Lysophosphatidic acid (LPA)-induced vasodilator-stimulated phosphoprotein mediates lamellipodia formation to initiate motility in PC-3 prostate cancer cells

doi:10.1016/j.molonc.2008.03.009

Journal List > NIHPA Author Manuscripts

Mol Oncol. Author manuscript; available in PMC 2008 December 9.

Published in final edited form as:

Mol Oncol. 2008 June; 2(1): 54–69.

doi: 10.1016/j.molonc.2008.03.009.

PMCID: PMC2597858

NIHMSID: NIHMS53664

Lysophosphatidic acid (LPA)-induced vasodilator-stimulated phosphoprotein mediates lamellipodia formation to initiate motility in PC-3 prostate cancer cells

Yutaka Hasegawa,^a^b Mandi Murph,^a^b Shuangxing Yu,^a Gabor Tigyi,^c and Gordon B. Mills^a^*

^aDepartment of Systems Biology, The University of Texas M. D. Anderson Cancer Center, 7435 Fannin Street, Houston, TX 77054

^cDepartment of Physiology, The University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN 38163

^bThese authors contributed equally to this manuscript.

^*Address correspondence to: Gordon Mills, M.D., Ph.D., Department of Systems Biology, The University of Texas M. D. Anderson Cancer Center, 7435 Fannin Street, Houston, TX 77054, Tel. 713-563-4212; Fax. 713-563-4235; E-Mail: gmills/at/mdanderson.org

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Abstract

Prostate cancer remains the most frequently diagnosed malignancy and the second leading cause of cancer mortality among men in the United States. Hormone refractory, metastatic disease has no molecular therapeutics to date and survival is poor. Lysophosphatidic acid (LPA) is a bioactive lipid exhibiting motility, invasive, growth, proliferative and survival effects in multiple cancer cell lineages. Cells express different combinations of LPA-specific G protein-coupled receptors, LPA₁, LPA₂ LPA₃, and LPA₄ as well as other LPA receptors, which bind LPA and thereby regulate lipid signaling. The role of specific LPA receptors in functional outcomes of lysolipid signaling remains to be fully elucidated in prostate cancer. We hypothesized that LPA can initiate cell migration through specific LPA receptors by activating actin-associating proteins involved in motility, including the vasodilator-stimulated phosphoprotein (VASP). In the present study, we demonstrate that LPA-induced lamellipodia formation in cells is dependent on LPA receptor-mediated phosphorylation of VASP, demonstrating a previously unknown regulation by LPA. LPA induces phosphorylation of VASP at Ser(157), through protein kinase A (PKA) since the stimulation was abrogated by PKA inhibition. In addition, we found the effects of LPA-induced lamellipodia formation and migration were reduced by knockdown of either VASP or LPA receptor expression, suggesting that LPA receptor-induced VASP phosphorylation is a critical mediator of migration initiation. Thus the LPA₂ and LPA₃ receptors, in addition to the previously implicated LPA₁ receptor, play a role in cellular motility potentially contributing to invasion and metastases. Emerging drugs targeting the LPA pathway may be beneficial for the treatment of metastatic progression in prostate cancer.

INTRODUCTION

In 2007 approximately 218,890 men in the U.S. were diagnosed with prostate cancer, ranking it as the most commonly diagnosed malignancy among men (Jemal et al.). Treatment options after diagnosis include radical prostatectomy, radiation therapy, chemotherapy or hormone therapy and each year 27,050 men will die from disease (Jemal et al.). The routine screening blood test for elevation of prostate-specific antigen (PSA) is controversial and concerns surround its ability to accurately predict disease. Unfortunately once prostate cancers metastasize some become incurable even with the available treatments, thus more drugs are needed to slow and prevent metastasis.

The bioactive lipid lysophosphatidic acid (LPA; 1- or 2-acyl-sn-glycerol 3-phosphate) mediates growth factor-like actions in cells, including proliferation, survival, motility, invasion, and interleukin production (Chettibi et al.; Eder et al.; Erickson et al.; Fang et al.; Fang et al.; Levine et al.; Mills et al.). LPA is present at low levels in normal human plasma due to feedback inhibition of autotaxin, the enzyme which produces LPA (Stracke et al. 1992; Umezu-Goto et al. 2002). LPA is present at markedly elevated levels in the ascites of patients with ovarian and other gynecologic cancers (Fang et al.). In addition, LPA receptors and the enzymes that metabolize LPA are aberrant in multiple different cancer lineages including prostate cancer implicating LPA in the pathophysiology of cancer (Mills et al.; Murph et al.).

LPA has been reported to stimulate the migration of a number of different cancer cells, including ovarian (Lu et al.; Sawada et al.), colon (Shida et al.), glioma (Manning et al.), neuroblastoma (Van Leeuwen et al.), hepatoma (Iwasaki et al.) and prostate (Park et al.) and to contribute to invasiveness and metastases in vivo (Kim et al.). This shift from an epithelial to a mesenchymal phenotype can facilitate the transition of cancer from a primary tumor to a metastatic and invasive disease type. Such a negative functional outcome of LPA action could be mediated through the binding and activation of the G protein-coupled receptors LPA_1–4 (An et al.; An et al.; Bandoh et al.; Noguchi et al.) along with the internal nuclear transcription factor receptor peroxisome proliferator-activated receptor γ, which regulates metabolic functions (McIntyre et al. 2003) or newly-identified LPA receptors GPR92/LPA₅ (Kotarsky et al.; Lee et al.), GPR87/LPA₆ (Tabata et al.) and P2Y5/LPA₇ (Pasternack et al.).

