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Endocrinology Vol. 147, No. 10 4883-4892
Copyright © 2006 by The Endocrine Society

Expression and Function of Lysophosphatidic Acid LPA1 Receptor in Prostate Cancer Cells

Rishu Guo, Elizabeth A. Kasbohm, Puneeta Arora, Christopher J. Sample, Babak Baban, Neetu Sud, Perumal Sivashanmugam, Nader H. Moniri and Yehia Daaka

Departments of Surgery (R.G., E.A.K., P.A., C.J.S., P.S., N.H.M., Y.D.) and Pharmacology and Cancer Biology (Y.D.), Duke University Medical Center, Durham, North Carolina 27710; and Department of Pathology (B.B., N.S., Y.D.), Medical College of Georgia, Augusta, Georgia 30912

Address all correspondence and requests for reprints to: Yehia Daaka, Department of Pathology, CN3114, Medical College of Georgia, Augusta, Georgia 30912. E-mail: ydaaka{at}mcg.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The bioactive phospholipid lysophosphatidic acid (LPA) promotes cell proliferation, survival, and migration by acting on cognate G protein-coupled receptors named LPA1, LPA2, and LPA3. We profiled gene expression of LPA receptors in androgen-dependent and androgen-insensitive prostate cancer cells and found that LPA1 gene is differentially expressed in androgen-insensitive and LPA-responsive but not androgen-dependent and LPA-resistant cells. In human prostate specimens, expression of LPA1 gene was significantly higher in the cancer compared with the benign tissues. The androgen-dependent LNCaP cells do not express LPA1 and do not proliferate in response to LPA stimulation, implying LPA1 transduces cell growth signals. Accordingly, stable expression of LPA1 in LNCaP cells rendered them responsive to LPA-induced cell proliferation and decreased their doubling time in serum. Implantation of LNCaP-LPA1 cells resulted in increased rate of tumor growth in animals compared with those tumors that developed from the wild-type cells. Growth of LNCaP cells depends on androgen receptor activation, and we show that LPA1 transduces G{alpha}i-dependent signals to promote nuclear localization of androgen receptor and cell proliferation. In addition, treatment with bicalutamide inhibited LPA-induced cell cycle progression and proliferation of LNCaP-LPA1 cells. These results suggest the possible utility of LPA1 as a drug target to interfere with progression of prostate cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LYSOPHOSPHATIDIC ACID (LPA) is a naturally occurring simple phospholipid that regulates cell proliferation, survival, and migration (1, 2). LPA is produced mainly in the extracellular compartment via the hydrolysis of phosphatidic acid by soluble phospholipase A2 (3) or the hydrolysis of lysophosphatidylcholine by autotaxin/lysophospholipase D that, interestingly, was originally identified as a tumor motility factor (4, 5). Initially, LPA was thought to be secreted primarily by platelets during wound healing (6). However, the recent finding that autotaxin/lysophospholipase D is expressed in a variety of tissues, including brain, ovary, and kidney, suggests that LPA may be produced by many cell types (2). In humans, ovarian cancer patients, for example, produce elevated levels of LPA, leading to the hypothesis that it may contribute to tumor initiation and/or progression (7). In tissue culture, ovarian, pancreatic, and androgen-insensitive (AI) prostate cancer cells are all able to secrete LPA and to use it as an autocrine factor to regulate cell proliferation and migration (8, 9, 10).

LPA exerts its effects on target cells through the activation of cognate receptors LPA1, LPA2, and LPA3 (11, 12), which belong to the endothelial differentiation gene family of G protein-coupled receptors (GPCRs). A more recent study suggested existence of another LPA receptor, LPA4 (13). The LPA4 shows limited homology to LPA1, LPA2, and LPA3 and, distinctly, is more related to the purinergic family of GPCRs. Stimulation with LPA activates at least three distinct subfamilies of G proteins, namely Gi, Gq, and G12/13. In most cells, LPA1 couples to Gi and inhibits accumulation of cAMP (12, 14). Activated LPA2 and LPA3 couple primarily to Gq and induce activation of protein kinase C through the release of Ca2+ ions from intracellular stores (15, 16). Unexpectedly, activated LPA4 was suggested to couple to Gs and to promote the synthesis of cAMP (13). Notwithstanding, these results clearly demonstrate that individual LPA receptors activate distinct signaling pathways that, in turn, mediate specific cellular responses.

Different LPA receptors show distinct tissue distribution. The LPA1 gene is highly expressed in the brain and heart, is virtually undetectable in the liver, lung, thymus, and leukocytes, and is expressed at low levels in all other organs (17). Expression of the LPA2 and LPA3 genes is confined largely to the pancreas and testes (16), whereas the putative LPA4 gene is highly expressed in the ovary and can be detected at low levels in other organs (13). Importantly, the expression of LPA receptor genes is increased in cancer cells; LPA2 and LPA3 genes are markedly elevated in epithelial ovarian cancers (18), and high expression of LPA1 was demonstrated in several human colon carcinoma cell lines (19). These results reinforce the idea that specific LPA receptors may regulate the initiation and/or progression of certain cancers.

In the case of prostate cancer, which is the second-leading cause of male cancer deaths in the majority of Western countries (20), the cancerous gland contains multiple independent and genetically distinct lesions, suggesting heterogeneity of the disease (21). Accumulating evidence suggests that heterotrimeric G proteins and their associated GPCRs play an important, albeit poorly understood, role in prostate carcinogenesis (22). For example, the pan inhibition of G protein ß{gamma}-subunit signaling attenuates the serum-induced mitogenic signaling in prostate cancer cells (23) and prostate tumor growth in animals (24). In tissue culture systems, activated GPCRs, including the LPA receptors, elicit mitogenic responses and proliferation of prostate cancer cells (25). Specifically, stimulation with LPA induces the ERK1-, ERK2-, and nuclear factor {kappa}B-mediated proliferation (23, 24, 25) and survival (26), respectively, of AI prostate cancer PC3 cells. Intriguingly, LPA does not appear to exert measurable mitogenic effects on androgen-dependent (AD) prostate cancer LNCaP cells (27, 28).

