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[Cancer Research 59, 4732-4737, September 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 4732-4737, September 15, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Dual Mechanisms for Lysophospholipid Induction of Proliferation of Human Breast Carcinoma Cells1

Edward J. Goetzl2, Hana Dolezalova, Yvonne Kong and Li Zeng

Departments of Medicine and Microbiology-Immunology, University of California Medical Center, San Francisco, California 94143-0711


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) Edg-1, Edg-3, and Edg-5 bind sphingosine 1-phosphate (S1P), and Edg-2 and Edg-4 Rs bind lysophosphatidic acid (LPA). LPA and S1P initiate ras- and rho-dependent signaling of cellular growth. Cultured lines of human breast cancer cells (BCCs) express Edg-3 > Edg-4 > Edg-5 > or = Edg-2, without detectable Edg-1, by both assessment of mRNA and Western blots with rabbit and monoclonal mouse anti-Edg R antibodies. BCC proliferation was stimulated significantly by 10-9 M to 10-6 M LPA and S1P. Luciferase constructs containing the serum response element (SRE) of growth-related gene promoters reported mean activation of BCCs by LPA and S1P of up to 85-fold. LPA and S1P stimulated BCC secretion of type II insulin-like growth factor (IGF-II) by 2–7-fold, to levels at which exogenous IGF-II stimulated increased proliferation and SRE activation of BCCs. All BCC responses to LPA and S1P were suppressed similarly by pertussis toxin, mitogen-activated protein kinase kinase inhibitors, and C3 exoenzyme inactivation of rho, suggesting mediation by Edg Rs. Monoclonal anti-IGF-II and anti-IGFR1 antibodies suppressed proliferation and SRE reports of BCCs to LPA and S1P by means of up to 65%. Edg Rs thus transduce LPA and S1P enhancement of BCC growth, both directly through SRE and indirectly by enhancing the contribution of IGF-II.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The lysolipid phosphate mediators LPA3 and S1P are generated enzymatically from membrane lipid precursors of many different types of normal and malignant cells (1 , 2) . Extracellular LPA and S1P both stimulate cellular proliferation, differentiation, survival, adhesion, aggregation, and other specific functions (3, 4, 5) . A recently characterized subfamily of at least five G protein-coupled receptors, which are encoded by edgs, bind and transduce signals from LPA or S1P (6, 7, 8, 9, 10) . Two homology clusters with greater structural similarity and shared ligand specificity are composed of the edg-encoded G protein-coupled receptors (Edg Rs) Edg-1, Edg-3, and Edg-5 set of S1P Rs and Edg-2 and Edg-4 LPA Rs. The capacity of LPA and S1P to improve cellular survival is in part a result of suppression of apoptosis by several distinct mechanisms (11 , 12) . LPA and S1P stimulate cellular proliferation directly by eliciting the serum response factor and ternary complex factor transcription factors, which together bind to and activate the SRE in promoters of many immediate-early genes (13) . The involvement of SRE-dependent mechanisms in mediating LPA and S1P enhancement of proliferation has not been examined carefully in malignant cells, nor has the possibility of effects of LPA and/or S1P on polypeptide growth factors necessary for optimal tumor growth.