Molecular functions required for metastasis development from the primary tumor site include angiogenesis, invasion and cell migration. The control of cell migration is a complex process resulting in the forward extension and rear retraction of a cell to propel it in a specific direction occurring in response to environmental cues. Visible activity along the cell membrane such as membrane ruffling, protruding cytoskeletal extensions, actin reorganization, cell rounding and cell spreading can indicate motility. Both lamellipodia and filopodia formation at the leading edge of cells are also an indication of forward motion (Gov and Gopinathan; Ridley and Hall). Characteristically, lamellipodia look like flat, curved spreading membrane structures along the cell periphery, and filopodia, which can occur along the lamellipodia, are long, needle-like extensions of actin filaments.

Actin dynamics are regulated by the vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family, which facilitates directional cell motility (Rottner et al.). Because actin is the driving force behind cell migration, VASP is a crucial component to this process. VASP was originally identified as a common substrate for protein kinase A (PKA) and protein kinase G, but is also a substrate for protein kinase C (Chitaley et al.). Through its association with actin-filament barbed ends, VASP is able to enhance actin polymerization and extend spreading membranes, regulating cell migration (Barzik et al. 2005). The translocation of VASP to lamellipodia’s leading edge is critical to the rate of cell motility (Rottner et al.), and VASP phosphorylation indicates cell detachment from the extracellular matrix required for migration (Howe et al.).

In this study, we sought to understand how LPA affects malignant cell transformation by assessing migration and evaluating proteins that link receptor signaling to membrane spreading. Cell migration is an important process to study to identify factors that allow LPA to induce metastasis of the primary tumor. We hypothesized that LPA can initiate filoipodia formation through LPA receptors by activating VASP. We used the highly metastatic prostate cancer cell line PC-3 as well as manipulation of LPA receptors and VASP to analyze VASP activation after LPA treatment. We found that LPA stimulates LPA receptor- and PKA-dependent phosphorylation of VASP at Ser(157) and thereby induces filoipodia formation. This study reveals a formerly unappreciated function of the LPA: induction of VASP-dependent motility initiation in cells. Emerging compounds that inhibit cancer cell invasion and metastasic pathways driving LPA-induced tumor progression may have potency in the prevention of metastatases in prostate cancer (Baker et al.).

RESULTS

LPA stimulates migration of PC-3 cells

To evaluate the role of specific LPA receptors in the migration of androgen-independent PC-3 prostate cancer cells, we investigated the effects of LPA receptor-selective analogs on this process. We assessed 18:1 LPA, a pan-LPA receptor agonist and 14:0 LPA, which activates the LPA_1/2 receptors but not the LPA₃ receptor. Consistent with findings from previous studies (Panetti and Mosher), our results showed that 18:1 and 14:0 LPA stimulated the random migration of PC-3 cells (p < 0.001 vs. control for both agonists) (Fig. 1-A

). The 18:1 LPA species also induced directional migration in Boyden chamber assays; the most effective result occurred when 18:1 LPA was added to the lower chamber and acted as a chemoattractant for the cells in the upper chamber (p < 0.01 vs. control) (Fig. 1-B

Fig. 1

LPA induces directional cell migration and activation of VASP. Cells were serum starved overnight, and 2 × 10⁵ cells were put into the upper side of a Boyden chamber. Cells that migrated through an 8.0-µm-pore filter were fixed and stained (more ...)

To rule out the possibility that the Boyden chamber assay results measured proliferation in addition to motility, we performed several migration experiments in the presence of a pharmacological inhibitor of mitosis, olomoucine. We used LPA 14:0 to stimulate random migration and added olomoucine to the lower chamber to prevent cell division. Our results indicate that we were unable to measure a difference between treatment groups (Fig. 1-C

). Similarly, we also examined LPA-mediated migration in the presence of olomoucine and did not detect a difference (Fig. 1-D

). Further evidence suggesting that LPA stimulates the migration of PC-3 cells was achieved by measuring in vitro gap filling of confluent monolayers on either side in the presence or absence of LPA (p < 0.001 vs. control) (Fig. 1-E

). Taken together, these data suggest that LPA stimulates both random and directed migration in PC-3 cells and that this process is not merely attributable to cell division.

We assessed 18:1 LPA-induced processes in another prostate carcinoma line, DU145, to determine whether the results achieved in PC-3 cells could be generalizable to other prostate cells. Indeed, LPA-induced proliferation and migration were nearly identical between PC-3 and DU145 (p < 0.05 or p < 0.01 vs. control for each condition) (Fig. 1-F

). These cells differed, however, between the extent of LPA-induced invasion, although both were able to mediate this process.