Information regarding expression profile and function of specific LPA receptor subtypes in commonly used prostate cancer cell lines or human prostate biopsies is limited. Here, we profiled expression of LPA1, LPA2,and LPA3 genes, using Northern blot analysis and mRNA in situ hybridization in AD LNCaP and AI PC3 and DU145 prostate cancer cells as well as in human clinical specimens containing prostate cancer and matched benign controls. We found LPA1 gene to be expressed in the AI but not AD prostate cancer cells and that expression of LPA1 gene was increased in the cancer compared with the benign prostate specimens. The stable expression of LPA1 in LNCaP cells rendered them responsive to treatment with LPA to proliferate, and implantation of these cells resulted in an increase in the size of tumors in animals compared with those tumors that develop from empty vector-transfected LNCaP cells. Moreover, we show that LPA1 transduces the G{alpha}i-mediated translocation of androgen receptor (AR) to the nucleus and promotes the AR-regulated cell cycle progression and cell proliferation. These findings suggest that targeted inhibition of LPA1 signaling may be beneficial for treatment of men with prostate cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and proliferation
The AD (LNCaP) and AI (PC3 and DU145) human prostate cancer cell lines were purchased from American Type Culture Collection (Bethesda, MD). The PC3 and DU145 cells were maintained in RPMI 1640 culture medium supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. The cell culture for LNCaP cells additionally contained 10 mM HEPES buffer (pH 7.5), 1 mM sodium pyruvate, and 1.26 g/liter glucose. Stable expression of LPA1 gene in the LNCaP cells was done by standard transfection methods using DMRIE-C (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Agonist-regulated cellular proliferation was determined using two independent assays: counting cells using trypan blue and a hemocytometer and the measurement of mitochondrial dehydrogenase activity with WST-1 (24, 26). To estimate cell doubling time, 1 x 106 cells were seeded into 60-mm plates and cultured to 80% confluence. Cells were harvested and counted, and cell doubling time was calculated using the formula Td = (ln2 x T)/ln(Nf/Ni) where Td is doubling time, T is total time (in hours) to reach 80% confluence, Nf is final cell number, and Ni is the initial number of seeded cells. To determine cell cycle progression, cells were treated with propidium iodide together with RNase A and analyzed by flow cytometry using a four-color Becton Dickinson FACScan machine.

DNA constructs
The three human LPA receptor clones (all in pcDNA3.1) were obtained from Guthrie DNA Resource Center (Sayre, PA). The LPA receptor cDNAs were released [LPA1: BamHI/XbaI (nt 704-1577); LPA2: BamHI/XbaI (nt 704-1821); and LPA3: HindIII/XbaI (nt 826-1921)] and cloned into a PCRII-TOPO vector (Invitrogen). After linearization, in vitro transcription and production of the antisense and sense RNA probes was done using T7 and SP6 RNA polymerases, respectively. The RNA probes were labeled using a digoxigenin-UTP RNA-labeling kit (Roche, Indianapolis, IN). Green/yellow fluorescent protein (GFP/YFP) fused to ß-arrestin2 (GFP-ß-arrestin2) and AR (YFP-AR) cDNAs were obtained from R. J. Lefkowitz (Duke University) and A. M. Thorburn (Wake Forest University), respectively. LNCaP cells were transiently transfected using the appropriate cDNAs and DMRIE-C (Invitrogen). Experiments were performed 2 d after transfection and cells were serum starved overnight in culture medium containing 10 mM HEPES (pH 7.5) and 0.1% BSA.

Northern analysis
Total RNA was prepared from monolayer cultures of DU145, PC3, or LNCaP cells using the RNeasy kit (QIAGEN, Valencia, CA). Poly A+ RNA was obtained from total RNA using Oligotex mRNA Isolation kit (QIAGEN). Approximately 1 µg of poly A+ RNA was separated on a 1.2% formaldehyde-agarose gel and transferred to a Hybond N+ membrane (Amersham Biosciences, Piscataway, NJ) by overnight upward capillary transfer. The RNA was cross-linked to the membrane by UV irradiation, and prehybridization was carried out for 1 h using PerfectHyb Plus buffer (Sigma Chemical Co., St. Louis, MO) with 100 µg/ml sheared salmon sperm DNA (Invitrogen). Probes were obtained by labeling cDNAs for LPA1, LPA2, and LPA3 with [{alpha}-32P]dCTP using Rediprime II Random Prime labeling system (Amersham). Hybridization was carried out overnight in the prehybridization buffer with the denatured probes. After high-stringency washes (0.1x saline sodium citrate [SSC], 0.1% SDS), the bands were visualized by autoradiography.

Tissue specimens
Prostate specimens were obtained from patients undergoing radical prostatectomy for biopsy-proven adenocarcinoma at Duke University Medical Center in accordance with guidelines of the Institutional Review Board for handling human materials. Specimens were obtained from the operating room immediately after resection, and samples were fixed in 10% buffered neutral formalin and embedded in paraffin. The tissue microarray slides were obtained from the National Cancer Institute Tissue Array Research Program (http://ccr.cancer.gov/tech-initiatives/trap). Each slide contained 130 prostate cancer samples with matched 64 normal-appearing prostate samples derived from the same patient and were made from paraffin-embedded, formalin-fixed tissue blocks.

In situ hybridization
Tissue sections of 5 µm thickness were deparaffinized and rehydrated in graded alcohol. Prehybridization was performed with 2x SSC/50% formamide for 30 min at 37 C. Hybridization was performed at higher stringency with 2 ng/µl of denatured probe at 42 C for 16 h in 50 µl hybridization mix (containing 50% deionized formamide, 100 µg/ml salmon sperm DNA, 50 µg/ml yeast tRNA, 10% dextran sulfate, and 2.5 µl of 50x Denhardt’s solution) on an inverted coverslip in a moist chamber. For every specimen, alternate sections were routinely examined in parallel with sense and antisense cRNA probes. All reactions were performed under RNase-free conditions. After hybridization, excess probe was removed by washing three times in 2x SSC/50% formamide for 20 min at 42 C. The specificity of the signal was also confirmed by digestion of the tissue sections with RNase A for 30 min at 37 C or by omission of the probe in the hybridization mixture. This was followed by three high-stringency washes in 0.1x SSC for 20 min at 42 C.

Immunological detection of the signal was done using a DIG Nuclear Acid Detection Kit (Roche) following the manufacturer’s instructions. Briefly, after incubation with 1% blocking reagent for 30 min, the sections were incubated with sheep antidigoxigenin alkaline phosphatase conjugate diluted at 1:200 for 2 h at ambient temperature. After three washes [0.1 M Tris-HCl (pH 7.5), 0.1 M NaCl, 0.05 M MgCl2], the signal was developed using 5-Br-4-Cl-3-indolyl phosphate and nitroblue tetrazolium salt, which produces an insoluble blue precipitate. Sections were counterstained with methyl green.

