Cross-talk between LPA1 and epidermal growth factor receptors mediates up-regulation of sphingosine kinase 1 to promote gastric cancer cell motility and invasion

Abstract

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lysophospholipid mediators of diverse cellular processes important for cancer progression. S1P is produced by two sphingosine kinases, SphK1 and SphK2. Expression of SphK1 is elevated in many cancers. Here, we report that LPA markedly enhanced SphK1 mRNA and protein in gastric cancer MKN1 cells but had no effect on SphK2. LPA also up-regulated SphK1 expression in other human cancer cells that endogenously express the LPA(1) receptor, such as DLD1 colon cancer cells and MDA-MB-231 breast cancer cells, but not in HT29 colon cancer cells or MDA-MB-453 breast cancer cells, which do not express the LPA(1) receptor. An LPA(1) receptor antagonist or down-regulation of its expression prevented SphK1 and S1P(3) receptor up-regulation by LPA. LPA transactivated the epidermal growth factor receptor (EGFR) in these cells, and the EGFR inhibitor AG1478 attenuated the increased SphK1 and S1P(3) expression induced by LPA. Moreover, down-regulation of SphK1 attenuated LPA-stimulated migration and invasion of MNK1 cells yet had no effect on expression of neovascularizing factors, such as interleukin (IL)-8, IL-6, urokinase-type plasminogen activator (uPA), or uPA receptor induced by LPA. Finally, down-regulation of S1P(3), but not S1P(1), also reduced LPA-stimulated migration and invasion of MKN1 cells. Collectively, our results suggest that SphK1 is a convergence point of multiple cell surface receptors for three different ligands, LPA, EGF, and S1P, which have all been implicated in regulation of motility and invasiveness of cancer cells.

Figure 1

LPA up-regulates SphK1 and S1P₃ expression. A, MKN1 cells were stimulated with LPA (10 μmol/L) for the indicated times. RNA was isolated and reverse transcribed, and SphK1, SphK2, and GAPDH mRNA levels were measured by real-time PCR. Data are expressed as fold change after normalization to GAPDH. B, left, MKN1 cells were stimulated without or with LPA (10 μmol/L) for the indicated times, cell lysates were prepared, and equal amounts of protein were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against SphK1 and phospho-SphK1 (P-SphK1). Blots were stripped and reprobed with anti-tubulin to insure equal loading and transfer. Asterisks, nonspecific immunostained bands; arrowheads, SphK1. Right, MKN1 cells transfected with siControl, siSphK1, or siSphK2 were lysed, and equal amounts of proteins were immunoblotted with anti-SphK1 or anti-tubulin antibodies. SphK1 and phospho-SphK1 levels were normalized to tubulin, and the ratios relative to untreated control (left) or siControl (right) are indicated. C, MKN1 cells were stimulated without or with LPA (10 μmol/L) for the indicated times and SphK1 and SphK2 activities were measured in whole-cell lysates with isozyme-specific assays as described in Materials and Methods. Columns, mean; bars, SD. Data are expressed as pmol S1P produced per mg protein per min. Similar results were obtained in two additional experiments. *, P ≤ 0.01. D, MKN1 cells were stimulated with the indicated concentrations of LPA for 6 h. RNA was isolated and reverse transcribed, and SphK1, SphK2, S1P_1-3, and GAPDH mRNA levels were measured by real-time PCR. Data are expressed as fold change after normalization to GAPDH.

Figure 2

LPA up-regulates SphK1 and S1P₃ expression via the LPA₁ receptor. A, DLD1 and HT29 colon cancer cells and MDA-MB-231 and MDA-MB-453 breast cancer cells were stimulated without or with 10 μmol/L LPA for 6 or 9 h. RNA was isolated and reverse transcribed, and SphK1 and GAPDH mRNA levels were measured by real-time PCR. Data are expressed as fold change after normalization to GAPDH. B, MKN1 cells were pretreated with vehicle (DMSO) or Ki16425 (Ki; 1 or 10 μmol/L) for 30 min and then stimulated without or with the indicated concentrations of LPA for 6 h. RNA was isolated and reverse transcribed, and SphK1 and GAPDH mRNA levels were measured by real-time PCR. Data are expressed as fold change after normalization to GAPDH. C and D, MKN1 cells were transfected with ON-TARGETplus SMARTpool siRNA targeted to LPA₁,LPA₂, and LPA₃ receptors or control siRNA. C, RNA was isolated and reverse transcribed, and LPA₁, LPA₂, LPA₃, and GAPDH mRNA levels were measured by real-time PCR. Data are expressed as fold change after normalization to GAPDH. Bottom, duplicate cultures were stimulated without or with the indicated concentrations of LPA for 6 h. RNA was isolated and reverse transcribed, and SphK1 and GAPDH mRNA levels were measured by real-time PCR. Data are expressed as fold change after normalization to GAPDH. D, MKN1 cells were pretreated with vehicle (DMSO) or Ki16425 (1 or 10 μmol/L) for 30 min. Duplicate MKN1 cultures were transfected with control siRNA (white columns) or siRNA targeted to LPA₁ (stippled columns). Cells were then stimulated without or with 10 μmol/L LPA for 6 h. RNA was isolated and S1P₃ and GAPDH mRNA levels were measured by real-time PCR. Data are expressed as fold change after normalization to GAPDH.