Actin-associated proteins and actin dynamics are required for cellular motion. We investigated the actin-associated VASP and found that VASP became phosphorylated at Ser(157) after 5 min of 18:1 LPA stimulation in PC-3 cells and that this phosphorylation was maintained through 180 min (Fig. 1-G

). This phosphorylation was seen both in the mobility shift in apparent molecular weight from approximately 46 kDa to 50 kDa in the total VASP analysis and with the use of an antibody directed against activated pVASP Ser(157). Phosphorylation at this VASP residue indicates cellular spreading and detachment from the substratum (Howe et al.). We also evaluated the phosphorylation of VASP Ser(239) after 1h incubation with 18:1 LPA and found levels also increased in response to LPA (Fig. 1-H

). Cholera toxin (CTX) was used as a positive control to stimulate the phosphorylation of VASP Ser(239) and visually compare the substantial response to the moderate response generated by LPA. In contrast to the pVASP Ser(157) staining, both bands on the gel represent pVASP Ser(239). Because the individual functions of VASP phosphorylation sites are not well understood or characterized, and may only differ in their activation kinetics, we focused on the Ser(157) site for our studies based on its role in regulation of motility.

We next evaluated the dose response in PC-3 cells of LPA-mediated VASP activation with the use of the three LPA agonists, including OMPT, which we previously demonstrated to be a non-hydrolyzable LPA analog selective for the LPA₃ receptor (Hasegawa et al. 2003). All three induced a shift in the molecular weight and phosphorylation of VASP Ser(157) (Fig. 2

). The most robust shifts were mediated by 1 µM 14:0 LPA and 10 µM 18:1 LPA. OMPT at a concentration of 0.1 µM preferably binds only LPA₃ (Erickson et al.) and induced modest VASP phosphorylation (Fig. 2

). There were no notable differences in the phosphorylation of the mitogen-activated protein kinase protein ERK1/2 by the different LPA receptor agonists. Using quantitative RT-PCR, we also measured the levels of LPA receptor expression in PC-3 cells and confirmed the expression of high levels of LPA₁, moderate levels of LPA₃, low levels of LPA₂, and extremely low or undetectable levels of the LPA-binding receptor GPR23 (data not shown). In addition, PC-3 cells have no detectable levels of LPA₅ (Valentine et al. 2007). The fact that OMPT had modest effects on the phosphorylation of VASP at Ser(157) suggests that LPA_1/2 could more efficiently mediate the phosphorylation of VASP than LPA₃.

Fig. 2

LPA analogues induce the phosphorylation of VASP. Effects of LPA agonists were assessed on the protein expression levels of total VASP, pVASP Ser(157), phosphorylated ERK1/2, and total ERK. PC-3 cells were starved in serum-free medium for 24 h and stimulated (more ...)

LPA stimulates the translocation of VASP to the leading edge of the spreading membrane

To better understand the function of VASP in this system, we visualized LPA-activated VASP in PC-3 cells. Because VASP has a central role in actin-associated functions, we also determined the localization of both VASP and actin after LPA stimulation. In quiescent, serum-deprived cells, total VASP was diffusely distributed throughout the cytoplasm, whereas pVASP was not readily detected (Figure 3-A

, top row). After treatment with 18:1 or 14:0 LPA, the appearance of VASP increased dramatically, pVASP was translocated to the edge of the spreading plasma membrane, and the cells became rounder and more symmetrical (Fig. 3-A

, middle and bottom rows). Similar results were obtained using OMPT (data not shown).

Fig. 3

LPA stimulates the translocation of VASP to the leading edge of the spreading membrane. (A) PC-3 cells were plated on glass coverslips, starved in serum-free medium for 24 h, stimulated with nothing, 18:1 LPA (10 µM), or 14:0 LPA (1 µM) (more ...)

We also assessed whether VASP localized to the tip of actin filaments in LPA-stimulated PC-3 cells. Confocal microscopy detected pVASP Ser(157) in the tip of actin filaments on the leading edge of the spreading membrane (Fig. 3-B

). After cell starvation in serum-free medium, minimal pVASP was detected on actin filaments at the edge of the spreading membrane, and the cells were asymmetrical and polarized. However, after treatment with 18:1 LPA, the cells became rounder and developed spreading edges, where pVASP was clearly colocalized with actin filaments (Fig. 3-C

, arrows indicating areas of colocalization in lower right quadrant of figures). Microscopic analysis revealed that both translocated VASP and pVASP were localized on longer, parallel actin filaments in the leading edge in the spreading membrane. These observations indicated that LPA activates lamellipodia formation, which would cause an extension of parallel actin filaments to push the membrane forward.

VASP is important for efficient LPA-induced migration

To determine whether VASP was required for LPA-induced migration, we knocked down VASP expression by transfecting VASP siRNA into PC-3 cells. VASP expression was knocked down 48 h after the transfection (Fig. 4-A

). The expression levels of total AKT and phosphorylated AKT Ser(473), which reflect cell viability, were unchanged (Fig. 4-A

). Knockdown of VASP expression abrogated VASP phosphorylation at Ser(157) after LPA stimulation (Fig. 4-B

). However, the activation of Rac1/Cdc42 Ser(71) was only modestly decreased by transfection with siRNA against VASP. On the basis of these results, we assessed the migration of VASP-knockdown PC-3 cells and found that the LPA-induced migration of PC-3 was strongly inhibited by reduction of VASP protein expression (p < 0.05) (Fig 4-C

). Our findings indicated that VASP protein is critical to the LPA-induced migration in PC-3 cells.

Fig. 4

VASP is required for LPA-induced migration in PC-3 cells. VASP protein expression was knocked down in PC-3 cells by siRNA transfection with Lipofectamine 2000 in serum-free medium for 48 h. (A) Cells were lysed and assessed for total VASP protein by Western (more ...)