In situ mRNA hybridization was initiated by seeding cells on glass coverslips in a humidified atmosphere of 95% air and 5% CO2 at 37 C for 24 h. Cells were washed once in PBS, fixed with 4% paraformaldehyde in 10% acetic acid for 10 min at ambient temperature, and permeabilized by overnight treatment with 70% ethanol at 4 C. After two washes with PBS, cells were prehydrated for 5 min in 2x SSC/50% formamide and hybridized with the specific LPA receptor cRNAs, exactly as described above.

Immunoblotting
Appropriately treated cells were lysed in radioimmunoprecipitation buffer [150 mM NaCl, 50 mM Tris-HCl (pH 8), 1 mM EDTA, 0.25% (wt/vol) sodium deoxycholate, 0.1% (vol/vol) Nonidet P-40, 1 mM NaF, 1 mM sodium pyrophosphate, 100 µM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.7 µg/ml pepstatin] and analyzed by SDS-PAGE and Western blotting. Anti-phospho-GSK3ß antibody (Cell Signaling Technology, Beverly, MA) was used at a dilution of 1:1000.

Fluorescence microscopy
Cells on glass coverslips were labeled with mouse monoclonal anti-HA antibodies at a dilution of 1:200, washed with PBS, and mounted. Expression of the HA-tagged LPA1 and subcellular localization of GFP-ß-arrestin2 and YFP-AR proteins were conducted on blinded samples and were inspected under a Zeiss Axiovert 200 fluorescence microscope (28, 29, 30). 4'-6-Diamidino-2-phenylindole was used for nuclear staining.

Tumor growth
All experiments involving animals were performed in accordance with the National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee at Duke University. Xenografts were established by injection of 1 x 106 cells suspended in a mixture of 50 µl culture medium and 50 µl Matrigel sc into the flanks of 6- to 8-wk-old BALB/c athymic male mice (24, 29). Tumors were measured using calipers, and tumor volume was determined using the following formula: {pi}/6 x larger diameter x (smaller diameter)2.

Statistical analyses
The significance of LPA-induced cellular proliferation and difference in the positive staining rate for LPA1, LPA2, and LPA3 mRNAs in the prostate cancer, prostatic intraepithelial neoplasia (PIN), and matched benign prostate tissues in the tissue microarray was analyzed using the two-tailed Student’s t test. Significance was implied at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPA is the major mitogen present in serum (1), and we have previously shown that it induces mitogenic ERK activation in stromal (29) and AI (23, 25) but not AD (28) epithelial prostate cancer cells. We compared the effect of LPA stimulation on the cell proliferation using model AI (PC3 and DU145) and AD (LNCaP) prostate cancer cells. Similar to the situation of ERK phosphorylation, LPA induced an actual increase in the number of AI PC3 and DU145 prostate cancer cells (Fig. 1Go). Distinctly, the stimulation with LPA did not affect proliferation of AD LNCaP cells (Fig. 1Go). Because LPA exerts its effects on target cells by activating its cognate receptors, these results suggested the possibility that AD and AI prostate cancer cells express distinct subtypes of LPA receptors. Accordingly, we profiled expression of the three well-established LPA receptors, namely LPA1, LPA2, and LPA3. Because of the lack of reliable LPA receptor antibodies, we used Northern blot analysis to document gene expression of the LPA receptors (Fig. 2AGo). The AD LNCaP, AI PC3, and DU145 cells exhibited distinct expression patterns for the three LPA receptor mRNAs. Whereas the LPA1 gene was detected in PC3 and DU145 but not in LNCaP cells, the LPA2 gene was expressed in all three prostate cancer cell lines (Fig. 2AGo). The LPA3 gene was expressed in both PC3 and LNCaP but not in DU145 cells, in agreement with previous results (16). In all extracts, a single band was observed on autoradiograms after exposure to 32P-labeled LPA1 and LPA2 probes, with relative migration of 4.0 and 1.9 kb, respectively. In LNCaP extracts, the autoradiogram showed two bands, one prominent (4.0 kb) and another minor (1.9 kb), after exposure to the 32P-labeled LPA3 probe.


Figure 1
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  		FIG. 1. LPA induces proliferation of AI but not AD prostate cancer cells. Cells were seeded in six-well plates and cultured in serum-free media for 2 d. The cells were treated or not with LPA (10 µM) for an additional 2 d, harvested, and mixed with 0.1% trypan blue stain. Cells excluding the dye were counted under light microscopy. Each point represents mean ± SD of values obtained from three independent experiments performed in duplicate. *, P < 0.05 vs. controls. NS, Not stimulated.

 

Figure 2
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  		FIG. 2. Expression of LPA receptor genes in prostate cancer cells. A, Northern blot analysis to detect LPA1, LPA2, and LPA3. mRNA was isolated from PC3, DU145, and LNCaP cells and used for Northern blot analysis with 32P-labeled LPA1, LPA2, and LPA3 probes. Images are representative of three separate experiments. B, Detection of LPA1, LPA2, and LPA3 mRNAs by in situ hybridization. Cell monolayers were grown on glass coverslips and subjected to in situ hybridization using antisense LPA1-as, LPA2-as, and LPA3-as or sense LPA2-s probes. The positive nuclei are stained in dark color.

 
Northern blot analysis is a widely used method for the detection of gene expression. However, this methodology is limiting when RNA is not readily extractable, such as the case in solid tumor biopsies. Therefore, we employed in situ hybridization, which can be applied to both cultured cells and tissue samples, to further analyze expression of the LPA receptor genes. We confirmed the similar expression pattern of the LPA receptor genes using Northern blot (Fig. 2AGo) and in situ mRNA hybridization (Fig. 2BGo) methods. The data show that PC3 cells express all three LPA receptor genes, DU145 cells express LPA1 and LPA2 but not LPA3 gene, and LNCaP cells express LPA2 and LPA3 but not LPA1 gene. Interestingly, expression of the three LPA receptor mRNAs was observed only in some PC3 cells (Fig. 2BGo), suggesting cell heterogeneity, which is in agreement with previous studies (30).