Functional Edg receptors and proliferative responses to LPA and S1P thus were characterized in the ER-positive MCF-7 cultured line of human BCCs and the MDA-MB-453 ER-negative line of BCCs. The relative contributions of direct SRE-dependent induction of transcription and of enhancement of production of IGF-II in proliferative responses to LPA and S1P also were determined in these BCCs.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemical Reagents and Antibodies.
The sources of chemicals were: S1P and sphingosine (Biomol, Plymouth Meeting, PA); LPA, phosphatidic acid, 1-ß-D-galactosyl-sphingosine (psychosine), and fatty acid-free BSA (Sigma Chemical Co., St. Louis, MO); and human IGF-II (Peprotech, Inc., Rocky Hill, NJ). Cells were treated with PTX (Calbiochem, Inc., La Jolla, CA), recombinant Clostridium botulinum C3 ADP-ribotransferase (C3 exoenzyme; List Biological Laboratories, Inc., Campbell, CA), which ADP-ribosylates rho specifically, and the MEK inhibitor 2'-amino-3'-methoxyflavone (PD98059; Calbiochem) as described (10 , 14) . Mouse monoclonal antibodies specific for substituent peptides of human Edg-3 (amino acids 1–21), Edg-4 (amino acids 9–27), and Edg-5 (amino acids 303–322) have been described (12 , 15) , the immunogens for which were selected from sequences of high homology among humans and rodents. The expected cross-reaction with corresponding rodent Edg Rs has been confirmed by the identical recognition of human and rat Edg-5 Rs. The cross-reactivity of each antibody with heterologous Edg proteins was <1%, as determined by Western blots of 0.1–100 µg of membrane proteins isolated from HTC4 rat hepatoma cells stably transfected with human Edg-2, Edg-3, Edg-4, or Edg-5 (12 , 15) . Each monoclonal IgG was purified by protein A affinity-chromatography (Pierce Chemical Co.) and used to develop Western blots at 0.1–0.3 µg/ml (15) . A mouse monoclonal IgG1 that specifically neutralizes activity of human/rat IGF-II, but not IGF-I (Upstate Biotechnology, Inc., Lake Placid, NY), and a mouse monoclonal antibody, termed {alpha}-IR3, which blocks binding of IGF-II to IGFR1 (Oncogene Science, Cambridge, MA), were purchased. A rabbit polyclonal antiserum to rodent and human Edg-2 was kindly provided by Dr. Jerold Chun (University of California–San Diego, San Diego, CA).

Cell Culture and Quantification of Cellular Proliferation.
Layers of ER-positive MCF-7 (ATCC # HTB-22) and ER-negative MDA-MB-453 (ATCC# HTB-131) human BCCs were cultured in DMEM with 4.5 g/100 ml of glucose, 10% FBS, 100 units/ml of penicillin G, and 100 µg/ml of streptomycin (complete DMEM) to 100% confluence and relayered every 3–4 days to 25–30% confluence. To assess proliferation, replicate layers of 1 x 104 BCCs were cultured in 48-well plates in complete DMEM for 4 h, washed once, and cultured for 20 h in serum-free DMEM. Some wells were pretreated with PTX for 6 h, C3 exoenzyme for 30 h, or MEK inhibitor for 2 h. Antisera were added, followed in 1 h by lipid stimuli and incubation for 72 h. Then wells were washed two times with Ca2+- and Mg2+-free Hanks’ solution, and the cells were harvested in 0.2 ml of EDTA-trypsin solution for staining with trypan blue and eosin and quantification by microscopic counting of 10 1-mm3 fields in a hemocytometer.