The LPA receptors play a critical role in lamellipodia formation

To assess whether one LPA receptor was the major mediator of VASP phosphorylation induced by LPA, we began by generating stably expressing V5-LPA₁, LPA₂, and LPA₃ receptor SKOV-3 human ovarian cancer cell lines using Lentiviral expression (Fig. 5-A

). These cells were chosen to confirm the extent and involvement in LPA receptor-mediated VASP activation across different types in a cell line that is highly responsive to LPA stimulation, expresses all LPA receptors and has high expression of the LPA₂ receptor (LPA₂ > LPA₁ [dbl greater-than sign]

LPA₃

LPA₄), the latter in contrast to PC-3 cells. After serum-starvation, the stable SKOV-3 cells were treated with 10 µM 18:1 LPA and discrete structures representing pVASP appeared along the outer edges of the spreading membrane (Fig. 5-B

). In all LPA receptor-stable cells pVASP was apparent, although SKOV-3 cells overexpressing the LPA₂ receptor were rounder and had more lamellipodia and more intense and frequent staining of pVASP along the spreading edges. We quantified these differences to illuminate our findings in respect to the LPA₂ receptor (Fig 5-C

). To confirm LPA was mediating motility in SKOV-3 cells, we examined both LPA (18:1, 10 µM) stimulated migration and invasion using the Boyden chamber assay. Indeed, LPA was able to induce a statistically significant increase in these processes (Fig. 5-D

). We also determined that LPA (18:1, 10 µM) stimulates two-dimensional migration in SKOV-3 cells (Fig. 5-E

). These data together suggest that LPA mediates motility and VASP activation in SKOV-3 cells.

Fig. 5

LPA-stimulated LPA receptor activation generates VASP activation and lamellipodia. (A) SKOV-3 cells were induced to stably-express individual LPA receptors using Lentiviral vectors. Whole-cell lysates from cells grown in culture were harvested and processed (more ...)

To confirm these findings in PC-3 cells, we assessed the effect of the LPA receptors on VASP phosphorylation by transfecting the PC-3 cells with siRNA for the LPA receptors. Without the existence of adequate antibodies capable of recognizing individual LPA receptors by Western blotting, we measured mRNA to demonstrate that the siRNA was reducing receptor expression (Fig. 5-F

). We next assessed which receptor was affecting the phosphorylation of VASP Ser(157) through siRNA transfection and treatment with 18:1 LPA (10 µM). Knockdown of all three LPA receptors reduced the level of total VASP protein and the mobility shift to 50 kDa (Fig. 5-G

). Likewise, all three LPA receptors had some effect on the phosphorylation of VASP Ser(157), although LPA₂ had the greatest inhibition on the size shift of VASP (Fig.5-G

, top band) but all three receptors were equivalent in the extent of VASP Ser(157) phosphorylation changes (Fig. 5-H

). Similar results were seen with the phosphorylation of VASP Ser(239) such that siRNA against each receptor reduced activation with LPA₂ and LPA₃ showing the strongest effects (data not shown). The observations could not be attributed to a dramatic effect on cell viability ensuing from transfection conditions or the consequence of knocking down individual LPA receptors (Fig. 5-I

Because our results suggested that all three LPA receptors are capable of mediating LPA-induced VASP phosphorylation and the LPA₂ and LPA₃ receptors have unappreciated roles in motility, we determined what role each receptor plays in the motility of PC-3 cells using 18:1 LPA as a chemoattractant in the lower wells of a Boyden chamber. Knockdown of each of the three receptors significantly reduced the ability of PC-3 cells to migrate towards LPA (Fig. 5-J

). Although the average number of migratory PC-3 cells in the LPA₂ receptor knockdowns is slightly above the LPA₁ and LPA₃ receptor knockdowns, this result is remarkable considering the distribution of LPA receptors in these cells (LPA₁ [dbl greater-than sign]

LPA₃ > LPA₂) (Kishi et al.) and the fact that LPA₂ is present in significantly lower amounts yet contributes a similar affect. These results suggested that all three LPA receptors have a role in the LPA-mediated signaling activation of VASP and subsequent lamellipodia formation along the spreading membrane in multiple cancer cell lines. It is possible that the LPA₁ and LPA₃ receptors contribute to motility through cross talk with LPA₂ (Zaslavsky et al.) or alternatively contribute to motility through non-VASP mediated mechanisms such as activation of Cdc42 or Rac1.

PKA is required for LPA-induced shape change, VASP activation, and migration in PC-3 cells

To identify the kinase pathway responsible for linking LPA signaling to VASP, we treated PC-3 cells with LPA for 3 h and then measured PKA activity. LPA induced a rapid and sustained activation of PKA in intact PC-3 cells (Fig. 6-A

). PKA activity was maximal by 5 min, decreased slightly at 10–15 min, and then returned to maximal for at least 3 h. Our PKA results corresponded with the kinetics of VASP activation by LPA (Fig. 1-C

), so we assessed whether PKA would also activate VASP after LPA stimulation. We pretreated PC-3 cells with the PKA inhibitor H-89, stimulated them with LPA, and assessed total VASP expression. In the presence of LPA, increasing concentrations of H-89 caused a linear reduction in pVASP as indicated by the upper band that appears in the total protein blot, which represents the mobility shift of phosphorylated VASP (Fig. 6-B

). This marked reduction was analogous to the results we achieved when we assessed phosphorylated Rac1/Cdc42 Ser(71) levels in the cells and treated them with increasing amounts of H-89. Two additional PKA inhibitors, KT5720 and Rp-CAMPs, also decreased pVASP Ser(157) and Ser(239) in the presence of LPA (data not shown) consistent with a role for PKA in LPA induced phosphorylation of VASP.

