SPHINGOSINE 1-PHOSPHATE RECEPTOR 4 USES HER2 (ERBB2) TO REGULATE EXTRACELLULAR SIGNAL REGULATED KINASE-1/2 IN MDA-MB-453 BREAST CANCER CELLS

We demonstrate here that the bioactive lipid sphingosine 1-phosphate (S1P) uses the sphingosine 1-phosphate receptor 4 (S1P 4 ) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 This was based on several of evidence.

2 tumourigenesis, and blocked apoptosis of MCF-7 cells induced by anti-cancer drugs, sphingosine and tumour necrosis factor alpha (16). SK1 and S1P are also required for EGF-induced MCF-7 migration, proliferation and cell survival (18). S1P also stimulates breast cancer cell growth through activation of the serum response element and indirectly by enhancing IGF-II synthesis and function (19).
The HER2/neu/c-erbB-2 gene encodes a 185 kDa transmembrane receptor tyrosine kinase, which is related to other members of the EGF receptor family (20). Moreover, the over-expression of HER2/neu is found in up to 30% of primary breast cancers and increased tumour invasion, poor prognosis and therapeutic resistance is correlated with its expression (21). A soluble ligand for HER2 has not been identified, although HER2 operates as a shared receptor subunit of other ErbBs. In this regard, HER2 is a heterodimerisation partner of the EGF receptor (22). HER2 delays EGF dissociation from it receptor, improves coupling of EGF receptor and stimulation of the ERK-1/2 pathway, and impedes EGF receptor down-regulation. Thus, HER2 is a master regulator that drives epithelial cell proliferation. An example of this is evident from studies which demonstrate that the ectopic expression of HER2/neu in MCF-7 (estrogen receptor (ER) positive/HER2 negative) cells stimulates the PI3K/Akt pathway and down regulates p53 (23), which increases the cell survival.
In this study, we have used MDA-MB-453 cells and HER2 + /ERbreast cancer tumour samples in order to investigate the role of S1P in regulating the ERK-1/2 pathway, which is well established as having a role in cancer metastasis.

Experimental Procedures
Materials-All general biochemicals were from Sigma-Aldrich (Poole, UK).
BioScript™ was from Bioline (London, UK). HER2 and S1P 2 and S1P 4 siRNA and anti-phosphorylated ERK1/2 antibody were from Santa Cruz (California, USA). EGF receptor siRNA was a gift from V. Natarajan (University of Chicago, USA). Anti-ERK2 and anti-HRP tyrosine phosphate antibodies were from BD Transduction Laboratories (Oxford, UK). Anti-HER2 antibody was from New England Biolabs (UK) Ltd (Hitchin, UK). S1P and phyto-S1P were from Avanti Polar Lipids ( Quantitative values were obtained from the threshold cycle value (Ct). GAPDH was quantified as an internal RNA control, and each sample was normalized on the basis of its GAPDH content. Samples were run in quadruplicate. Data are representative of three independent experiments. Transfection-HEK 293 and MDA-MB-453 cells were transfected with HA-tagged S1P 4 plasmid constructs using Lipofectamine TM 2000 reagent according to the manufacturer's instruction. Transfection was performed for 24 hours at 37°C before serum starvation for a further 24 hours prior to harvesting in Laemmli buffer and analysis by SDS-PAGE and western blotting. Western Blotting-Analysis of proteins by SDS-PAGE and western blotting was performed as previously described by us (24) using anti-phosphorylated ERK1/2, anti-ERK2, anti-HER2 and anti-HRP phosphotyrosine antibodies. Calcium Assays-Monitoring