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. 2020 Jan 25;12(2):289.
doi: 10.3390/cancers12020289.

GPER Activation Inhibits Cancer Cell Mechanotransduction and Basement Membrane Invasion via RhoA

Alistair Rice  1 , Ernesto Cortes  1 , Dariusz Lachowski  1 , Philipp Oertle  2 , Carlos Matellan  1 , Stephen D Thorpe  3 , Ritobrata Ghose  1   4 , Haiyun Wang  5 , David A Lee  3 , Marija Plodinec  2 , Armando E Del Río Hernández  1
Affiliations

GPER Activation Inhibits Cancer Cell Mechanotransduction and Basement Membrane Invasion via RhoA

Alistair Rice et al. Cancers (Basel). .

Abstract

The invasive properties of cancer cells are intimately linked to their mechanical phenotype, which can be regulated by intracellular biochemical signalling. Cell contractility, induced by mechanotransduction of a stiff fibrotic matrix, and the epithelial-mesenchymal transition (EMT) promote invasion. Metastasis involves cells pushing through the basement membrane into the stroma-both of which are altered in composition with cancer progression. Agonists of the G protein-coupled oestrogen receptor (GPER), such as tamoxifen, have been largely used in the clinic, and interest in GPER, which is abundantly expressed in tissues, has greatly increased despite a lack of understanding regarding the mechanisms which promote its multiple effects. Here, we show that specific activation of GPER inhibits EMT, mechanotransduction and cell contractility in cancer cells via the GTPase Ras homolog family member A (RhoA). We further show that GPER activation inhibits invasion through an in vitro basement membrane mimic, similar in structure to the pancreatic basement membrane that we reveal as an asymmetric bilayer, which differs in composition between healthy and cancer patients.

Keywords: G protein-coupled receptors; cancer biomechanics; metastasis; tumour microenvironment.

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Conflict of interest statement