We examined five human prostate biopsies, obtained from patients undergoing radical prostatectomy for biopsy-proven adenocarcinoma for the expression of LPA receptor genes using in situ hybridization. The different areas of invasive carcinoma, PIN, and benign tissue from the same patient revealed distinct expression profiles of the LPA receptor genes. The signals were observed mostly in epithelial cells, with low expression levels in the stromal compartment (Fig. 3Go). Higher expression of LPA1 mRNA was observed in regions with invasive carcinoma and PIN in comparison with normal-appearing tissues from the same sample (Fig. 3AGo). We could also show that expression of LPA2 and LPA3 genes was more in the malignant compared with the benign tissues (Fig. 3AGo).


Figure 3
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  		FIG. 3. Expression of LPA receptor genes in human prostate biopsies. A, Detection of LPA receptor genes in individual human prostate biopsies. Different regions representing invasive carcinoma (PCa), PIN, and normal-appearing (benign) prostate samples were used for determining gene expression using in situ hybridization to detect LPA1, LPA2, and LPA3 genes. Positively stained nuclei have dark color. The last column is a control where hybridization was done using the sense LPA2-s probe. Magnification, x200. B, Detection of LPA receptor expression in tissue microarrays by in situ mRNA hybridization. Tissue microarrays were employed to study a large number of samples for LPA receptor gene expression. The image depicts signals obtained for the three LPA receptors in cancerous (top) and matched benign (bottom) human prostate biopsies. The last column is a control where tissues were hybridized using sense LPA2-s probe. Original magnification, x100; inset, x400. C, Summary of the LPA receptor gene expression obtained from the tissue microarrays. The number of positive nuclei was counted in equal fields in 40 prostate cancer, 23 PIN, and 39 benign samples, and each data point represents the mean ± SD. *, P < 0.05 vs. matched benign samples.

 
We observed distinct expression patterns for the different LPA receptor genes in the prostate cancer cell lines (Fig. 2Go) and in a limited number of human prostate biopsies (Fig. 3AGo). To gain more confidence in these results, we increased the number of prostate samples by using tissue microarrays. Each slide contained 130 prostate cancer samples with 64 corresponding benign samples from the same patients. Specificity of the signal was confirmed by using contiguous sections for in situ hybridization with LPA1, LPA2, and LPA3 mRNA probes and by using sense cRNA probes as negative controls. Expression of the LPA receptor genes was localized largely to the cancerous epithelial cells (Fig. 3BGo). Levels of all three LPA receptor transcripts were elevated in about 70% of the tumor samples (90 of 130) compared with the respective sections of benign tissues. In all cases, there was only little LPA receptor gene expression in regions of normal-appearing tissue (Fig. 3Go, A and B). In 7% of the prostate cancer samples, expression of the LPA receptor genes was either very weak or totally negative (data not shown), and about 23% of the samples could not be studied because of tissue loss or damage. To quantitate the differences seen in LPA receptor gene expression in the tissue microarrays, we counted the number of positive nuclei for each receptor in an equal number of fields for the tumor, PIN, and benign samples. Expression of the three LPA receptor genes was similar among the tumor and PIN samples (Fig. 3CGo). Benign prostate samples expressed all three receptor genes, albeit at a significantly lower level than that observed in the cancerous tissues.

The DU145 cells express LPA1 and LPA2 but not LPA3 gene (Fig. 2Go) and proliferate in response to LPA stimulation (Fig. 1Go), suggesting LPA3 is not critical for the LPA-induced cell proliferation. On the other hand, the LNCaP cells express LPA2 and LPA3 but not LPA1 gene (Fig. 2Go) and do not proliferate in response to LPA stimulation (Fig. 1Go), implying LPA1 may relay the mitogenic actions of LPA. Importantly, LPA1 expression is higher in human cancer and PIN tissues compared with benign prostate samples (Fig. 3Go). To begin to investigate the contribution of LPA1 to mitogenic signaling and prostate cancer cell proliferation, we established lines of human LNCaP cells that stably overexpress LPA1 protein tagged at its amino terminus with a peptide derived from the influenza virus hemagglutinin (HA) protein (Fig. 4AGo, lower panel). FACScan analysis using anti-HA antibodies revealed expression of the LPA1 protein on the cell surface, and stimulation with LPA resulted in the internalization of about 50% of cell surface-expressed LPA1 (data not shown), demonstrating that the ectopically expressed LPA1 protein is capable of binding LPA and subsequently internalizing into the intracellular compartment, in agreement with previous results (31).


Figure 4
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  		FIG. 4. Expression and function of LPA1 in LNCaP cells. A, Representative fluorescence microscopy images of empty vector-transfected LNCaP (upper image) and LNCaP-LPA1 cells stably expressing HA-LPA1 fusion protein (lower image). The HA-LPA1 protein was visualized using a primary anti-HA antibody and a secondary fluorescein-isothiocyanate-conjugated antibody. Note the fluorescence signal is expressed on the plasma membrane, and no signal is detected in the control LNCaP cells. B, LPA1 mediates subcellular redistribution of GFP-ß-arrestin2 protein. LNCaP and LNCaP-LPA1 cells transiently expressing GFP-ß-arrestin2 were stimulated or not with LPA (10 µM) for 5 min and fixed, and fluorescence signal was acquired with an Axiovert 200 Zeiss microscope. In unstimulated cells, GFP-ß-arrestin2 protein is distributed uniformly throughout the cytoplasm (left column). After LPA stimulation, GFP-ß-arrestin2 protein relocalizes displaying a punctated distribution in LNCaP-LPA1, but not empty vector-transfected LNCaP (right column) cells.

 
Agonist-induced activation of plasma membrane-anchored GPCRs leads to a change in the subcellular localization of specific proteins, including the recruitment of ß-arrestin2 protein to the plasma membrane that mediates, among other things, GPCR internalization (32). We used this characteristic to further illustrate functionality of the ectopically expressed LPA1 protein. Stimulation with LPA induced redistribution of a GFP-ß-arrestin2 fusion protein to the plasma membrane (as demonstrated by the formation of puncta) in LNCaP-LPA1 cells (Fig. 4BGo, lower right), but not in control LNCaP cells (Fig. 4BGo, upper right). It is not clear why stimulation of the wild-type LNCaP cells (which express LPA2 and LPA3 genes) with LPA does not lead to a detectable redistribution of GFP-ß-arrestin2 protein to the plasma membrane. It is possible that LPA2 and LPA3 genes are not translated into functional proteins (and we could not verify this possibility because of the lack of available specific LPA receptor antibodies). Another possibility is that levels of translated LPA2 and LPA3 proteins are low, such that they do not support the detectable LPA-regulated translocation of GFP-ß-arrestin2 protein. The latter suggestion is consistent with the original work by the Caron group who showed that isoproterenol-mediated translocation of GFP-ß-arrestin2 to the plasma membrane in HEK293 cells requires overexpression of ß2 adrenergic receptor (33). More recent studies showed that certain GPCRs differentially recruit ß-arrestin1 and ß-arrestin2 proteins (34), and therefore, it is possible that LPA1, but not LPA2 or LPA3, specifically recruits ß-arrestin2 protein to the plasma membrane.