Figure 3

Activation of EGFR is involved in LPA-induced SphK1 expression. A, MKN1 cells were stimulated with 10 μmol/L LPA for the indicated times and lysed, and equal amounts of protein were analyzed by immunoblotting with antibodies against phosphotyrosine (PY) or phospho-EGFR (Tyr¹⁰⁶⁸). Blots were stripped and reprobed with EGFR antibody to confirm equal loading. B, MKN1 cells were stimulated with the indicated concentrations of EGF for 6 h or 10 ng/mL EGF for the indicated times. RNA was isolated and reverse transcribed, and SphK1 and GAPDH mRNA levels were measured by real-time PCR. Data are expressed as fold changes after normalization to GAPDH. C, MKN1 cells were pretreated with vehicle (DMSO; white columns) or 0.5 μmol/L AG1478 (black columns) for 30 min and then stimulated with 10 μmol/L LPA or 10 ng/mL EGF for 6 h. RNA was isolated and reverse transcribed, and SphK1, S1P₃, and GAPDH mRNA levels were measured by real-time PCR. SphK1 and S1P₃ mRNA levels are expressed as fold changes after normalization to GAPDH. *, P ≥ 0.01.

Figure 4

Down-regulation of SphK1 attenuates LPA-induced migration and invasion of MKN1 cells. A, MKN1 cells transfected with ON-TARGETplus SMARTpool siRNA targeted to SphK1 (gray columns) or control siRNA (white columns) were stimulated without or with 10 μmol/L LPA for 4 h, as indicated. mRNA levels of SphK1 and GAPDH were determined and data are expressed as fold change. SphK1 protein expression was analyzed by Western blotting with specific SphK1 antibodies. Blots were stripped and reprobed with anti-tubulin to insure equal loading and transfer. Asterisk, nonspecific immunostained bands; arrowhead, SphK1. B, MKN1 cells were transfected with ON-TARGETplus SMARTpool siRNA targeted to SphK1 (siSphK1 #1; hatched columns), control siRNA #1 (white columns), siSphK1 #2 (gray columns), or control siRNA #2 (black columns). Cultures were wounded and migration of cells into the wound was measured 6 h after stimulation without or with 1 μmol/L LPA or with 10 ng/mL EGF. Columns, mean of migrating cells per field; bars, SD. Left, representative images of a typical experiment. C and D, MKN1 cells transfected with control siRNA or siRNA targeted to SphK1 were allowed to migrate for 6 h through fibronectin-coated filters (C) or invade through Matrigel-coated filters (D) toward vehicle (None), 1 μmol/L LPA, 1 μmol/L S1P, or 10 ng/mL EGF. Columns, mean number of migrating cells per field; bars, SD. *, P ≥ 0.01; **, P ≥ 0.05.

Figure 5

Down-regulation of SphK1 has no effect on LPA-induced expression of IL-8, IL-6, uPA, or uPAR. MKN1 cells transfected with control siRNA (white columns) or siRNA targeted to SphK1 (gray columns) were stimulated with 10 μmol/L LPA for the indicated times. RNA was isolated and reverse transcribed, and IL-8, IL-6, uPA, uPAR, SphK1, and GAPDH mRNA levels were measured by real-time PCR. Data are expressed as fold change after normalization to GAPDH.

Figure 6

Involvement of S1P₃ receptor in chemotaxis toward LPA. A, MKN1 cells were pretreated for 30 min with vehicle (white columns), 0.3 μmol/L VPC23019 (VPC; gray columns), or 10 μmol/L VPC23019 (stippled columns) and then allowed to migrate for 6 h through fibronectin-coated filters or invade through Matrigel-coated filters toward vehicle (None), 1 μmol/L LPA, or 1 μmol/L S1P. Columns, mean number of migrating cells per field; bars, SD. B and C, MKN1 cells were transfected with control siRNA (white columns), siS1P₁ (hatched columns), or siS1P₃ (black columns). B, RNA was isolated and reverse transcribed, and S1P₁, S1P₃, and GAPDH mRNA levels were measured by real-time PCR. Data are expressed as fold change after normalization to GAPDH. C, cells were allowed to migrate for 6 h through fibronectin-coated filters or invade through Matrigel-coated filters toward vehicle (None), 1 μmol/L LPA, or 1 μmol/L S1P. Columns, mean number of migrating cells per field; bars, SD. *, P ≥ 0.01. D, scheme depicting the complex signaling interplay between LPA receptors and EGFRs and up-regulation of SphK1 and their roles in LPA-induced migration and invasion of gastric cancer cells. See text for more details. For simplicity, many other known signaling pathways downstream of LPA receptors and EGFRs are not shown. It is unlikely that SphK1 is the sole mechanism by which LPA induces migration. HB-EGF, heparin-binding EGF; MMP, matrix metalloproteinase.