of S1P-evoked changes in intracellular Ca 2+ was done in HTC4 cells stably expressing S1P 1 , S1P 2 , S1P 3 , or S1P 4 receptors originally described by An et al. (10) which were generously provided by Dr. Edward Goetzl (UCSF). The assay protocol was identical to that described in our previous publication (25). Wild type HTC4 cells do not respond to S1P with changes in (Ca 2+ ) i . HTC4 cells stably expressing each receptor were plated onto black-wall clear-bottom 96-well plates (Corning Incorporated Life Sciences, Acton, MA) at a density of 5 X 10 4 cells/well and cultured overnight. The following day, the culture medium was replaced with modified Krebs buffer (120 mM NaCl, 5 mM KCl, 0.62 mM MgSO 4 , 1.8 mM CaCl 2 , 10 mM HEPES, 6 mM glucose, pH 7.4), and the cells were serum starved for 4 hours. Subsequently, cells were loaded with Fura-2 AM (Invitrogen, Carlsbad, CA) for 30 minutes in modified Krebs buffer containing 2% (v/v) pluronic acid. After incubating the cells with Fura-2 AM, the cells were rinsed with Krebs buffer and changes in the intracellular Ca 2+ concentration were monitored by determining the ratio of emitted light intensities at 520 nm in response to excitation at 340 and 380 nm using FLEXstation II (Molecular Devices, Sunnyvale, CA). Each well was monitored for 80 seconds. To test antagonist activity of the inhibitors, JTE013 and CAY10444, increasing concentrations of the inhibitors were mixed with a constant concentration of S1P, and added automatically after 15 seconds of baseline measurement. Each test was performed in quadruplicate. Significant difference between two experimental groups was determined by the Student's t-test at a P value of 0.05. Data were plotted and fitted a sigmoid function by using the nonlinear curve-fitting feature of KaleidaGraph (Synergy Software, Essex Junction, VT). Immunoprecipitation-The medium was removed and cells lysed in ice-cold immunoprecipitation buffer (1 ml) containing 20 mM TRIS/HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 1% (v/v) Nonidet P-40 (NP-40), 10% (v/v) glycerol, 1 mg/ml bovine serum albumin, 0.5 mM sodium orthovanadate, 0.2 mM phenylmethylsulfonyl fluoride, leupeptin and aprotinin (all protease inhibitors were at 10 µg/ml pH 8) for 10 min at 4 o C. The material was harvested, centrifuged at 22 000 g for 5 min at 4 o C and 200-400 µL of cell lysate supernatant (equalized for protein, 0.5-1 mg/ml) combined with 20 µl of protein A or G Sepharose and incubated for 20 min at 4 o c. The samples were centrifuged at 22 000 g and the supernatant taken for immunoprecipitation with anti-HER2 or anti-ERK-1/2 antibody (5 µg of anti-HER2 antibody or 3 µg of anti-ERK-1/2 antibody and 20-40 µl of one part immunoprecipitation buffer and one part protein A or G Sepharose CL4B respectively). After agitation for 2 h at 4 o C, the immune complex was by guest on July 9, 2020 http://www.jbc.org/ Downloaded from collected by centrifugation at 22 000 g for 15 s at 4 o C. Immunoprecipitates were washed twice with buffer A containing 10 mM HEPES, pH 7, 100 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml aprotinin and 0.5% (v/v) NP-40 and once in buffer A without NP-40. The immunoprecipitates were then combined with boiling sample buffer containing 62 mM TRIS HCl, pH 6.7, 1.25% (w/v) sodium dodecyl sulfate, 10% (v/v) glycerol, 3.75% (v/v) mercaptoethanol and 0.05% (w/v) bromophenol blue. The samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting.