In vitro, LPA induces cell proliferation through activation of diverse, cell type-specific mechanisms. We tested whether mitogenic actions of LPA are transduced via LPA1 using the LNCaP-LPA1 cells, and we used two independent assays to measure cell proliferation: actual count of cell number and mitochondrial dehydrogenase activity. Initial screening of the effect of LPA stimulation on growth of several LNCaP-LPA1 monoclonal lines and a polyclonal LNCaP-LPA1 line revealed no significant differences among them (data not shown). Subsequent experiments were done using a monoclonal LNCaP-LPA1 line. Stimulation with LPA induced a significant increase in the rate of cell proliferation (Fig. 5Go, A and B), which is in contrast to the lack of LPA effect on proliferation of wild-type LNCaP cells (Fig. 1Go). LPA is present in serum and is thought to be the major mitogen in serum (1). To gain more confidence in the conclusion that LPA1 mediates the mitogenic action of LPA, we compared the doubling time in serum of empty vector-transfected LNCaP and LNCaP-LPA1 cells. Figure 5CGo shows that the expression of LPA1 conferred growth advantage to target cells; on average, the doubling time of control empty vector-transfected LNCaP cells was 78.5 ± 6.6 h, and that of LNCaP-LPA1 cells was 63.6 ± 3.2 h. Together, these results suggest that LPA1 relays LPA-induced cell growth signals.


Figure 5
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  		FIG. 5. LPA1 mediates mitogenic action of LPA in LNCaP cells. LNCaP-LPA1 cells proliferate in response to LPA stimulation. A, Trypan blue cell count. Cells were seeded in 60-mm plates, treated, and counted exactly as described for Fig. 1Go. B, Mitochondrial dehydrogenase assay. Cells in 96-well plates were treated as described, and WST-1 was added (2 d after LPA treatment) for 30 min. Absorbance at A450 was determined using an ELISA reader. C, LPA1 increases rate of LNCaP cell division. LNCaP and LNCaP-LPA1 cells were cultured in serum-containing media to 80% confluence, harvested with trypsin, and counted after staining with trypan blue. Doubling time was calculated using the formula Td = (ln2 x T)/ln(Nf/Ni), where Td is doubling time, T is total time (in hours) to reach 80% confluence, Nf is final cell number, and Ni is the initial number of seeded cells. For A–C, each point represents mean ± SD of values obtained from three to five independent experiments. *, P < 0.05 vs. controls. NS, Not stimulated. D, LPA1 increases rate of LNCaP tumor formation in mice. Cells (1 x 106) were injected sc with Matrigel into the flanks of 6- to 8-wk-old BALB/c athymic mice. Tumor volume was measured using calipers and calculated using the following formula: volume = {pi}/6 x (long dimension) x (short dimension)2. Shown are curves representing the average tumor size of 10 mice for each of the LNCaP and LNCaP-LPA1 groups. E, Effect of LPA stimulation on GSK3ß phosphorylation. LNCaP and LNCaP-LPA1 cells were stimulated with LPA (10 µM) for the indicated time. Cell monolayers were washed with PBS and lysed, and cell lysates were analyzed by Western blotting using anti-phospho-GSK3ß antibody (upper panel). The filter was stripped of Ig and reblotted with anticlathrin antibody to demonstrate equal protein loading (lower panel). Data shown are representative of three experiments.

 
To further investigate the possible physiological impact of LPA1 expression, we established a xenograft model to assess LPA1 effect on tumor growth in mice. LNCaP-LPA1 (or empty vector-transfected LNCaP) cells were coinjected with Matrigel sc in the flanks of athymic male mice. Tumors arising from both cell types were detected within 1 month after implantation (Fig. 5DGo). Although tumor take was similar in animals injected with the control and LNCaP-LPA1 cells (data not shown), the growth rate of the LNCaP-LPA1 tumors was consistently faster in comparison with those tumors arising from the control LNCaP cells (Fig. 5DGo). Together with the in vitro cell growth data (Fig. 5Go, A–C), these observations reinforce the idea that LPA1 provides selective growth advantage to AD LNCaP cells.

We have previously shown that LPA induces growth of PC3 cells (which express endogenous LPA1) through activation of the ERK pathway (23, 25), and we tested the idea that LPA induces growth of the LNCaP-LPA1 cells through ERK signaling. Similar to the situation in wild-type LNCaP cells (27, 28), the stimulation with LPA failed to activate ERK in the LNCaP-LPA1 cells (data not shown). As a control, we used epidermal growth factor and observed that it promoted (the similar) ERK activation in both LNCaP and LNCaP-LPA1 cells. A recent report showed that LPA induced growth of colon cancer cells through activation of glycogen synthase kinase 3ß (GSK3ß) pathway (35). Stimulation of wild-type LNCaP cells with LPA induced the time-dependent 2- to 3-fold increase in GSK3ß phosphorylation (Fig. 5EGo). However, the stimulation of LNCaP-LPA1 cells with LPA showed no consistent increase in the GSK3ß phosphorylation (Fig. 5EGo). Together, these data suggest that LPA1 promotes proliferation of LNCaP-LPA1 cells via ERK- and GSK3ß-independent mechanisms.

Growth of LNCaP cells requires a functioning AR, and our data show that LPA1 mediates LNCaP cell proliferation (Fig. 5Go). Upon activation, AR translocates to the nucleus and binds specific DNA elements to regulate expression of genes involved in cell growth and differentiation. Importantly, AR can be transactivated by growth factors other than androgen to mediate the cell response (28, 36). To address just how LPA1 might regulate LNCaP cell proliferation, we tested the effect of LPA stimulation on subcellular localization of AR. Stimulation with AR agonist 5{alpha}-dihydrotestosterone (DHT) induced the similar nuclear translocation of a YFP-AR fusion protein in LNCaP and LNCaP-LPA1 cells (Fig. 6AGo). Distinctly, the stimulation with LPA induced nuclear expression of YFP-AR in LNCaP-LPA1 but not control LNCaP cells (Fig. 6AGo), demonstrating LPA1 transduces signals to transactivate the AR. In support of this idea, we find that stimulation of PC3 cells (which endogenously express LPA1, LPA2, and LPA3) with LPA induces nuclear translocation of ectopically expressed AR (data not shown). Activated AR acts as a transcription factor that regulates expression of specific genes, such as prostate-specific antigen (PSA), and we show that stimulation of LNCaP-LPA1, but not LNCaP cells with LPA induced the AR-regulated increase in the number of cells that produce PSA (Fig. 6BGo). Furthermore, we find that LPA treatment prompted cell cycle progression (Fig. 6CGo) and cell proliferation (Fig. 6DGo) in a bicalutamide-inhibitable manner. Together, these results demonstrate that LPA1 transduces the AR-regulated signals to promote growth of LNCaP-LPA1 cells.