Reverse Transcription-PCR Analysis of Edg Rs.
Total cellular RNA was extracted by the TRIzol method (Life Technologies, Inc., Grand Island, NY), from suspensions of BCCs and lines of stably transfected rat HTC4 hepatoma cells, that all had low background expression of native Edg Rs, and each overexpressed one recombinant human Edg R. A Superscript kit (Life Technologies, Inc.) was used for reverse transcription synthesis of cDNAs. PCR began with a "hot start" at 94°C for 3 min; Taq DNA polymerase was added, and amplification was carried out with 35 cycles of 30 s at 94°C, 2 min at 55°C, and 1 min at 72°C. Two µCi of [{alpha}-32P]dCTP were added to some sets of reaction mixtures to allow quantification of mRNA encoding each Edg R relative to that of the standard G3PDH (16) . Oligonucleotide primer pairs were: G3PDH, 5'-dCCTGGCCAAGGTCATCCATGACAAC and 5'-dTGTCATACCAGGAAATGAGCTTGAC; Edg-1, 5'-CTACACAAAAAGCTTGGATCACTCA and 5'-CGACCAAGTCTAGAGCGCTTCCGGT (1100 bp); Edg-2, 5'-dGCTCCACACACGGATGAGCAACC and 5'-GTGGTCATTGCTGTGAACTCCAGC (621 bp); Edg-3, 5'-dCAAAATGAGGCCTTACGACGCCA and 5'-dTCCCATTCTGAAGTGCTGCGTTC (701 bp); Edg-4, 5'-dAGCTGCACAGCCGCCTGCCCCGT and 5'-dTGCTGTGCCATGCCAGACCTTGTC (775 bp); and Edg-5, 5'-CTCTCTACGCCAAGCATTATGTGCT and 5'-ATCTAGACCCTCAGACCACCGTGTTGCCCTC (500 bp). PCR products were resolved by electrophoresis in a 2 g/100 ml agarose gel with ethidium bromide staining. G3PDH and Edg R cDNA bands were cut from gels and solubilized for ß-scintillation counting in 0.5 ml of sodium perchlorate solution at 55°C for 1 h (Elu-Quick; Schleicher and Schuell, Keene, NH). Initially, the G3PDH cDNA templates in several different-sized portions of each sample were amplified to determine volumes that would result in G3PDH bands of equal intensity for each sample. Relative quantities of cDNA encoding each Edg R also were calculated by the ratio of radioactivity to that in the corresponding G3PDH band (16) .

Western Blots.
Replicate suspensions of 1 x 107 BCCs, which had been incubated without or with LPA or S1P for 16 h, were washed three times with 10 ml of cold Ca2+- and Mg2+-free PBS, resuspended in 0.3 ml of cold 10 mM Tris-HCl (pH 7.4) containing a protease inhibitor mixture (Sigma Chemical Co., St. Louis, MO), 0.12 M sucrose, and 5% glycerol (v/v). After homogenization with a Teflon pestle on ice for 2 min at 250 rpm, each sample was centrifuged at 400 x g for 5 min at 4°C, and the supernatant was centrifuged at 300,000 x g for 30 min at 4°C. Each 300,000 x g pellet was resuspended in 0.2 ml of 10 mM Tris-HCl (pH 7.4) with 1% (v/v) NP40, 5% glycerol, and protease inhibitor mixture and rehomogenized and incubated at 4°C for 2 h prior to centrifugation again at 300,000 x g. Aliquots of supernatant containing 1–100 µg of protein were mixed with 4x Laemmli’s solution, heated to 100°C for 3 min, and electrophoresed in an SDS-12% polyacrylamide gel for 20 min at 100 V and 1.5 h at 140 V, along with a rainbow prestained set of molecular weight markers (DuPont NEN, Boston, MA or Amersham, Inc., Arlington Heights, IL). Proteins in each gel were transferred electrophoretically to a nitrocellulose membrane (Hybond; Amersham) for sequential incubation with 5 g% reconstituted nonfat milk powder to block unspecific sites, dilutions of mouse monoclonal anti-Edg R antibody, and then horseradish peroxidase-labeled goat anti-mouse IgG, prior to development with a standard ECL kit (Amersham).

RIA and Dot-Blot Quantification of IGF-II.
RIAs were conducted according to the instructions of Research and Diagnostic Antibodies, Inc. (Berkeley, CA), after removal of some IGF binding proteins by Sep-Pak chromatography (Millipore Corp., Milford, MA), as directed (17) . Dot-blot quantification of IGF-II was performed using a method in which binding proteins do not alter immunoreactivity of IGF-II in unprocessed cellular secretions (18) .