MBP
Kinase Assay-Immunoprecipitates were combined with a phosphorylation cocktail containing 20mM Hepes pH 7, 5mM-MgCl 2 , 25µM ATP and 5µCi [γ-32P]ATP and 1µg Myelin Basic Protein and incubated at 30 o C for 10 min. Incubations were terminated by addition of boiling sample buffer and subjected to SDS-PAGE. Immunohistochemistry-The cell pellet slides were first dewaxed and rehydrated through a series of xylene and alcohol washes. Antigen retrieval was performed for S1P 2 by microwaving the slides under pressure in a TRIS EDTA buffer for 5 minutes (pH 8.0) and for S1P 4 in 10mM Citrate Buffer at 96 o C in for 20 minutes. Endogenous peroxidase was blocked using 3% hydrogen peroxide for 20 minutes and non-specific background staining was reduced by blocking with a 1:20 concentration of horse serum diluted in TRIS buffered saline for 30 minutes. The sections were incubated with the primary antibody for S1P 2 and S1P 4 (Exalpha, Shirley, MA, USA). Each antibody was incubated at a dilution of 1:100 at 4 0 C overnight. EnVision-HRP conjugate (DAKO, Cambridgeshire, UK) was used for signal amplification and positive staining was identified using 3, 3'-diaminobenzidine (DAB) chromagen (Vector Laboratories). The slides were then counterstained with haematoxylin and Scott's Tap Water Substitute before dehydration and mounting. Densitometry-Densitometric quantification of western blots was performed using the Molecular Analyst Software (Bio-Rad Laboratories Ltd).
Statistical analysis was performed with the GraphPad Prism software, using one-way ANOVA followed by Newman-Keuls post-hoc test.
Treatment of MDA-MB-453 cells with S1P stimulated the phosphorylation/activation of ERK-1/2, which was reduced by pre-treating these cells with pertussis toxin (PTX, which uncouples GPCR from G i ) (Fig 2a, b). These data suggest that S1P uses a heterotrimeric G-protein coupled receptor to regulate the ERK-1/2 pathway.
PTX did not significantly reduce EGF-induced activation of ERK-1/2 ( Fig. 2a, b, P>0.05 for EGF + PTX versus EGF). The identity of the S1P receptor involved in regulating the ERK-1/2 pathway was evaluated using pharmacological agents that demonstrate selectivity at S1P receptors. We used JTE013, which is reported to be an S1P 2 selective antagonist (28), and CAY10444 (29), which is an S1P 3 antagonist.
To further characterise the pharmacological specificity of JTE013 and CAY10444 we used HTC4 cells in which S1P 2, 3, 4 were separately stably expressed and intracellular calcium mobilisation was measured using the Fura-2 indicator dye. We found that JTE013 reduced S1P-stimulated calcium mobilisation in cells over-expressing S1P 2 , while CAY10444 was very weakly effective at concentrations >1μM (Fig. 3a). In contrast JTE013 had no effect on calcium mobilisation induced by S1P in cells over-expressing S1P 3 , while CAY10444 reduced this response (Fig. 3a), thereby confirming specificity of CAY10444 at S1P 3 . Surprisingly, we found that JTE013 can also function as an S1P 4 antagonist, as evidenced by results showing that JTE013 potently reduced S1P-stimulated calcium mobilisation (Ki=236.9 nM) in cells over-expressing S1P 4 , while CAY10444 was without effect (Fig. 3a). As there are no selective S1P 4 antagonists available, we used JTE013 as a tool to investigate the role of S1P 2/4 in mediating the effect of S1P on the activation of ERK-1/2 in MDA-MB-453 cells. We found that the S1P-induced activation of ERK-1/2 was substantially reduced by JTE013 and was not affected by CAY10444 (Fig. 3b, c). Confirmation that MDA-MB-by guest on July 9, 2020 http://www.jbc.org/ Downloaded from 453 cells lack functional S1P 1 was evidenced by the finding that the S1P 1 selective agonist SEW2871 was without effect on ERK-1/2 activation (Fig. 3d).
We also used specific siRNA approaches to knock down expression of the S1P 4 receptor in MDA-MB-453 cells. In this regard, we found that siRNA knock down of S1P 4 reduced the S1P-stimulated activation of ERK-1/2 ( Fig. 4a, b). We confirmed by QRT-PCR that S1P 4 siRNA reduced S1P 4 mRNA transcript and had no effect on S1P 2 or S1P 3 mRNA transcript (Fig. 4c). We also detected S1P 4 protein (M=42kDa) in lysates of MDA-MB 453 cells on western blots probed with anti-S1P 4 antibody and found that S1P 4 siRNA partially reduced expression (55 ± 12% reduction, n=5) of the protein (Fig. 4c). In addition, IHC of MDA-MB-453 cells with anti-S1P 4 antibody demonstrated that siRNA knock down of S1P 4 reduced immunostaining (Fig. 4d). We could also confirm increased immunostaining when MDA-MB-453 cells were transfected with S1P 4 plasmid construct (Fig.  4d). Collectively, these data confirm expression of S1P 4 protein in MDA-MB-453 cells, specificity of the antibody and effectiveness of the S1P 4 siRNA treatment.
