tumorigenesis. The epidermal growth factor receptor (EGFR) can both activate and interact with PLD2. Murine lymphoma EL4 cells lacking endogenous PLD2 present a unique model to elucidate the role of PLD2 in signal transduction. In the current study, we investigated effects of PLD2 on EGF response. Western blotting and RT-PCR were used to establish that both parental cells and PLD2 transfectants express endogenous EGFR. Levels of EGFR protein are increased in cells expressing active PLD2, as compared to parental cells or cells expressing inactive PLD2. EGF stimulates proliferation of EL4 cells transfected with active PLD2, but not parental cells or cells transfected with inactive PLD2. EGF-mediated proliferation in cells expressing active PLD2 is dependent on the activities of both the EGFR and the PI3K/Akt pathway, as demonstrated by studies using protein kinase inhibitors. EGF-induced invasion through a synthetic extracellular matrix is enhanced in cells expressing active PLD2, as compared to parental cells or cells expressing inactive PLD2. Taken together, the data suggest that PLD2 acts in concert with EGFR to enhance mitogenesis and invasion in lymphoma cells.