Fig. 6

PKA is activated by LPA and is required for PC-3 cell migration. (A) Cells were starved in serum-free medium for 24 h exposed to LPA, and cell lysates were collected. PKA activity was determined by phosphorylation reaction using 100 µM biotinylated (more ...)

We then determined whether inhibiting PKA would affect the functional outcome in LPA-stimulated PC-3 cells. We measured cell migration in the presence and absence of H-89 and LPA and found that inhibiting PKA did not affect basal cell migration; however, H-89 pretreatment of PC-3 cells abrogated LPA-stimulated cell migration (p < 0.001) (Fig. 6-C

). This suggests that PKA is a major mediator of LPA-induced motility in PC-3 cells.

To corroborate these results, we treated the cells with CTX, a potent activator of the cAMP/PKA pathway. We examined the extent of pVASP Ser(157) in lamellipodia structures after treatment with either 18:1 LPA (10 µM) or CTX (2U/ml) for 1 h. LPA induced VASP activation in lamellipodia along the spreading membrane, although CTX produced a more robust effect (Fig. 6-D

) that was nearly three times that of LPA alone (Fig. 6-E

) and consistent with pVASP Ser(239) activation in Fig. 1-D

. Taken together, these data suggest that PKA is a dominant pathway for LPA-induced cell migration in PC-3 cells.

To assess whether the PKA pathway was also responsible for LPA-induced lamellipodia formation and morphologic changes in the dynamic membrane in multiple cell types, we monitored shape change in the presence of PKA inhibitors and LPA. Different inhibitors, other than H-89, were used in this experiment to confirm previous results. After starvation in serum-free medium, untreated PC-3 cells were elliptical, asymmetrical and polarized with no pVASP Ser(157) staining along the outer membrane (Fig. 7-A

, Untreated). Approximately 1 h after exposure to 18:1 LPA (10 µM), the cells became rounded, lost their polarity, and had aggressively activated, spreading membranes marked by dramatic staining of lamellipodia along the outer membrane (Fig. 7-A

, LPA 10 µM). Robust lamellipodia was not apparent in cells treated with KT5720 (2 µM) nor Rp-CAMPS (100 µM). Treatment with these PKA inhibitors caused a decrease in pVASP Ser(157) staining intensity in PC-3 cells (Fig. 7-B

). Similar results were obtained in HT-29 cells treated with 18:1 LPA (10 µM) and Rp-CAMPS (100 µM) (Fig. 9-C and D). These data suggest that the PKA pathway is involved in LPA-induced lamellipodia formation in PC-3 cells.

Fig. 7

Inhibition of PKA blocks LPA-induced morphologic changes and lamellipodia formation in multiple cell types. (A) PC-3 cells or (C) HT-29 cells were plated on glass coverslips, starved with serum-free medium for 24 h and exposed to 18:1 LPA (10 µM) (more ...)

To assess whether inhibiting LPA availability in culture medium affects directed cell motility we utilized a compound, 2-ccPA 16:1, that is under development as an autotaxin inhibitor (Baker et al., 2006); autotaxin is the enzyme which hydrolyzes a precursor molecule to produce LPA. We also used LPA as a chemoattractant for PC-3 cells in a Boyden chamber assay for directional motility. Cells that were exposed to 2-ccPA 16:1 (10 µM) in the upper chamber were significantly reduced in their capacity to migrate towards LPA as compared to cells without this compound in the upper chamber (untreated) (Fig. 8

). Equal numbers of cells were plated in 1% serum in the upper chambers. This suggests an involvement for LPA production and/or LPA-mediated receptor activation in response to directional motility in PC-3 cells.

Fig. 8

The cyclic phosphatidic acid analogue, 2-ccPA 16:1, inhibits LPA-directed motility of PC-3 cells. Approximately 6.5×10⁵ PC-3 cells were plated in each 10 cm dish and grown in 1% serum overnight with either no additive (untreated groups) or 10 (more ...)

DISCUSSION

Our findings suggest that LPA stimulation of all three Edg-family LPA receptors can activate VASP through the PKA pathway to initiate directional cell migration and the formation lamellipodia along the outer membrane. Several lines of evidence support this conclusion. First, LPA increased the phosphorylation of VASP in a time- and dose-dependent manner. Second, in multiple cell lines that express varying amounts of different LPA receptors, LPA initiated the appearance of lamellipodia and pVASP Ser(157) along the cell periphery. Third, siRNA of the Edg-family LPA receptors reduced the ability of LPA to stimulate motility and VASP phosphorylation. Fourth, PKA inhibitors abrogated these processes in PC-3 cells.