The authors declare no conflict of interest

Figures

Figure 1
Figure 1
G protein-coupled oestrogen receptor (GPER) expression and correlation with survival in cancer (a) RNA-seq data from cancer types downloaded from TCGA cBioPortal (http://www.cbioportal.org/index.do), 11/2017 and 2/2018. Gene abundance quantified as RNA-seq by Expectation Maximisation). p values from Wilcoxon rank-sum test. BRCA—breast invasive carcinoma, LIHC—liver hepatocellular carcinoma, LUAD—lung adenocarcinoma, STAD—stomach adenocarcinoma, UCEC—uterine corpus endometrial carcinoma, KICH—kidney chromophobe, STES—stomach and esophageal carcinoma, and COADREAD—colorectal adenocarcinoma. Number of patients/normal—BRCA (1093/112), LIHC (371/50), LUAD (515/59), STAD (415/35), UCEC (545/35), KICH (66/25), STES (599/46), COADREAD (623/51). (b) Survival curves for cancer patients, divided into high and low expression determined using median gene expression of GPER. p values from Kaplan–Meier statistical test. PDAC—pancreatic ductal adenocarcinoma, and KIRC—Kidney renal clear cell carcinoma. For PDAC, BRCA, UCEC and KIRC, n = 177, 1090, 543, 532 patients respectively. (c) Relapse-free probability curves for PDAC and KIRC cancer patients. High and low expression determined using median gene expression of GPER. p value from Kaplan–Meier statistical test, For PDAC, KIRC n = 138, 434 patients. (d) Immunofluorescence images of GPER (green), actin (red), and DAPI (blue) in Suit2-007 cells. Scale bar = 25 µm. (e) Immunofluorescence images of GPER (green), actin (red), and DAPI (blue) in PC-3 cells. Scale bar = 25 µm. (f) Immunofluorescence images of GPER (green), cytokeratin 19 (red) and DAPI (blue) in PDAC patients. Scale bar = 100 µm. (g) Western blot for GPER and total protein in untreated SUIT2 cells (Control), SUIT2 cells with siRNA to GPER (siGPER) and HEK293 cells. Quantification of GPER (ab154069) normalised to total protein. Mean ± s.e.m. with individual values overlaid (n = 3); one-way ANOVA with Dunnett pairwise comparisons. ** p < 0.01, *** p < 0.001. Full blot images in Supplementary Figure S1.
Figure 2
Figure 2
Mechanosensing in Suit2-007 cells is inhibited by GPER activation. (a) Diagram of magnetic tweezers setup with fibronectin-coated magnetic beads attached to cells, and tweezers placed at 40 µm lateral distance from bead. (b) Relative bead displacement for 1st and 12th pulses for bead profiles where displacement values for 12th pulse < 1st pulse, indicating mechanotransduction. For 1st pulse, error bars = s.e.m. for normalised displacement of bead during 1st pulse of pulsatile regime. For 12th pulse, error bars = s.e.m. for normalised bead displacement values relative to their respective 1st pulse (i.e., values between 0 and 1). For both 1st and 12th pulse, for control, G1, siRNA GPER + G1, and G1 + RhoA (Ras homolog family member A) rescue, n = 25, 16, 28 and 30 cells respectively. Mann–Whitney test comparing each 12th pulse mean to G1 12th pulse mean, * p < 0.05, ** p < 0.01. (c) Representative traces for bead displacement and reinforcement for control, G1, siRNA GPER + G1, and G1 + RhoA rescue conditions. Green points represent the maximum displacement in a pulse and red points the starting displacement in a pulse, which increase due to bead drift. Black downwards arrow indicates 1st pulse and red arrow indicates 12th pulse. (d) Immunofluorescence images of YAP (yes-associated protein 1) nuclear localisation in Suit2-007 under control, G1 (1 µM, 24 h), and G1 + G15 (1 µM and 2 µM respectively, 24 h) conditions. Green represents YAP, and blue represents DAPI staining of the nucleus. White arrow indicates nuclear localisation of YAP, orange arrow indicates cytoplasmic localisation of YAP. Scale bar = 25 µm. (e) Quantification of percentage cells containing nuclear YAP. For control, G1, G1 + G15, siRNA GPER + G1 and G1 + RhoA rescue, n = 24, 24, 20, 12, 12 regions of interest from three independent samples respectively. *** represents Mann–Whitney test between each individual condition and G1 condition, p < 0.001. (f) Quantitative PCR analysis of the YAP-target genes CTGF and ANKRD1, normalised to expression of RPLP0. Dotted line represents y = 1.0. For Mann–Whitney test between each condition and G1 condition, ** p < 0.01, *** p < 0.001. n = three independent samples. (g) Western Blot analysis of YAP and pYAP (phosphorylated on Ser127) for control, G1 and G1 + G15 conditions. n = two independent samples. All experiments with siRNA and RhoA were conducted 72 h after transfection. For experiments with G1, cells were treated with 1 µM G1 for 24 h before analysis. For experiments with G15, cells were treated with 2 µM G15 for 24 h before analysis.