In addition, we used FTY720 (which is phosphorylated by SK2 to FTY720 phosphate) and FTY720 phosphate, which is an agonist at S1P 1, 3, 4, 5 but not S1P 2 , and which stimulated activation of ERK-1/2 in MDA-MB-453 cells (Fig. 4f). Finally, we used phyto-S1P, which is an agonist at S1P 4 (30) and demonstrated that this agent also induced activation of ERK-1/2 (Fig. 4g), which was reduced by siRNA knockdown of S1P 4 (Fig. 4g), but not S1P 2 (Fig. 4e). S1P 4 knock down was more effective against phyto-S1P compared with S1P. This apparent anomaly might be explained by a model in which there is higher fractional receptor occupancy with phyto-S1P compared with S1P for ERK-1/2 activation, and where efficacy for each phyto-S1P bound S1P 4 receptor is less than for each S1P bound S1P 4 receptor. We conclude that S1P 4 mediates the effect of S1P on ERK-1/2 activation in MDA-MB-453 cells. S1P Receptor Functional Interaction with HER2. Evidence that a functional interaction occurs between S1P and HER2 was obtained by the finding that S1P, but not EGF, stimulated the tyrosine phosphorylation of HER2 and this was severely reduced by JTE013 (Fig. 5a, b). Evidence for the involvement of HER2 in regulating S1P 4 signaling to the ERK-1/2 pathway was demonstrated by results showing that siRNA knock down HER2 expression reduced S1P-stimulation of ERK-1/2 by ~ 50% compared to cells treated with scrambled siRNA (Fig. 6a, b). The incomplete reduction in ERK-1/2 activation might be due to residual HER2 expression after siRNA treatment. To confirm data obtained by western blotting with antiphospho ERK-1/2 antibody, we immunoprecipitated ERK-1/2 with anti-ERK-1/2 antibody and measured ERK-1/2 activity against MBP in the immunoprecipitates. This assay confirmed that S1P or EGF stimulated ERK-1/2 activity (Fig. 6a). Moreover, siRNA knock down of HER2 reduced the stimulation of ERK-1/2 activity by S1P and had no effect on the response to EGF (Fig. 6a). HER2 siRNA had no effect on cell integrity as assessed using an MTT assay (Fig. 6a). We also used the HER2 inhibitor, ErbB2 inhibitor II (4-(3-Phenoxyphenyl)-5-cyano-2H-1,2,3triazole), which is a cell permeable HER2 ATP binding kinase inhibitor and reduces phosphorylation of HER2 in MDA-MB-453 cells but not that of overexpressed EGFR in MDA-MB-468 cells, even at concentrations as high as 100µM (31). The inhibitor was discovered from a computer aided drug design approach and searched from molecule libraries. Modelling has also been used to demonstrate binding of the inhibitor in the HER2 ATP binding site (31).
We found that the pre-treatment of MDA-MB-453 cells with the ErbB2 inhibitor II reduced basal ERK-1/2 phosphorylation (Fig. 6c) suggesting a tonic influence of HER2 on this pathway. However, this does not explain the involvement of HER2 in S1P 4 receptor signaling, as S1P-stimulated ERK-1/2 activation was almost completely abolished by ErbB2 inhibitor II while the response to EGF was unaffected (Fig. 6c). The ability of ErbB2 inhibitor II to reduce basal ERK-1/2 activation differs from HER2 siRNA, which had no significant effect (Fig. 6a, b). These findings suggest that the knockdown of HER2 with siRNA, which is incomplete, might not be sufficient to ablate basal ERK-1/2 activation, but is able to reduce S1P signaling via S1P 4 . Thus, basal and S1Pstimulated ERK-1/2 activation may have different requirements for HER2 e.g. less HER2 is required to sustain the basal ERK-1/2 activation compared with the S1P-stimulated activation of ERK-1/2.