Some of the data from our study are consistent with previous studies that have shown serum can induce the phosphorylation of VASP in vascular smooth muscle cells (Chitaley et al.) and LPA is abundant in serum. In addition, PKA activation was previously implicated in LPA-induced migration and LPA-induced migration of MDA-MB-435 cells occurs through a cAMP-dependent PKA pathway (O'Connor and Mercurio). Further, LPA-induced neurite retraction in PC-12 cells is insensitive to pertussis toxin and requires cAMP-dependent PKA activation (Tigyi et al.). Given our results, it is likely that the phosphorylation of VASP by LPA through PKA begins at the plasma membrane whereby the LPA_1/2 receptors couple to G_α13 to transduce the signal to the VASP pathway. This pathway, beginning with G_α13 and leading to VASP activation, was recently demonstrated in other systems (Bian et al.; Profirovic et al. 2005).

The purpose of having multiple LPA receptors within the same cell type is unknown. From the results in our studies, we hypothesize that there is a significant amount of redundancy in the system to stimulate a critical ligand-mediated response, like motility. Alternatively, there may be different tasks assigned to LPA₁, LPA₂, and LPA₃ receptors to achieve other functional outcomes, like growth or survival. However, these different roles could be contextual dependent on the actions of other factors or required for the kinetic organization of complex functions such as migration and invasion. In light our results, we speculate that LPA stimulates organizational activity through the LPA receptors and that converges TRIP6 (Xu et al.), VASP, zyxin, vinculin, focal adhesion kinase, and other required proteins at focal adhesions to assemble the components necessary to initiate cell migration. Studies by Chen et al. (Chen et al. 2007) were in agreement with this hypothesis and suggested that both the LPA₁ and LPA₂ receptors contribute to chemotaxis in BT-20 breast cancer cells. Other studies by Chan et al (Chan et al. 2007) emphasized a role for the LPA₃ receptor in the migration of bone marrow-derived mouse dendritic cells and showed a 50% inhibition of migration with cells from mice lacking this receptor.

Some of our data differ from those in previous studies with respect to the involvement of LPA receptor participation in cell migration. It is important to note, however, that while the LPA₂ receptor appears to be sufficient for lamellipodia formation and migration, it appears that other LPA receptors can mediate this function. For example, Hama et al. (Hama et al. 2004) emphasized a key role for the LPA₁ receptor in motility and lamellipodia formation in newborn knockout mouse skin fibroblast cells. Hao et al. (Hao et al. 2007) also emphasized the LPA₁ receptor as the dominant mediator of LPA-induced PC-3 cell motility through ERK and p38 MAPK. In contrast, our study results also implicate LPA₂ and LPA₃ in LPA-induced motility, at least in the formation of lamellipodia in PC-3 cancer cells. These differences might represent lineage-specific characteristics, or LPA-induced cellular migration might be context specific, as several different LPA receptors have the potential to mediate LPA-induced cellular migration. Importantly HT-29 cells, which express only the LPA₂ receptor, do not migrate in response to LPA suggesting that while the LPA₂ receptor can induce lamellipodia formation and activate VASP, subsequent coordination of the process by the LPA₁ receptor is required for efficient migration. Thus coordinate activation of multiple LPA receptors functions to mediate efficient cellular outcomes.

The therapeutic benefit that may be extenuated from our conclusions is that targeting LPA, an initiator of cell migration, could reduce the incidence of metastasis in prostate cancer. In support of this idea studies using such targeted compounds, analogues of cyclic phosphatidic acid (cPA), to inhibit the production and action of LPA demonstrated therapeutic efficacy by inhibiting invasion and metastasis in vivo (Baker et al.; Mukai et al.; Murakami-Murofushi et al.). In vitro, cPA analogues also inhibit the LPA-induced invasion or migration of rat hepatoma (Uchiyama et al.), mouse melanoma, human pancreatic, human lung and human fibrosarcoma cells (Mukai et al.). Likewise, our in vitro studies show the ability of 2-ccPA 16:1 to significantly reduce PC-3 cell motility. Emerging cPA analogues under investigation inhibit autotaxin, the enzyme that hydrolyzes an LPA precursor molecule to produce LPA, along with the LPA receptors, generating a “one-two punch” against LPA activation (Jiang et al.). Theoretically, cPA analogues could be used as part of a combinatorial anti-cancer drug regimen to prevent metastasis or control further secondary spread of disease in prostate cancer patients.

In conclusion, we found that LPA and VASP together may contribute to the etiology of androgen-independent prostate cancer and enhance tumor metastasis by increasing lamellipodia formation initiating the processes required for motility and invasion. Our data bridge these different observations and fill a gap in knowledge by revealing a functional outcome mediated by LPA receptors other than LPA₁. These data may also explain the observed up-regulation of the LPA₂ receptor in cancer cells (Fang et al.; Fujita et al.). Dertsiz et al. showed a significant increase in VASP expression in lung cancer cells compared with normal lung cells (Dertsiz et al. 2005) suggesting that these could be linked processes. Thus, simultaneous targeting of VASP and LPA receptor signaling could decrease tumor migration and the subsequent metastasis of prostate cancer cells.