Figure 6
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  		FIG. 6. LPA1 transduced the AR-regulated cell proliferation. A, LPA1 promotes nuclear expression of AR. LNCaP and LNCaP-LPA1 cells were transiently transfected with YFP-AR, allowed to recover for 1 d, and then incubated in culture medium containing charcoal-stripped serum for an additional 2 d. Cells were treated or not with LPA (10 µM) or DHT (10 nM) for 30 min. Subcellular localization of YFP-AR was determined by fluorescence microscopy. B, LPA induces the AR-regulated increase in cell number expressing PSA. LNCaP-LPA1 cells were stimulated with LPA (10 µM) or DHT (10 nM) in the presence or absence of bicalutamide (10 µM) for 2 d. Methanol-fixed cells were labeled with monoclonal anti-PSA antibodies, washed, and then incubated with fluorescein-isothiocyanate-conjugated antimouse IgG. To analyze the PSA-expressing cells, gates were set between distinct populations of stained and unstained cells, and the percentage of gated and PSA-expressing cells was recorded. Data shown are representative of three experiments. C, Bicalutamide inhibits LPA-mediated LNCaP-LPA1 cell cycle progression. Cells were stained with propidium iodide, and cytograms were recorded by flow cytometry employing a 15-mW argon-ion laser tuned to 488 nm and analyzed using CellQuest software. D, Bicalutamide inhibits LPA-mediated LNCaP-LPA1 cell proliferation. Cells were treated as above, and cell number was determined as in Fig. 1Go. Each point represents the mean ± SD of values obtained from three independent experiments performed in duplicate. *, P < 0.05 vs. controls. NS, Not stimulated.

 
A remaining issue is identification of intermediates that connect the LPA1 and AR. LPA1 has been shown to activate heterotrimeric Gi proteins (16), and we used pertussis toxin (PTX) as a diagnostic tool to selectively inhibit Gi-mediated cell signaling. Treatment of LNCaP-LPA1 cells with PTX abrogated the LPA-mediated translocation of AR to the nucleus but had no effect on the DHT-mediated AR translocation (Fig. 7AGo). Importantly, treatment of LNCaP-LPA1 cells with PTX inhibited the LPA-mediated cell proliferation (Fig. 7BGo). Together, these results show that LPA1 transduces the Gi-mediated nuclear expression of AR and cell growth.


Figure 7
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  		FIG. 7. LPA1 transduces Gi-mediated signal to activate AR. A, PTX inhibits LPA-induced translocation of AR to the nucleus. LNCaP and LNCaP-LPA1 cells were pretreated overnight with PTX (50 ng/ml), stimulated with LPA (10 µM) or DHT (5 nM) for 30 min, and analyzed for nuclear YFP-AR expression, as described in Fig. 6AGo. Data are presented as percentage of cells expressing nuclear AR, and for each treatment more than 100 randomly selected cells were counted. *, P < 0.05 vs. cells not treated with PTX. NS, Not stimulated. B, Effect of PTX on LPA-induced LNCaP-LPA1 cell proliferation. Cells were seeded in 60-mm plates, treated or not with PTX (50 ng/ml), and stimulated with LPA (10 µM) for 2 d. Cell number was determined as described in Fig. 1Go, and each data point represents mean ± SD of values obtained from three independent experiments. *, P < 0.05 vs. unstimulated cells; **, P < 0.05 vs. cells treated with LPA in the absence of PTX.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LPA, a principal mitogen in serum (1), regulates cellular responses by activating its cognate G protein-coupled LPA receptors (12). The LPA receptors share high sequence homology but differ markedly in their tissue distribution as well as the repertoire of downstream effectors that control cell proliferation, survival, or migration (2). The existence of several LPA receptor subtypes together with the findings that LPA exerts a multiplicity of cellular responses suggest that each LPA receptor subtype may mediate a specific cellular function. The major finding of this study is that expression of LPA receptor genes is significantly higher in prostate cancer compared with benign tissues and that LPA1 confers selective growth advantage to AD prostate cancer cells. These data suggest that targeted inhibition of LPA1 signaling may be beneficial for treatment of patients with prostate cancer.

In addition to being expressed in epithelial prostate cancer cells, our results show that LPA receptor genes are also expressed in prostate stromal cells. These observations may be significant in light of recent findings that stromal cells play a critical role in the incidence of prostate tumor take and growth rate in animal model systems (29, 37). Stimulation of human prostate stromal cells with LPA induces secretion of growth factors that, in turn, induce the mitogenic signaling and proliferation of epithelial prostate cancer cells (29). Hence, LPA may act directly on the epithelial prostate cancer cells to regulate their growth or indirectly by activating stromal cells (to secrete factors that, in turn, regulate epithelial prostate cancer cell mitogenic responses). Consistent with this idea, we find that AR is activated in response to cell stimulation with conditioned medium obtained from LPA-stimulated stromal prostate cells (P.S. and Y.D., data not shown).

Accumulating evidence suggests that LPA receptors play an important role in tumor initiation and progression. The LPA receptors are widely expressed in normal tissues and are up-regulated in cancers, including those of the pancreas and ovaries (17, 18, 19). Our results demonstrate that expression of LPA receptor genes is higher in human prostate cancer compared with benign tissues. The results also show an increased expression of the LPA receptor genes in PIN. Because PIN lesions are viewed as precursors to prostate cancer (38), our findings suggest that aberrant LPA receptor expression is an early event in cancer development. Specifically, the data suggest that LPA1 is the critical mediator of LPA-induced mitogenic signaling and prostate cell proliferation. Consistent with this conclusion is the finding that LPA1 mediates the effect of LPA in colon cancer cells; cells that express LPA1 but not LPA2 or LPA3 migrate after stimulation with LPA, and conversely, cells that express LPA2 and LPA3, but not LPA1 do not migrate in response to LPA stimulation (19).