EGF Regulation of ERK-1/2 in MDA-MB-453 and MCF-7 Cells. HER2 is an orphan receptor tyrosine kinase and is the preferred dimerisation partner of the EGF receptor. However, we have demonstrated here that the EGF-induced activation of ERK-1/2 was not reduced by the siRNA knock down of HER2 (Fig. 6a,  b).
Thus, S1P and EGF function in a mutually exclusive manner. In addition, S1P does not use the EGF receptor tyrosine kinase to regulate ERK-1/2. Thus, the activation of ERK-1/2 by EGF was reduced by the EGF receptor tyrosine kinase inhibitor AG 1478 (Fig. 7a, b) and by siRNA knockdown of EGF receptor expression (Fig. 7c, d). However, the S1P-stimulated activation of ERK-1/2 was not modulated by AG 1478 (Fig. 7a, b) or by siRNA knock down of EGF receptor (Fig. 7c, d). Further evidence to support divergent signaling by S1P and EGF with respect to HER2 was the finding that unlike S1P, EGF did not induce the tyrosine phosphorylation of HER2 (Fig. 5a, b). S1P 3 has no functional role in terms of regulating ERK-1/2 signaling in response to S1P in MDA-MB-453 cells (Fig. 3b). On the contrary S1P 4 signaling appears to predominate. We therefore asked the question: what is the molecular organisation of signaling from the S1P 3 receptor in other breast cancer cell types that lack S1P 4 ? For this purpose we used a breast cancer cell line, ER + MCF-7 cells, where S1P 3 mRNA is abundantly expressed but where S1P 4 mRNA is absent (Fig. 8a). Moreover, S1P stimulated activation of ERK-1/2 is known to be mediated by the S1P 3 receptor and involves transactivation of the EGF receptor (S1P increases the tyrosine phosphorylation of the EGF receptor) in these cells (32). Indeed, we have confirmed that siRNA knockdown of S1P 3 or use of the S1P 3 antagonist, CAY10444, reduced the S1Pstimulated activation of ERK-1/2 in both MCF-7 Neo (express the Neo vector) and MCF-7 HER2 cells (HER218 cells, stably expressing HER2) by >90% (data not shown). We show here that the stimulation of ERK-1/2 by S1P in MCF-7 Neo and MCF-7 HER2 cells was reduced by pre-treating cells with AG 1478 (Fig. 8b, c). The higher basal ERK-1/2 activation in MCF-7 HER2 cells compared with MCF-7 Neo cells might be due to EGF receptor, as this was also reduced by AG 1478 (Fig. 8c). In conclusion, S1P/S1P 3 stimulation of ERK-1/2 is characterised by a requirement for EGF receptor tyrosine kinase activity in MCF-7 cells. In addition the S1P stimulation of ERK-1/2 in MCF-7 Neo (which lack HER2) and MCF-7 HER2 cells was unaffected by the HER2 kinase inhibitor, AG 879 (Fig. 8b, c).

DISCUSSION
We demonstrate here that S1P stimulates the ERK-1/2 pathway via a mechanism that involves HER2 and S1P 4 in MDA-MB-453 cells. This novel mechanism is based on several lines of evidence. First, we demonstrated that the S1P-induced activation of ERK-1/2 was reduced by JTE013, but not by CAY10444, an S1P 3 selective antagonist. We have shown here for the first time, that in addition to being an S1P 2 receptor antagonist, JTE013 is also a potent antagonist of S1P 4 , and can block S1P-induced mobilisation of calcium in cells over-expressing this receptor. This is an important finding because JTE013 is used widely and is considered to have a high-degree of specificity for S1P 2 . Second, siRNA knock down of S1P 4 reduced the activation of ERK-1/2 by S1P. Third, phyto-S1P, which is a selective S1P 4 agonist stimulated ERK-1/2 activation indicating that S1P 4 specific ligands are able to activate this kinase pathway in MDA-MB-453 cells. Moreover siRNA knock down of S1P 4 reduced the activation of ERK-1/2 by phyto-S1P. A role for S1P 2 in the regulation of ERK-1/2 is excluded based on results showing that: (i) siRNA knock down of S1P 2 had no effect on the stimulation of ERK-1/2 by either S1P or phyto-S1P; (ii) FTY720 (the phosphorylated equivalent of which does not bind to S1P 2 ) stimulated the activation of ERK-1/2. Three additional results provide evidence for a role of HER2 in regulating S1P 4 signaling in by guest on July 9, 2020 http://www.jbc.org/ Downloaded from

MDA-MB-453 cells.