Transfections and Reporter Assay.
Replicate suspensions of 0.3–1 x 105 MCF-7 and MDA-MB-453 BCCs in 1 ml of complete DMEM were cultured in 12-well plates for 24 h to establish monolayers of 40–50% confluency. The monolayers were washed twice and covered with 1 ml of serum-free DMEM and lipotransfected with 100 ng/well of a SRE firefly luciferase reporter plasmid (8) and 5 ng/well of pRL-CMV Renilla luciferase vector (Promega Corp., Madison, WI) using FuGENE 6 (Boehringer Mannheim Corp., Indianapolis, IN). After 30 h of incubation, medium was replaced with fresh serum-free DMEM and anti-IGFR1 or anti-IGF-II mouse monoclonal antibodies or IgG1 isotype control was added, followed in 2 h by 10-10 M to 10-6 M LPA, S1P, or other lipids in serum-free DMEM with 0.1 mg/ml of fatty acid-free BSA. Some wells were pretreated with PTX for 6 h, C3 exoenzyme for 30 h, or MEK inhibitor for 2 h. After 4 h of incubation at 37°C, the luciferases were extracted in Reporter lysis buffer (Promega), and their activities were quantified sequentially by luminometry using Luciferase Assay and Stop & Glo reagents (Promega), with integration of light emitted during the 15 s after addition of each reagent (EG & G Berthold microplate luminometer, model LB96V). Firefly luciferase values were corrected for differences in apparent transfection efficiency by expression as a ratio with Renilla luciferase signals in the corresponding samples.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
BCC Expression of Edg Receptors.
mRNA encoding individual Edg Rs had been detected by Northern blotting in some human tumor cells (7, 8, 9) . The relative levels of mRNA encoding each of the Edg Rs in BCCs now have been semiquantified by RT-PCR (Fig. 1)Citation . Several different amounts of first-strand cDNAs prepared from MCF-7 and MDA-MB-453 BCCs were amplified initially to allow selection of a volume of each that provided equally intense cDNA bands for the internal standard G3PDH. With this standard approach, the mRNA from both human BCC lines was found to encode similarly high levels of Edg-3 R but had no detectable Edg-1 R message (Fig. 1)Citation . The ER-negative MDA-MB-453 BCCs had higher levels of mRNA encoding the Edg-2 R, whereas the ER-positive MCF-7 BCCs had higher levels of mRNA for Edg-4 and Edg-5.



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  		Fig. 1. RT-PCR semiquantification of mRNA encoding Edg Rs in MCF-7 and MDA-MB-453 cells. The volume of cDNA mixture from each type of BCC was selected to equalize the level of amplified G3PDH cDNA product. Lanes 1, 3, 5, 7, and 9 are from MDA-MB-453 cells, and Lanes 2, 4, 6, 8, and 10 are from MCF-7 cells. Lanes 1 and 2, Edg-1; Lanes 3 and 4, Edg-2; Lanes 5 and 6, Edg-3; Lanes 7 and 8, Edg-4; Lanes 9 and 10, Edg-5. The number below each lane represents the ratio of 32P in cDNA for an Edg R to that for G3PDH.

 
RadioPCR has been used to assess levels of mRNA specific for other G protein-coupled Rs, but not Edg Rs (16) . Thus, an initial study examined mRNA from four lines of rat HTC4 hepatoma cells, which were stably transfected with individual human Edg Rs 2 to 5 (Table 1)Citation . The rank order of levels of mRNA for endogenous Edg Rs in HTC4 cells prior to transfection was Edg-2 >> Edg-3 > Edg-4 > Edg-5, without detectable Edg-1 mRNA. The level of mRNA for the transfected Edg R in each line was much higher than background (Table 1)Citation . In this frame of reference, the levels of BCC mRNA encoding Edg-3 were nearly as high as the index transfectant and > Edg-4 > Edg-5 >= Edg-2, without any Edg-1 mRNA. The differences in relative amounts of mRNA for each Edg R between the two lines of BCCs were the same as for standard PCR (Fig. 1)Citation .