Forced expression of LPA1 renders LNCaP cells responsive to the mitogenic actions of LPA. How does LPA1 regulate prostate cell proliferation? In model RH7777 hybridoma cells, which do not express endogenous LPA receptors, ectopically expressed LPA1 couples primarily to Gi (16). Furthermore, the LPA-induced mitogenic signaling in AI PC3 cells is inhibited by PTX (23), again arguing for a role for Gi proteins. In noncancerous fibroblast NIH3T3 cells, LPA induces mitogenic signaling predominantly via G12/13 (39). Our data show that activated LPA1 transduces the PTX-inhibitable nuclear expression of AR and cell proliferation. A recent report showed that forced expression of the constitutively active {alpha}-subunit of Gi does not alter AR activity (40). It is possible that Gß{gamma}, and not G{alpha}, subunits derived from Gi are the actual mediators of LPA1-induced AR transactivation and subsequent cell proliferation. Indeed, ß{gamma}-subunits of Gi transduce the mitogenic signals in response to activation of Gi-coupled receptors (22), and expression of a Gß{gamma} inhibitor attenuates PC3 prostate tumor growth in animals (24). Alternatively, a recent report showed that stimulation of {delta}-opioid receptor in model HEK293 cells promotes the ß-arrestin1-mediated gene regulation (34). Hence, it is possible that LPA1 transduces mitogenic signals via G{alpha} and Gß{gamma} subunits as well as ß-arrestin proteins.

In summary, the data demonstrate that LPA1 mediates the mitogenic action of LPA on target LNCaP prostate cancer cells, LPA1 increases rate of LPA- and serum-induced cell proliferation in vitro and tumor growth in animals. Mechanisms involved in the LPA1-mediated increase in prostate cancer cell proliferation remain incomplete but may involve Gi and AR proteins. Furthermore, LPA1 gene expression is increased in PIN and cancer compared with benign human prostate tissues, suggesting its possible utility as a diagnostic marker as well as a drug target to interfere with disease progression.


   Footnotes
 
This work was supported by the Department of Defense Prostate Cancer Research Program (DAMD17-01-0053) and the National Institutes of Health (AG17952 and DK60917). Y.D. is a Georgia Cancer Coalition Distinguished Cancer Scholar.

Disclosure of potential conflicts of interest: All authors have nothing to declare.

First Published Online June 29, 2006

Abbreviations: AD, Androgen dependent; AI, androgen independent; AR, androgen receptor; DHT, 5{alpha}-dihydrotestosterone; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; GSK3ß, glycogen synthase kinase 3ß; LPA, lysophosphatidic acid; PIN, prostatic intraepithelial neoplasia, PSA, prostate-specific antigen; PTX, pertussis toxin; SSC, saline sodium citrate; YFP, yellow fluorescent protein.

Received December 21, 2005.