Figure 3
Figure 3
Force generation in Suit2-007 cells is inhibited by GPER activation. (a) Immunofluorescence staining for total MLC-2 (myosin light chain-2) and pMLC-2 (phospho-myosin light chain-2) for Suit2-007 cells. Scale bar = 25 µm. (b) Quantification of MLC-2 and pMLC-2 staining intensities relative to control condition. For MLC-2 intensity, control, G1 (1 µM, 24 h), G1 + G15 (1 µM and 2 µM respectively, 24 h), siRNA GPER + G1 (1 µM, 24 h), G1 (1 µM, 24 h) + RhoA rescue, n = 35, 29, 36, 36, 36 cells across three independent experiments respectively. Kruskal-Wallis analysis indicates no significant differences between median values. For pMLC-2 intensity, control, G1, G1 + G15, siRNA GPER + G1. G1 + RhoA rescue, n = 30, 24, 20, 36, 36 cells across three independent experiments respectively. Mann–Whitney test comparing each individual condition to G1-treated conditions, ** p < 0.01, *** p < 0.001. (c) Heat maps indicating force generation of cells on elastic pillars. Each point represents one pillar, with intensity equal to the maximum force applied to that pillar over 1 min of imaging. Scale bar = 20 µm. (d) Quantification of maximum mean force exerted by Suit2-007. For control, G1, G1 + G15, siRNA GPER + G1, G1 + RhoA rescue, n = 46, 44, 25, 54, 43 cells across two independent experiments respectively. Mann–Whitney test comparing each individual condition to G1-treated conditions, *** p < 0.001. (e) Young’s modulus values for control, G1 and G1 + G15 treated cells. For control, G1 and G1 + G15, n = 51, 50, and 48 cells across three independent samples respectively. * represents Mann–Whitney test, p < 0.05, *** p < 0.001. All experiments with siRNA and RhoA were conducted 72 h after transfection. For experiments with G1, cells were treated with 1 µM G1 for 24 h before analysis. For experiments with G15, cells were treated with 2 µM G15 for 24 h before analysis.
Figure 4
Figure 4
GPER activation inhibits epithelial–mesenchymal transition in Suit2-007 cells. (a) Left: Immunofluorescence images of Suit2-007 cells stained for β-catenin, DAPI and actin, for control and 1 μM G1 (GPER agonist) after 24 h of culture. White arrow indicates nuclear localisation of β-catenin, orange arrow indicates cytoplasmic localisation of β-catenin. Scale bar = 25 μm. Right: Quantification of β-catenin localisation. For Suit2-007, control and G1, n = 18 and 18 regions of interest respectively. For PC-3, control and G1, n = 17 and 16 regions of interest respectively. Mann–Whitney test for statistical significance, * p < 0.05, *** p < 0.001. (b) Left: Immunofluorescence images of Suit2-007 cells stained for vimentin, for control and G1 (1 µM) after 24 h of culture. Scale bar = 25 μm. Right: Quantification of vimentin immunofluorescence images respectively. For control and G1, n = 30 and 30 regions of interest respectively. Mann–Whitney test for statistical significance, * p < 0.05, *** p < 0.001. (c) Immunofluorescence images of Suit2-007 cells stained for β-catenin, DAPI and actin, for control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ICI 182780 (ER antagonist), 5 µM tamoxifen + 2 µM G15 (GPER antagonist) for 72 h of culture. White arrows indicate nuclear localisation of β-catenin, orange arrows indicate cytoplasmic localisation of β-catenin. Scale bar = 25 μm. (d) Quantification of β-catenin localisation. For Suit2-007, control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ER Ant, 5 µM tamoxifen + 2 µM GPER Ant (all for 72 h), n = 15, 11, 15, 18 regions of interest respectively. For PC-3, control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ER Ant, 5 µM tamoxifen + GPER Ant (all for 72 h), n = 16, 15, 10, 11 regions of interest respectively. Mann–Whitney test for statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001. (e) Immunofluorescence images of Suit2-007 cells stained for vimentin, for control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ER Ant, 5 µM tamoxifen + 2 µM GPER Ant for 72 h of culture. (f) Quantification of immunofluorescence intensity for vimentin. For Suit2-007, control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ER Ant, 5 µM tamoxifen + GPER Ant (all for 72 h), n = 16, 10, 10, 17 regions of interest respectively. For PC-3, control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ER Ant, 5 µM tamoxifen + 2 µM GPER Ant (all for 72 h), n = 17, 15, 16, 15 cells regions of interest respectively. Mann–Whitney test for statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001. (g) Quantitative PCR analysis of the YAP-target genes for vimentin and E-cadherin, normalised to expression of RPLP0. For Mann–Whitney test between each condition and G1 condition, ** p < 0.01, *** p < 0.001. n = three independent samples. (h) Left: Crystal Violet stained cells on Transwells for control and G1-treated conditions. Right: Count of invaded cells in imaged region for control and G1 (1 µM, 24 h) treated cells. For control and G1, n = 23 and 21 regions respectively. *** represents Mann–Whitney test, p < 0.001. Scale bar = 100 µm.