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  		Table 1 Relative levels of mRNA encoding Edg receptors in BCCs

 
Western blots developed with polyclonal anti-Edg-2 R and monoclonal anti-Edg-3, anti-Edg-4, and anti-Edg-5 antibodies showed one predominant protein of expected size in extracts of each of the four lines of HTC4 cell transfectants (Fig. 2)Citation . Electrophoresis of over three times more protein from untransfected control HTC4 cells than transfectants did not show Edg-3, Edg-4, or Edg-5 protein antigen, but a faint band of Edg-2 protein was detected that might reflect the higher endogenous levels of mRNA encoding this R (Table 1)Citation . The results of BCC Western blots confirmed expression of Edg proteins representing both LPA R and S1P R subtypes, with a predominance of Edg-3 R in both BCC lines (Fig. 2)Citation . In contrast to expectations from PCR results, however, MCF-7 BCCs had higher levels of Edg-2 as well as Edg-4 and Edg-5 proteins than MDA-MB-453 BCCs. The Edg-4 R protein of both BCC lines was consistently Mr 2000–3000 smaller than the recombinant human Edg-4 R protein, but the basis for the difference has not yet been elucidated.



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  		Fig. 2. Western blot analysis of the expression of Edg-2, Edg-3, Edg-4, and Edg-5 Rs by MCF-7 and MDA-MB-453 BCCs. The four samples analyzed for content of each Edg R are: H, 3 µg of protein extracted from HTC4 rat liver cells that were stably transfected with the respective Edg Rs; C, 10 µg of protein from control untransfected HTC4 cells; CF, 10 µg of protein from MCF-7 BCCs; and DA, 10 µg of protein from MDA-MB-453 BCCs. Blots were developed with rabbit anti-Edg-2 antiserum and anti-Edg-3, anti-Edg-4, and anti-Edg-5 mouse monoclonal antibodies. The marginal lines show the positions of Mr 45,000 and Mr 66,000 protein molecular weight markers.

 
Functional and Biochemical Responses of BCCs to LPA and S1P.
The proliferation of both lines of BCCs was assessed by counting viable cells after 72 h (Fig. 3)Citation . Proliferation of MCF-7 BCCs was increased significantly by 10-8 M to 10-6 M LPA and S1P to maximum levels similar to those attained by 2% FBS. In parallel studies of MDA-MB-453 BCCs, proliferative responses to LPA and S1P were similar to those of MCF-7 BCCs, with significant increases evoked by 10-8 M to 10-6 M LPA and S1P (Fig. 3)Citation .



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  		Fig. 3. Stimulation of proliferation of MCF-7 and MDA-MB-453 BCCs by LPA and S1P. Columns, means of the results of three studies performed in duplicate; bars, SD. FBS is the 2% FBS-positive control. The serum-free medium alone controls (100%) were 1.5, 1.4, and 1.5 x 104/well in the three studies of MCF-7 BCC proliferation and 1.1, 1.2, and 1.1 x 104/well in the three studies of MDA-MB-453 BCC proliferation. The levels of significance of increases above medium control proliferation were determined by a paired Student t test; *, P < 0.01.

 
Activation of SRE in the promoters of diverse growth-related genes is a fundamental characteristic of the growth-promoting potential of LPA and S1P. BCCs thus were transfected with an SRE-firefly luciferase construct and 1/20 the amount of a Renilla luciferase-CMV construct as an internal standard for consistency of transfection. LPA and S1P increased the mean levels of standardized luciferase luminometric activity in ligand concentration-dependent relationships by maxima of up to 37-fold and 85-fold, respectively, in MCF-7 BCCs (Fig. 4)Citation . Similar responses to the same concentrations of LPA and S1P were detected in MDA-MB-453 BCCs, where the respective mean maxima were 24-fold and 26-fold.



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  		Fig. 4. SRE reporter assay of LPA and S1P stimulation of human BCCs. Columns, means of the results of three studies performed in duplicate; bars, SD. The medium alone control values were 1272, 957, and 352 luminometer units for MCF-7 BCCs and 269, 715, and 1401 for MDA-MB-453 BCCs. The statistical methods and symbols are the same as in Fig. 3Citation , except that + = P < 0.05.

 
Pharmacological inhibitors known to suppress one or more components of the pathways by which Edg Rs signal nuclear events were applied in BCCs transfected with the SRE-luciferase reporter. Suppression of Gi protein activity by PTX, the ras-mitogen-activated protein kinase pathway by a MEK inhibitor, and the rho pathway by C3 exoenzyme all substantially decreased nuclear signals from Edg receptors in both types of BCCs (Table 2)Citation .