EXPERIMENTAL PROCEDURES

Cell culture and transfection

PC-3 human prostate cancer cells were maintained in RPMI 1640 medium (Invitrogen/Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum and transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. PC-3 cells stably expressing FLAG-tagged LPA₂ were created by transiently transfecting 1 µg of pCDNA3-FLAG- LPA₂ or pCDNA3 expression plasmid in a 6-well culture dish. Cells were then selected by growth in the presence of the antibiotic G418 (50 mg/ml). LPA receptor expression was confirmed by indirect immunofluorescence microscopy using an affinity-purified rabbit anti-FLAG antibody (Sigma, St. Louis, MO). DU145 human prostate cancer cells were maintained in DMEM (CCSG Media Preparation Facility, The University of Texas M. D. Anderson Cancer Center, Houston, TX) supplemented with 10% fetal bovine serum. SKOV-3 human ovarian cancer cells derived from ascites fluid were maintained in RPMI 1640 medium (CCSG Media Preparation Facility) supplemented with 10% fetal bovine serum and blasticidin S HCl (10 µg/ml; Invitrogen) to maintain stable transfection. SKOV-3 cells stably expressing the V5 epitope were created by transiently transfecting V5-expressing LPA receptors into dishes of SKOV-3 cells and selecting for growth in the presence of the antibiotic blasticidin. Receptor expression was confirmed by Western blot analysis for the V5 epitope by using mouse monoclonal immunoglobulin G anti-V5 antibody (Invitrogen).

Materials

LPA (14:0 and 18:1 species) was purchased from Avanti Polar Lipids (Alabaster, AL) and reconstituted in chloroform. For incubation with cells, LPA was added in 0.1% charcoal-stripped BSA. The LPA₃-selective agonist, 1-oleoyl-2-O-methyl-rac-glycerophosphothionate (Mills et al.), was synthesized at Oxford Asymmetry (Oxford, UK) under contract with LXR Biotechnology (Richmond, CA). The purity of OMPT was analyzed by electrospray ionization mass spectrometry as previously described (Hasegawa et al.). The PKA inhibitors H-89 and Adenosine 3′,5′-cyclic Phosphorothioate-Rp (Rp-cAMPS), and cholera toxin were purchased from Calbiochem (San Diego, CA), KT5720 was purchased from VWR (West Chester, PA), antibody against GAPDH was purchased from Ambion (Austin, TX), and antibodies for detecting total and activated AKT, ERK and VASP were purchased from Cell Signaling Technology (Beverly, MA).

Boyden chamber-based cell migration and invasion assay

Serum-starved PC-3 cells were plated into the upper wells of a membrane chamber and incubated at 37°C for 30 min to allow attachment to the filter membrane. The upper and lower wells of this 24-well chemotaxis chamber with an 8.0-µm polycarbonate filter membrane (Fisher) were then filled with serum-free RPMI 1640 medium containing the indicated LPA species (18:1 or 14:0) and incubated at 37°C for 24 h (migration) or incubated on matrigel in the Boyden chamber for 48 h (invasion). Cells remaining on the upper filter were carefully scraped, and cells on the lower filter were fixed, stained with crystal violet, and counted.

Two-dimensional cell motility

PC-3 or SKOV-3 cells were seeded in triplicates in 24-well plates and grown to confluency. Cells were then rinsed repeatedly in serum-free medium and serum starved for 6 h. ‘Wounds’ were created using the scratch assay were a pipette tip runs through the middle of the well from top to bottom creating a gap of equivalent width in all wells. Photomicrographs are taken at the beginning of the experiment to maintain equivalency between all conditions. LPA was then added to the wells where indicated, either 18:1 at 10 µM or 14:0 at 1 µM, and incubated for 24 h. Additional photomicrographs were taken of the wells, approximately six per well in triplicate wells, and measurements were calculated for the remaining distance between confluent monolayers. Data was pooled for each condition and results reflect the combined results from three repeated assays, all showing reproducible results.

Cell proliferation and viability assay

PC-3 or DU145 cells were seeded in triplicates in 96-well plates (5000–10,000 cells/well). Cells were then serum starved and incubated either untreated or with 10 µM LPA for 48 h. For proliferation, cells were rinsed with ice-cold PBS, stained using 0.5% crystal violet with 20% methanol for 30 min, washed and the remaining crystal violet was extracted in Sorenson's buffer for 1 h before the absorbance was measured at 570 nm using a microplate reader. For viability, cells were incubated with CellTiter™ Blue reagent (Promega, Madison, WI) for 3 h at 37°C prior to measuring the absorbance at 604 nm with a fluorescence plate reader. The OD is directly proportional to the number of viable cells that are able to metabolize product. In order to assess the reduction in cell viability, results were compared to untreated controls and calculated as reduction of cell viability (%) = (Absolute value (OD untreated control − OD condition) / OD untreated control) × 100.

Western blot analysis

PC-3 cells were grown in 60-mm dishes, serum starved for 24 h, exposed to LPA, pelleted, and lysed in ice-cold lysis buffer [1% Triton X-100, 50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 100 mM NaF, 10 mM pyrophosphate, 1 mM Na₃VO₄, 10% glycerol, 1 mM PMSF, and 10 µg/ml aprotinin] or SDS sample buffer. Whole-cell lysate was then resolved by SDS-PAGE and transferred to an Immobilon [poly(vinylidene difluoride)] membrane (Fisher, Pittsburgh, PA) for immunoblotting. Protein bands were visualized with an enhanced chemiluminescence detection kit (Amersham Biosciences, Piscataway, NJ) using horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA). Densitometry of protein bands was performed using the public domain software NIH Image 1.63 which was developed by the National Institutes of Health for image processing and analysis. Values listed in the figures below the corresponding row of scanned Western blotting images represent the ratio of peak values for the experimental versus the control group.