First, the S1P-stimulated activation of ERK-1/2 was almost completely abolished by treatment of cells with ErbB2 inhibitor II. Second, the S1P-and phyto-S1P-stimulated activation of ERK-1/2 was reduced by the siRNA knock down of HER2 expression. Third, S1P stimulated the tyrosine phosphorylation of HER2, which was reduced by JTE013.
We also found that S1P stimulation of ERK-1/2 via S1P 4 in MDA-MB-453 cells does not involve participation of the EGF receptor, thereby excluding EGF release as a possible mechanism mediating the effects of S1P on this pathway. This contrasts with a role for EGF receptor down-stream of S1P 3 in MCF-7 cells (this study and (32)). Regarding S1P 4 /ERK-1/2 signalling in MDA-MB-453 cells, the lack of participation of EGF receptor is based on the finding that the EGF receptor tyrosine kinase inhibitor, AG 1478 and siRNA knock down of EGF receptor expression reduced the EGF-dependent activation of ERK-1/2, but failed to modulate the response to S1P. Moreover, EGF receptor does not require HER2 to activate the ERK-1/2 pathway based on results showing that EGF-stimulated activation of ERK-1/2 was not reduced by ErbB2 inhibitor 2 or siRNA knock down of HER2. Furthermore, EGF failed to stimulate the tyrosine phosphorylation of HER2. Taken together, these findings demonstrate that S1P and EGF function in a mutually exclusive manner with S1P using S1P 4 and HER2 to regulate ERK-1/2, while the EGF stimulation of this kinase pathway does not involve HER2.
Transactivation of the EGF receptor represents the paradigm for cross-talk between GPCRs and RTK signaling pathways leading to regulation of ERK-1/2. Geschwind et al (33) demonstrated in a variety of squamous cell carcinoma cell lines of the head and neck, that treatment with the GPCR agonists LPA, bradykinin, thrombin, and carbachol resulted in rapid tyrosine phosphorylation of the EGF receptor and concomitant activation of the ERK-1/2 pathway. In addition, these authors reported that the activation of the ERK-1/2 pathway by LPA was dependent both on metalloproteinase function and EGF receptor tyrosine kinase activity (33). Furthermore, Schaffer et al. (34) demonstrated that GPCR ligands induced tyrosine phosphorylation of EGF receptor as well as downstream signaling events such as recruitment of the adapter protein Shc and activation of ERK-1/2, JNK and p38. Moreover, these authors reported that EGF receptor transactivation involves amphiregulin, HB-EGF and TGFα. These growth factors are released as a consequence of the action of metalloproteinases ADAM 10, 15 and 17 and are cell context specific. Shida and colleagues (35) demonstrated that S1P induces rapid and transient phosphorylation of the EGF receptor via a G i -and matrix metalloproteinase-dependent mechanism in MKN28 and MKN74 cells. S1P also induces rapid tyrosine phosphorylation of the HGF receptor (c-Met) in these cells, although, in contrast with EGF receptor transactivation, this involves a matrixmetalloproteinase-and G i -independent mechanism. JTE013 reduced the S1P-stimulated tyrosine phosphorylation of EGF receptor and c-Met. However, our findings concerning the specificity of JTE013, suggest that there is a need to evaluate whether the S1P 4 receptor is involved this process. The EGF receptor tyrosine kinase inhibitor, AG 1478 was shown to have no effect on the S1P-stimulated tyrosine phosphorylation of c-Met, thereby excluding EGF receptor as an intermediate between S1P 2 and c-Met. Shida and colleagues also demonstrated that LPA and S1P induce the tyrosine phosphorylation of HER2 in MKN28 and MKN74 cells and this was dependent upon metalloproteinase-dependent release of EGF receptor ligands (36).