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  		Table 2 Pharmacological inhibition of LPA and S1P signaling to the SRE-Luciferase reporter in BCCs

 
Enhancement of BCC Secretion of IGF-II by LPA and S1P.
Functional prominence of the IGF-II/IGFR1 system in many breast cancers suggested the possibility that part of the stimulation of proliferation of some lines of BCCs by LPA and/or S1P might be attributable to augmentation of secretion of IGF-II by one or both lysophospholipid mediators. Secretion of radioimmunoreactive IGF-II by MCF-7 BCCs was enhanced significantly by 10-9 M to 10-6 M LPA and S1P in concentration-dependent relationships where the maximal effects were attained by 10-6 M LPA and 10-8 M and 10-7 M S1P (Fig. 5)Citation . In two of the studies, neither 10-10 M LPA nor S1P affected release of IGF-II. At 10-6 M, but not 10-8 M, the phosphatidic acid and sphingosine biochemical precursors of LPA and S1P enhanced secretion of IGF-II with marginal statistical significance. A dot-blot immunoassay for IGF-II, which eliminates the blocking activity of IGF-binding proteins, gave similar results for MCF-7 BCCs. With 10-7 M LPA, 10-6 M LPA, 10-8 M S1P, and 10-7 M S1P, MCF-7 BCC-derived IGF-II was increased to respective means of 2.6-, 3.2-, 4.7-, and 5.5-fold above a mean unstimulated level of 2.2 ng/ml. Stimulation of MCF-7 BCC secretion of IGF-II by LPA and S1P was inhibited by PTX, MEK inhibition, and C3 exoenzyme sufficiently to implicate Gi and both the ras and rho pathways of signaling by the Edg receptors (Table 3)Citation . A greater involvement of signaling through the ras-raf-mitogen-activated protein kinase pathway than rho pathways may be predicted based on the higher effectiveness of the MEK inhibitor than C3 exoenzyme.



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  		Fig. 5. Stimulation by LPA and S1P of MCF-7 BCC secretion of IGF-II. Columns, means of the results of three studies; bars, SD. Secretion of IGF-II in medium alone was 1.7, 3.0, and 4.1 ng/ml in the three studies. The statistical methods and symbols are the same as in Fig. 4Citation , except that # = P = 0.05.

 

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  		Table 3 Pharmacological inhibition of LPA and S1P enhancement of MCF-7 BCC secretion of IGF-II

 
The level of secretion of IGF-II by LPA- and S1P-stimulated MDA-MB-453 BCCs was much lower than that by MCF-7 cells, and it was not possible to quantify accurately the very low IGF-II concentrations attained by unstimulated MDA-MB-453 cells. With 10-6 M LPA and 10-7 M S1P, the levels of IGF-II secreted by MDA-MB-453 BCCs attained means of 1.2 and 2.0 ng/ml, respectively. Because stimulated levels of IGF-II from MDA-MB-453 BCCs were only one-fifth of those from MCF-7 BCCs or lower and unstimulated levels were not reliably detectable, subsequent studies focused only on IGF-II mechanisms in MCF-7 BCCs. The capacity of human synthetic IGF-II to stimulate BCC proliferation, at concentrations in the range attained by incubation of MCF-7 BCCs with LPA and S1P, was examined to assess functional relevance of the observed endogenous increases. IGF-II increased MCF-7 BCC proliferation significantly, as determined by increases in cell counts after 72 h. MCF-7 BCC counts were increased by 1, 3, 10, and 30 ng/ml of IGF-II to respective means of 152, 234, 316, and 388% (n = 2) of serum-free medium control. The same range of concentrations of synthetic IGF-II also activated SRE in MCF-7 BCCs, as detected in the reporter assay (Table 4)Citation . The increases in SRE signal above control level were significant for all concentrations of IGF-II examined, and the increment in SRE signal attained by each higher concentration compared with the next lower concentration also was significant. The reduction in LPA-induced SRE signal by immunoneutralization of IGF-II was similar in magnitude to the maximum increase elicited by IGF-II alone (Table 4)Citation .


