A role for L-alpha-lysophosphatidylinositol and GPR55 in the modulation of migration, orientation and polarization of human breast cancer cells

Abstract

Background and purpose: Increased circulating levels of L-alpha-lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G-protein-coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55.

Experimental approach: Quantitative RT-PCR was used to measure GPR55 expression in breast cancer cell lines. Cell migration and invasion were measured using a Boyden chamber chemotaxis assay and Cultrex invasion assay, respectively. Cell polarization and orientation in response to the microenvironment were measured using slides containing nanometric grooves.

Key results: GPR55 expression was detected in the highly metastatic MDA-MB-231 breast cancer cell line. In these cells, LPI stimulated binding of [(35)S]GTPgammaS to cell membranes (pEC(50) 6.47 +/- 0.45) and significantly enhanced cell chemotaxis towards serum. MCF-7 cells expressed low levels of GPR55 and did not migrate or invade towards serum factors. When GPR55 was over-expressed in MCF-7 cells, serum induced a robust migratory and invasive response, which was further enhanced by LPI and prevented by siRNA to GPR55. The physical microenvironment has been identified as a key factor in determining breast tumour cell metastatic fate. LPI endowed MDA-MB-231 cells with the capacity to detect shallow (40 nm deep) grooved slides and induced marked cancer cell polarization on both flat and grooved surfaces.

Conclusions and implications: LPI and GPR55 play a role in the modulation of migration, orientation and polarization of breast cancer cells in response to the tumour microenvironment.

Figure 1

GPR55 expression in breast cancer cell lines. (A) Histogram showing expression levels of GPR55 in MDA-MB-231 and MCF-7 cell lines. Data are expressed as the mean ± SEM (n= 3). **P < 0.01, analysed by unpaired t-test. (B) LPI stimulated [³⁵S]GTPγS binding to cell membranes from MDA-MB-231 cells with an E_max of 51 ± 8 % (pEC₅₀ 6.47 ± 0.45); an effect that was attenuated in the presence of CBD (pEC₅₀ 5.5 ± 0.38) (data are mean ± SEM, n= 6). (C) CBD alone had no effect on basal [³⁵S]GTPγS binding to cell membranes from MDA-MB-231 cells (1 nM–1 µM); at 10 µM the compound produced a stimulation of 27.5 ± 9%. CBD, cannabidiol; LPI, L-α-lysophosphatidylinositol.

Figure 2

Histograms showing the migration of MDA-MB-231 cells in a Boyden chamber transwell assay. (A) Cells migrating towards LPI or FBS placed in lower wells of the chamber; (B) cells that have been pre-incubated with LPI (1 µM) migrating towards FBS placed in lower chamber; (C) cells that have pre-incubated with LPI and CBD migrating towards FBS placed in the lower chamber; (D) cells that have been pre-incubated with CBD (1 µM) migrating towards FBS placed in the lower chamber. The data represent mean ± SEM (n= 3–4). *P < 0.05; ***P < 0.001; n/s, not significant; one-way anova followed by Newman–Keuls multiple comparison tests. CBD, cannabidiol; FBS, foetal bovine serum; LPI, L-α-lysophosphatidylinositol.

Figure 3

Histograms showing the migration of MCF-7 cells over-expressing GPR55 in a Boyden chamber transwell assay and Cultrex® 24-well BME invasion assay. (A) Detection of HA-GPR55 expression in MCF-7 cells by confocal microscopy using an anti-HA antibody. Green: HA-GPR55; Red: Nuclei. (B) Detection of HA-GPR55 over-expression in MCF-7 cells, and knock down using siRNA to GPR55, by flow cytometry. (C) MCF-7 cells over-expressing GPR55 migrated towards 10% FBS, whereas empty vector transfected cells did not. In GPR55 over-expressing cells, FBS-induced migration was significantly enhanced by pre-incubation of cells with LPI (1 µM), and was completely abolished using siRNA to GPR55. (D) MCF-7 cells over-expressing GPR55 invaded towards 10% FBS, whereas empty vector transfected cells did not. The data represent mean ± SEM (n= 3). *P < 0.05; ***P < 0.001, one-way anova followed by Newman–Keuls multiple comparison tests. FBS, foetal bovine serum; LPI, L-α-lysophosphatidylinositol.

Figure 5

Histograms showing that MDA-MB-231 cells (A) become polarized (elongated) in response to 520 nm deep grooves, with a significant enhancement of polarity in response to LPI on both the flat and grooved surface, (B) do not elongate in response to 40 nm deep grooves, but elongate in the presence of LPI, (C) almost fully align in response to 520 nm deep nanogrooved slides, and (D) do not align in response to 40 nm deep grooves when vehicle-treated, but align in response to these grooves following incubation with LPI. Data are the mean ± SEM, n= 58–97 cells (from three separate experiments). (E) Representative phase contrast images of MDA-MB-231 cells cultured on nanogrooved slides showing vehicle-treated and LPI-treated MDA-MB-231 cells on flat surface, and on 4 µm wide, 40 nm deep grooves. *P < 0.05; **P < 0.01; ***P < 0.001, one-way anova followed by Newman–Keuls multiple comparison tests. LPI, L-α-lysophosphatidylinositol.

Figure 4

(A) Image showing a polarized MDA-MB-231 cell on grooved substratum, red: actin, green: tubulin. (B) Diagram representing the analysis of the effects of grooved substrata on cancer cells. Cell 1 is orientated at 90° relative to groove direction and is unaligned with an orientation index (OI) of +1. Cell 2 is orientated at 0° relative to groove direction and is fully aligned with an OI of −1. Cell 3 is orientated at 45° relative to groove direction with an OI of 0. The cell elongation (polarization) is represented as the length/width ratio.

Figure 6

Histograms showing that (A) MCF-7 cells expressing GPR55 display a significantly greater degree of alignment to grooved substrata (1 µm wide, 520 nm deep) after 4 h than empty vector (EV) transfected cells, which display significantly less alignment than MDA-MB-231 cells. Data are the mean ± SEM, n= 39–76 cells. *P < 0.05; ***P < 0.001. (B) Polarization of MCF-7 cells expressing GPR55 is not significantly different from empty vector transfected cells; MDA-MB-231 cells display significantly greater elongation as compared with MCF-7 cells. ***P < 0.001, analysed by Students's unpaired t-test. (C) Representative images of MCF-7 cells artificially expressing GPR55, or EV transfected control cells, stained for HA-GPR55 (green) and actin (red). (i) HA-GPR55-expressing cells on flat surface, wide-view; (ii) vector-transfected cells on flat surface, wide-view; (iii and iv) HA-GPR55-expressing cells aligned along grooves; (v) HA-GPR55-expressing cells on flat surface; (vi and vii) vector-transfected cells on grooves. White arrows indicate groove direction.