Accepted for publication June 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Moolenaar WH 1995 Lysophosphatidic acid, a multifunctional phospholipid messenger. J Biol Chem 270:12949–12952[Free Full Text]
  2. Mills GB, Moolenaar WH 2003 The emerging role of lysophosphatidic acid in cancer. Nat Rev Cancer 3:582–591[CrossRef][Medline]
  3. Aoki J, Taira A, Takanezawa Y, Kishi Y, Hama K, Kishimoto T, Mizuno K, Saku K, Taguchi R, Arai H 2002 Serum lysophosphatidic acid is produced through diverse phospholipase pathways. J Biol Chem 277:48737–48744[Abstract/Free Full Text]
  4. Tokumura A, Majima E, Kariya Y, Tominaga K, Kogure K, Yasuda K, Fukuzawa K 2002 Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase. J Biol Chem 277:39436–39442[Abstract/Free Full Text]
  5. Stracke ML, Clair T, Liotta LA 1997 Autotaxin, tumor motility-stimulating exophosphodiesterase. Adv Enzyme Regul 37:135–144[CrossRef][Medline]
  6. Gerrard JM, Robinson P 1989 Identification of the molecular species of lysophosphatidic acid produced when platelets are stimulated by thrombin. Biochim Biophys Acta 1001:282–285[Medline]
  7. Xu Y, Shen Z, Wiper DW, Wu M, Morton RE, Elson P, Kennedy AW, Belinson J, Markman M, Casey G 1998 Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers. JAMA 280:719–723[Abstract/Free Full Text]
  8. Xu Y, Gaudette DC, Boynton JD, Frankel A, Fang XJ, Sharma A, Hurteau J, Casey G, Goodbody A, Mellors A 1995 Characterization of an ovarian cancer activating factor in ascites from ovarian cancer patients. Clin Cancer Res 1:1223–1232[Abstract]
  9. Xie Y, Gibbs TC, Mukhin YV, Meier KE 2002 Role for 18:1 lysophosphatidic acid as an autocrine mediator in prostate cancer cells. J Biol Chem 277:32516–32526[Abstract/Free Full Text]
  10. Yamada T, Sato K, Komachi M, Malchinkhuu E, Tobo M, Kimura T, Kuwabara A, Yanagita Y, Ikeya T, Tanahashi Y, Ogawa T, Ohwada S, Morishita Y, Ohta H, Im DS, Tamoto K, Tomura H, Okajima F 2004 Lysophosphatidic acid (LPA) in malignant ascites stimulates motility of human pancreatic cancer cells through LPA1. J Biol Chem 279:6595–6605[Abstract/Free Full Text]
  11. Contos JJ, Ishii I, Chun J 2000 Lysophosphatidic acid receptors. Mol Pharmacol 58:1188–1196
  12. Anliker B, Chun J 2004 Cell surface receptors in lysophospholipid signaling. Semin Cell Dev Biol 15:457–465[CrossRef][Medline]
  13. Noguchi K, Ishii S, Shimizu T 2003 Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family. J Biol Chem 278:25600–25606[Abstract/Free Full Text]
  14. Yoshida A, Ueda H 1999 Activation of Gi1 by lysophosphatidic acid receptor without ligand in the baculovirus expression system. Biochem Biophys Res Commun 259:78–84[CrossRef][Medline]
  15. An S, Bleu T, Zheng Y, Goetzl EJ 1998 Recombinant human G protein-coupled lysophosphatidic acid receptors mediate intracellular calcium mobilization. Mol Pharmacol 54:881–888[Abstract/Free Full Text]
  16. Im DS, Heise CE, Harding MA, George SR, O’Dowd BF, Theodorescu D, Lynch KR 2000 Molecular cloning and characterization of a lysophosphatidic acid receptor, Edg-7, expressed in prostate. Mol Pharmacol 57:753–759[Abstract/Free Full Text]
  17. Goetzl EJ, Dolezalova H, Kong Y, Hu YL, Jaffe RB, Kalli KR, Conover CA 1999 Distinctive expression and functions of the type 4 endothelial differentiation gene-encoded G protein-coupled receptor for lysophosphatidic acid in ovarian cancer. Cancer Res 59:5370–5375[Abstract/Free Full Text]
  18. Furui T, LaPushin R, Mao M, Khan H, Watt SR, Watt MA, Lu Y, Fang X, Tsutsui S, Siddik ZH, Bast RC, Mills GB 1999 Overexpression of edg-2/vzg-1 induces apoptosis and anoikis in ovarian cancer cells in a lysophosphatidic acid-independent manner. Clin Cancer Res 5:4308–4318[Abstract/Free Full Text]
  19. Shida D, Kitayama J, Yamaguchi H, Okaji Y, Tsuno NH, Watanabe T, Takuwa Y, Nagawa H 2003 Lysophosphatidic acid (LPA) enhances the metastatic potential of human colon carcinoma DLD1 cells through LPA1. Cancer Res 63:1706–1711[Abstract/Free Full Text]
  20. Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, Thun MJ 2005 Cancer statistics, 2005. CA Cancer J Clin 55:10–30[Abstract/Free Full Text]
  21. Bostwick DG, Shan A, Qian J, Darson M, Maihle NJ, Jenkins RB, Cheng L 1998 Independent origin of multiple foci of prostatic intraepithelial neoplasia: comparison with matched foci of prostate carcinoma. Cancer 83:1995–2002[CrossRef][Medline]
  22. Daaka Y 2004 G proteins in cancer: the prostate cancer paradigm. Science STKE 2004:re2
  23. Kue PF, Daaka Y 2000 Essential role for G proteins in prostate cancer cell growth and signaling. J Urol 164:2162–2167[CrossRef][Medline]
  24. Bookout AL, Finney AE, Guo R, Peppel K, Koch WJ, Daaka Y 2003 Targeting Gß{gamma} signaling to inhibit prostate tumor formation and growth. J Biol Chem 278:37569–37573[Abstract/Free Full Text]
  25. Kue PF, Taub JS, Harrington LB, Polakiewicz RD, Ullrich A, Daaka Y 2002 Lysophosphatidic acid-regulated mitogenic ERK signaling in androgen-insensitive prostate cancer PC-3 cells. Int J Cancer 102:572–579[CrossRef][Medline]
  26. Raj GV, Sekula JA, Guo R, Madden JF, Daaka Y 2004 Lysophosphatidic acid promotes survival of androgen-insensitive prostate cancer PC3 cells via activation of NF-{kappa}B. Prostate 61:105–113[CrossRef][Medline]
  27. Guo C, Luttrell LM, Price DT 2000 Mitogenic signaling in androgen sensitive and insensitive prostate cancer cell lines. J Urol 163:1027–1032[CrossRef][Medline]
  28. Daaka Y 2002 Mitogenic action of LPA in prostate. Biochim Biophys Acta 1582:265–269[Medline]
  29. Sivashanmugam P, Tang L, Daaka Y 2004 Interleukin 6 mediates the lysophosphatidic acid-regulated cross-talk between stromal and epithelial prostate cancer cells. J Biol Chem 279:21154–21159[Abstract/Free Full Text]
  30. Taub JS, Guo R, Leeb-Lundberg LM, Madden JF, Daaka Y 2003 Bradykinin receptor subtype 1 expression and function in prostate cancer. Cancer Res 63:2037–2041[Abstract/Free Full Text]
  31. Urs N, Jones KT, Salo PD, Severin JE, Trejo J, Radhakrishna H 2005 A requirement for membrane cholesterol in the ß-arrestin- and clathrin-dependent endocytosis of LPA1 lysophosphatidic acid receptors. J Cell Sci 118:5291–5304[Abstract/Free Full Text]
  32. Shenoy SK, Lefkowitz RJ 2003 Multifaceted roles of ß-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling. Biochem J 375:503–515[CrossRef][Medline]
  33. Zhang J, Barak LS, Anborgh PH, Laporte SA, Caron MG, Ferguson SS 1999 Cellular trafficking of G protein-coupled receptor/ß-arrestin endocytic complexes. J Biol Chem 274:10999–11100[Abstract/Free Full Text]
  34. Kang J, Shi Y, Xiang B, Qu B, Su W, Zhu M, Zhang M, Bao G, Wang F, Zhang X, Yang R, Fan F, Chen X, Pei G, Ma L 2005 A nuclear function of ß-arrestin1 in GPCR signaling: regulation of histone acetylation and gene transcription. Cell [Erratum (2006) 124:645] 123:833–847
  35. Yang M, Zhong WW, Srivastava N, Slavin A, Yang J, Hoey T, An S 2005 G protein-coupled lysophosphatidic acid receptors stimulate proliferation of colon cancer cells through the ß-catenin pathway. Proc Natl Acad Sci USA 102:6027–6032[Abstract/Free Full Text]
  36. Heinlein CA, Chang C 2004 Androgen receptor in prostate cancer. Endocr Rev 25:276–308[Abstract/Free Full Text]
  37. Tuxhorn JA, Ayala GE, Rowley DR 2001 Reactive stroma in prostate cancer progression. J Urol 166:2472–2483[CrossRef][Medline]
  38. DeMarzo AM, Nelson WG, Isaacs WB, Epstein JI 2003 Pathological and molecular aspects of prostate cancer. Lancet 361:955–964[CrossRef][Medline]
  39. Radhika V, Hee Ha J, Jayaraman M, Tsim ST, Dhanasekaran N 2005 Mitogenic signaling by lysophosphatidic acid (LPA) involves G{alpha}12. Oncogene 24:4597–4603[CrossRef][Medline]
  40. Kasbohm EA, Guo R, Yowell CW, Bagchi G, Kelly P, Arora P, Casey PJ, Daaka Y 2005 Androgen receptor activation by Gs signaling in prostate cancer cells. J Biol Chem 280:11583–11589[Abstract/Free Full Text]



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