Figure 5
Figure 5
Composition of basement membrane in healthy and PDAC patients. (a) Immunofluorescence images of laminin/collagen IV bilayer in healthy and PDAC patients. Scale bar = 10 µm. L = laminin 111-rich side. C = collagen IV-rich side. Upper panel = Lam111 + Col IV, lower left = Lam111 + DAPI, lower right = Col IV + DAPI. (b) Immunofluorescence images of laminin 332/collagen IV bilayer in PDAC. Scale bar = 10 µm. L = laminin 111-rich side. C = collagen IV-rich side. Upper panel = Lam332 + Col IV, lower left = Lam332 + DAPI, lower right = Col IV + DAPI. (c) Immunofluorescence images of collagen IV organisation in healthy and PDAC tissues. Scale bar = 50 µm. Yellow arrow indicates organised collagen IV, red arrow indicates disorganised collagen IV. (d) Immunofluorescence images of laminin 332 organisation in healthy and PDAC tissues. Scale bar = 50 µm. (e) Immunofluorescence images of laminin α3 organisation in healthy and PDAC tissues, using P3H9 monoclonal antibody. Scale bar = 50 µm.
Figure 6
Figure 6
GPER activation inhibits Suit2-007 invasion through basement membrane mimics. (a) Immunofluorescence images of a decellularised mesentery extracted from mice, stained for laminin 111 (green) and perlecan (magenta). Image from single plane (top down view) and maximum intensity projection from side view, indicating presence of bilayer. Scale bar = 20 µm. (b) Suit2-007 cells beginning invasion of mesentery (24 h). White arrows indicate filopodia. Scale bar = 20 µm. (c) Top down and side view of control and G1 (1 µM) treated cells on mesenteries after 1 day. Scale bar = 10 µm. (d) Top down and side view of control and G1 (1 µM) treated cells on mesenteries after 10 days. Scale bar = 10 µm. (e) Mesh representations of invading cells after 1, 5, or 10 days from volume analysis. G1 (1 µM) + RhoA rescue mesh representation on Day 1 only. Blue mesh = cells, grey plane = top layer of mesentery. Scale bar = 2 μm. Arrow represents direction of invasion. (f) Quantification of cell area below top layer of mesentery for 1, 5 and 10 days. For control, n = 53, 34 and 55 cells for day 1, 5, and 10 respectively. For G1, n = 56, 39 and 27 cells for day 1, 5, and 10 respectively. For G1 + RhoA, n = 80 cells. * represents Mann–Whitney test between control and G1-treated cells for each individual time point, *** p < 0.001. (g) Cumulative count of cells invaded through mesenteries attached to bottom of well in 24 well plate for control and G1 (1 µM) treated Suit2-007 cells. Mesenteries were transferred to a new well each day, and cells attached to the bottom of the old well, where mesenteries had been for 24 h, were counted. Cell count was normalised depending on the amount of mesenteries in a well, each with the same amount of cells seeded on top of them. Each point is the sum of the mean values for each day, with standard error for each day calculated as the sum of standard errors for all the days used in summation. For Day 1-10, control = 13, 21, 23, 13, 21, 10, 17, 12, 13, 16 regions. For Day 1–10, G1 = 22, 19, 13, 11, 12, 10, 5, 5, 5, 7 regions. p = 0.00032 for straight line slope comparison.
Figure 7
Figure 7
GPER inhibits membrane invasion via the RhoA/ROCK (Rho-associated protein kinase) system. The first step in the metastatic process is the invasion of neighbouring tissues, which requires the cells to breach the basement membrane. In pancreatic and prostate cancer cells, GPER can reduce their ability to breach the basement membrane by acting as a mechanoregulator. Pharmacological activation of GPER using G1 or Tamoxifen inhibits the activity of RhoA, preventing the subsequent activation of ROCK and the formation of phospho-myosin light chain 2 (pMLC-2). By controlling the activity of pMLC-2, GPER activation can modulate the contractile actomyosin machinery and regulate the mechanical activity of the cell.
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