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  		Table 4 Activation of SRE-Luciferase reporter in MCF-7 BCCs by IGF-II

 
Suppression of MCF-7 BCC Responses to LPA and S1P by Anti-IGF-II and Anti-IGFR1 Antibodies.
MCF-7 BCCs were preincubated with a range of concentrations of an IgG1 mouse neutralizing monoclonal anti-IGF II antibody, prior to introduction of 10-7 M LPA and S1P. The neutralizing antibody to IGF-II suppressed significantly both proliferative responses and SRE-luciferase reporter responses with antibody concentration dependence, whereas isotype-matched control IgG1 had no effect (Fig. 6)Citation . The effects of anti-IGFR1 antibody, which blocks binding of IGF-II to IGFR1, were examined in relation to the stimulatory effects of 10-7 M S1P on MCF-7 BCCs. At 1, 3, and 10 µg/ml, anti-IGFR1 antibody suppressed S1P-stimulated proliferation of MCF-7 BCCs, as assessed with cell counts, by means ± SD (n = 3) of 20 ± 4.6%, 32 ± 4.0%, and 41 ± 3.6% (P < 0.01 for all), respectively. At 3, 10, and 30 µg/ml, anti-IGFR1 antibody suppressed S1P-stimulated activation of the SRE-luciferase reporter in MCF-7 BCCs by means ± SD (n = 3) of 36 ± 7.8%, 47 ± 7.8%, and 51 ± 7.6% (P < 0.01 for all), respectively. In contrast, the IgG isotype control had no significant inhibitory effect, and anti-IGFR1 antibody did not suppress unstimulated proliferation of MCF-7 BCCs.



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  		Fig. 6. Suppression of MCF-7 BCC responses to LPA and S1P by a neutralizing anti-IGF-II mouse monoclonal antibody. Columns, means of the results of three studies performed in duplicate; bars, SD. The control (0% inhibition) responses to 10-7 M LPA and S1P are shown in Figs. 3Citation and 4Citation . The statistical methods and symbols are the same as in Fig. 4Citation .

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IGF-I and IGF-II potently stimulate proliferation of many types of normal and malignant cells (19 , 20) . The IGFR1 is a heterotetrameric complex with tyrosine kinase activity that binds and transduces signals from IGF-I and IGF-II similarly (21) . IGFR2 differs structurally from IGFR1, lacks signal transduction functions, and does not mediate cellular proliferation (22) . IGF-II is the predominant form in human cultured BCCs, stimulates BCC proliferation through IGFR1, and decreases the estrogen growth requirement of ER-positive BCCs (23) . Estrogen is a potent stimulus of proliferation of ER-positive BCCs that concurrently enhances expression and secretion of IGF-II by such lines of BCCs (19) . However, the possibility that the IGF system may not have a major role in estrogen enhancement of proliferation of some ER-positive BCCs was suggested by the lack of inhibition of estrogen stimulation when IGFR1 was blocked by a neutralizing monoclonal antibody (24) . In contrast, stimulation of proliferation of BCCs by the lysolipid phosphate growth factors LPA and S1P appears to be mediated in part by IGF-II but is not dependent on the expression of ERs.

The ER-positive MCF-7 cells and ER-negative MDA-MB-453 cells both express Edg-2 and Edg-4 Rs for LPA and Edg-3 and Edg-5 Rs, but not Edg-1 Rs, for S1P, with quantitative differences in the respective levels (Figs. 1Citation and 2Citation ; Table 1Citation ). Significant ligand concentration-dependent stimulation of BCC proliferation by LPA and S1P was observed with both lines, irrespective of ER status (Fig. 3)Citation . Signaling of transcription of growth-related genes, as assessed by prominent enhancement of SRE-coupled luciferase activity, was increased significantly by proliferation-stimulating concentrations of LPA and S1P in both MCF-7 and MDA-MB-453 BCCs (Fig. 4)Citation . The suppression of SRE-coupled reporter responses to LPA and S1P by PTX and by inhibition of MEK and rho, in a pattern characteristic of signal transduction by Edg Rs, confirms the presence of functional Edg Rs in both BCC lines (Table 2)Citation .