With respect to the studies described above our findings clearly describe a novel mechanism of HER2 transactivation and stimulation of the ERK-1/2 pathway in response to S1P in MDA-MB-453 cells. In this regard, we have previously demonstrated that GPCR and RTK can form functional signaling units that result in the RTK enhancing stimulation of the ERK-1/2 pathway by the respective GPCR ligand (24,37,38). For instance, we demonstrated that S1P 1 and PDGFβ receptor form a functional signaling unit, where the PDGFβ receptor tyrosine kinase enhances S1P stimulation of ERK-1/2 mediated by S1P 1 (24,37). It is therefore possible that S1P 4 and HER2 might form similar signaling units to regulate the ERK-1/2 pathway in MDA-MB-453 cells. In this case, the tyrosine phosphorylation of HER2 in response to S1P might produce a signaling platform that enables recruitment of regulatory/adaptor proteins via their SH2 interaction with phosphotyrosines on HER2 and which may facilitate stronger activation of the ERK-1/2 pathway in response to S1P. This possibility requires formal testing.
We have also shown that heregulin does not use either S1P 2/4 or S1P 3 to regulate the ERK-1/2 pathway. In addition, the potential release of heregulin in by guest on July 9, 2020 http://www.jbc.org/ Downloaded from response to S1P is unlikely because heregulin signaling is sensitive to AG 1478 and therefore requires EGF receptor, while S1P responses are insensitive to AG 1478.
In conclusion, we have demonstrated that the magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by an oncogene-HER2 and in an ERbreast cancer cell line. This is unusual as S1P 4 expression and function is largely restricted to lymphoid cells such as T-cells (39). Therefore, S1P 4 expression and function may exhibit some promiscuity in cancer cells. In addition, the interaction between S1P 4 and HER2 may provide new targets for drug intervention to reduce breast cancer progression. Further research is required to validate this possibility. In addition, JTE013 can be considered a potent antagonist of S1P 4 , providing both a useful tool for interrogating the function of this receptor in breast cancer, and also as a prototype for further compound optimisation and translational approaches to target S1P 4 in ER -/HER2 + breast cancer.  (a) The western blot shows the effect of PTX on S1P-and EGF-stimulated activation of ERK-1/2. Images are from the same western blot. Phosphorylated ERK-1/2 was detected on western blots probed with antiphosphorylated ERK-1/2 antibody. Blots were also probed with anti-ERK-1/2 antibody to ensure equal protein loading; (b) bar chart quantifying the effect of PTX on ERK-1/2 activation. Results are expressed P-ERK-1/2:ERK-2 ratios for n=3 experiments.  (d), phosphorylated ERK-1/2 was detected on western blots probed with anti-phosphorylated ERK-1/2 antibody. Blots were also probed with anti-ERK-1/2 antibody to ensure equal protein loading. In (c) results are expressed P-ERK-1/2:ERK-2 ratios for n=3 experiments. Fig. 4 The effect of S1P, FTY720, FTY720 phosphate and phyto-S1P on the ERK-1/2 pathway and role of S1P 2/4 receptor in MDA-MB-453 cells. MDA-MB-453 cells were treated with scrambled siRNA or S1P 4 siRNA (200nM, 48 hours) or S1P 2 siRNA (100nM, 48 hours) prior to stimulation with and without S1P (1μM). Cells were also stimulated with FTY720 or FTY720 phosphate or phyto-S1P (at the indicated concentration) for 10 minutes. Western blots showing: (a) the effect of siRNA knock down of S1P 4 on S1Pstimulated activation of