Fluorescence imaging

To assess the localization of VASP and phosphorylated VASP (pVASP) (Ser157), PC-3 cells were seeded on glass coverslips in 24-well plates for 24 h, serum starved, and treated with LPA where indicated. Before staining, cells were washed with cold phosphate-buffered saline (Mg²⁺ and Ca²⁺ free) and fixed with 4% paraformaldehyde. The cell membranes were then permeabilized with 0.2% saponin before incubation with anti-pVASP antibody (1:300) diluted in the blocking buffer at 4°C overnight. Oregon Green-conjugated goat anti-rabbit immunoglobulin G (1:500; Invitrogen/Molecular Probes) was applied at room temperature for 60 min. For actin staining, Texas Red-conjugated phalloidin was applied at room temperature for 20 min according to the manufacturer’s protocol (Invitrogen/Molecular Probes). Samples were observed using a DMLB imaging microscope (Leica Microsystems, Bannockburn, IL) or a FluoView FV500 confocal microscope (Olympus, Melville, NY) and a CoolSNAPx digital camera (Photometrics, Tuscon, AZ) using imaging software IP Lab version 3.5 (Scanalytics, Fairfax, VA) or merged images using FluoView version 4.3 (Olympus).

Analysis of pVASP Ser(157) was performed using the CellProfiler cell image analysis software designed to quantitatively measure cell phenotypes. This software was developed as an open source project in a non-profit institution and is found at www.cellprofiler.org (Carpenter et al.).

Lentivirus construct generation

To obtain the lentivirus constructs, we used the ViralPower™ Lentiviral Expression System (Invitrogen). Details for this method have been described previously (Fang et al. 2004 Lu, Mills JBC 2004). Briefly, LPA receptor cDNAs were isolated, restriction sites were incorporated, constructs were amplified by PCR and then cloned into a Gateway entry vector (pENTR4). Viral constructs were created by homologous recombination between pENTR4-LPA_1–3 and the lentiviral destination vector (pLenti6/V5-DEST). The final constructs contain V5-tagged LPA receptors, which were stably expressed in cells and subsequently confirmed by Western blot. Subsequent cell passages were grown in the presence of blasticidin (10 µg/ml) to maintain stable transfection.

Small interfering RNA (siRNA) transfection

We down-regulated VASP and LPA receptor expression by using sequence-specific siRNA. The siRNA target sequence for VASP (siVASP: 5′-AACTTCGGCAG CAAGGAGGAT-3′) was purchased from Qiagen (Valencia, CA). Non-targeting siRNA and siRNA SMART pool reagents targeting the LPA₁, LPA₂ and LPA₃ receptors were purchased from Dharmacon (Lafayette, Colorado). These cells were transfected with the Dharmacon II reagent according to the manufacturer’s protocol. Expression levels of knocked down protein were determined by Western blot analysis. Gene knock down was confirmed by real-time quantitative PCR.

Assessment of siRNA transfection

We extracted total cellular RNA from siRNA-tranfected cells with Trizol (Invitrogen) according to the manufacturer's protocol. The amount of LPA receptor mRNA knock down was quantified with Taqman probes designed by Ambion (San Jose, CA) and employing quantitative PCR using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The LPA receptor results were normalized to the level of the control reference, β-actin.

Assessment of PKA activity

After 24 h of serum starvation, PC-3 cells were treated with up to 10 µM of LPA. Whole-cell lysate was collected, and PKA activity was measured with the SignaTECT PKA assay system (Promega, Madison, WI) using Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly; Upstate USA, Charlottesville, VA) as a substrate for PKA. Briefly, cell lysate was incubated for 10 min at 37°C in a mixture containing 100 µM ATP, 40 mM Tris-HCl (pH 7.4), 20 mM MgCl₂, and 100 µg/ml bovine serum albumin. Phosphorylation was measured at 30°C for 5 min after 100 µM biotinylated Kemptide and 100 µM [γ-³²P]ATP (3000 Ci/mmol) were added into the reaction mixture. The reaction was terminated with 1.8 M guanidine hydrochloride, the ³²P-labeled biotinylated substrate was recovered from the reaction mix with the SAM2 biotin capture membrane (Promega), and acid-insoluble radioactivity was measured.

Statistical Analysis

Statistical differences were analyzed using an analysis of variance (ANOVA) followed by Bonferroni’s Multiple Comparison test or the Newman-Keuls Multiple Comparison test between groups, where indicated. *p<0.05 **p<0.01 and ***p<0.001 indicate the levels of significance.

ACKNOWLEDGEMENTS

Grant support: This research was supported by the MDACC Prostate Cancer Research Program and NIH SPORE in Prostate Cancer (5 P50 CA902703) to YH, a training fellowship from the Keck Center Pharmacoinformatics Training Program of the Gulf Coast Consortia to MM by NIH Grant 1 T90 070109-01, by the Cancer Center Support Grant to MDACC and GM by NIH/NCI 5 P30 CA16672 and by CA91260 support to GT. We thank Elizabeth Hess for critically reviewing this work and Adam Szafran for assistance with CellProfiler.


LPA	Lysophosphatidic acid

VASP	Vasodilator-stimulated phosphoprotein

PKA	Protein Kinase A

Footnotes

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