LPA and S1P both significantly enhanced secretion of immunoreactive IGF-II by MCF-7 cells up to respective peaks 4- and 5-fold higher than control levels (Fig. 5)Citation . IGF-II secretion evoked by 10-7 M LPA or S1P was suppressed significantly by PTX and MEK inhibition and less significantly by C3 exoenzyme inactivation of rho, which also is consistent with Edg R mediation (Table 3)Citation . The role of IGF-II was explored first by investigating the stimulation of proliferation and SRE-luciferase activity in MCF-7 BCCs by a range of concentrations of purified synthetic IGF-II (Table 4)Citation . At concentrations elicited by LPA or S1P, the synthetic IGF-II evoked greater proliferation and SRE-luciferase activity than at concentrations attained by unstimulated MCF-7 BCCs. The role of native IGF-II was confirmed by defining the effects of neutralizing antibodies to IGF-II and IGFR1 on growth and SRE-reporter responses to 10-7 M LPA and S1P (Fig. 6)Citation . Both responses of MCF-7 cells were inhibited by means of up to 55 and 65%, respectively, without an effect of non-antibody isotype-identical IgG (Fig. 6)Citation . Thus, a substantial part of the stimulation of growth of some BCCs by LPA and S1P depends on increased release of IGF-II and its capacity to induce BCC proliferation.

A tentative integration of the present findings suggests distinctive functions for lysolipid phosphate mediators in BCC biology. At concentrations usually attained in serum and in some inflammatory and malignant exudates and plasma (1 , 25 , 26) , LPA and S1P both exert dual effects on BCC proliferation. First, the SRE-luciferase responses not inhibited by anti-IGF-II or anti-IGFR1 neutralizing antibodies represent either direct nuclear signaling through Edg Rs or possibly the actions of other non-IGF protein growth factors elicited by the lysolipid phosphate mediators and capable of activating SRE. Second, LPA and S1P enhance generation and/or release of IGF-II by the BCCs, irrespective of ER expression. The results of preliminary analyses of LPA and S1P production by BCCs showed very low endogenous levels, which would not have functional relevance. The sources of LPA and S1P, therefore, are likely to be cells other than the target BCCs, and these lysolipid phosphate growth factors thus would not appear to be autocrine stimuli in breast cancer. Rather, this class of mediators may function both as paracrine growth factors and by setting thresholds for secretory responses of one or more autocrine protein growth factors.


   ACKNOWLEDGMENTS
 
We are grateful to Bethann Easterly for expert preparation of graphics.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 These studies were supported by Grant HL-31809 from the NIH and a grant (to E. J. G.) and fellowship (to H. D.) from the Department of the Army. Back

2 To whom requests for reprints should be addressed, at Immunology and Allergy, UB8B, Box 0711, University of California Medical Center, 533 Parnassus, San Francisco, CA 94143-0711. Phone: (415) 476-5339; Fax: (415) 476-6915; E-mail: egoetzl{at}itsa.ucsf.edu Back

3 The abbreviations used are: LPA, lysophosphatidic acid; S1P, sphingosine 1-phosphate; Edg, endothelial differentiation gene; SRE, serum response element; ER, estrogen receptor; BCC, breast cancer cell; MEK, mitogen-activated protein kinase kinase; PTX, pertussis toxin; IGF-II, type II insulin-like growth factor; IGFR, IGF receptor; FBS, fetal bovine serum; RT-PCR, reverse transcription-PCR; G3PDH, glyceraldehyde 3-phosphate dehydrogenase. Back

Received 5/ 6/99. Accepted 7/20/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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