ERK-1/2; (b) bar chart showing quantification of the effect of S1P 4 siRNA on the S1P-induced activation of ERK-1/2 activation; (c) quantification by QRT-PCR of S1P 2, 3, 4 mRNA expression in cells treated with scrambled or S1P 4 siRNA. Also shown is a western blot probed with anti-S1P 4 antibody to show siRNA knock down of S1P 4 in MDA-MB-453 cells (n=5 cell samples). HEK-HA-S1P 4 are samples from HA-S1P 4 over-expressing HEK 293 cell; (d) IHC staining showing the effect of S1P 2 siRNA and S1P 4 siRNA on the expression of S1P 2 and S1P 4 protein respectively in MDA-MB-453 cells. Cells were also costained with haematoxylin. AB denotes S1P 2 or S1P 4 antibody. Also shown is S1P 4 AB immunostaining of MDA-MB-453 cells transfected with S1P 4 plasmid construct; (e) the lack of effect of S1P 2 siRNA on S1P-or phyto-S1P (5μM)-stimulated activation of ERK-1/2; (f) the effect of FTY720 (5μM), (R) -and (S)-FTY720 phosphate (each at 5μM) on ERK-1/2 activation; (g) the effect of siRNA knock down of S1P 4 on phyto-S1P (5μM)-stimulated activation of ERK-1/2. Also included is a bar chart showing the quantification of the effect of S1P 4 siRNA on phyto-S1P-induced activation of ERK-1/2. Results in (b) and (g) are expressed P-ERK-1/2:ERK-2 ratios for n=3 experiments. Results in (a), (e), (f) and (g) are representative of three separate experiments. Phosphorylated ERK-1/2 was detected on western blots probed with anti-phosphorylated ERK-1/2 antibody. ERK-2 was also detected with anti-ERK-2 antibody either on reprobes or in the same samples run on a separate SDS-PAGE to ensure comparable protein loading.  MDA-MB-453 cells were pre-treated with scrambled siRNA or a HER2 siRNA (100 or 400nM, 48 hours) to knock down expression of HER2 or ErbB2 inhibitor II (at indicated concentrations) for 10 min prior to stimulation with and without S1P (at the indicated concentrations) or EGF (50ng/ml) or phyto-S1P (1 or 5μM) or FTY720 (5μM) for 10 minutes. (a) western blots showing the effect of siRNA (400nM) knock down of HER2 on S1P-(1μΜ and 5μΜ) and EGF-stimulated activation of ERK-1/2. In addition, cell lysates were immunoprecipitated with anti-ERK-1/2 antibody and analysed for ERK-1/2 activity with MBP as the substrate. The autoradiograph shows the siRNA knock down of HER2 reduces S1P-(5μΜ, 10 min) but not EGF-stimulated activation of ERK-1/2 (upper panel). Total ERK-2 inputs are also shown in the lower panel. The bar chart demonstrates quantification of the kinase assay using Cherenkov counting (p<0.01 for S1Pstimulated HER2 siRNA-treated cells versus S1P-stimulated scrambled siRNA-treated cells, n=4). Also shown is the siRNA knock down of HER2 (Mr=185kDa) detected on western blots probed with anti-HER2 antibody. The bar chart shows that siRNA knock down of HER2 had no effect on cell viability as assessed in the MTT assay; (b) bar chart showing quantification of the effect of siRNA knockdown of HER2 on S1P (1μΜ)-and EGF-stimulated ERK-1/2 activation; (c) western blot showing the effect of ErbB2 inhibitor II on S1P (5μM)-stimulated activation of ERK-1/2 and lack of effect on the response to EGF. Also shown; is a bar chart of the effect of ErbB2 inhibitor II on S1P-and EGF-stimulated ERK-1/2 activation; (d) western blot showing the effect of siRNA (100nM) knock down of HER2 on phyto-S1P-(1μΜ) stimulated activation of ERK-1/2. Also included is a bar chart showing quantification of the effect of HER2 siRNA on the phyto-S1P-induced activation of ERK-1/2 activation. These are representative results from three separate experiments.