Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine

Abstract

Acting downstream of many growth factors, extracellular signal-regulated kinase (ERK) plays a pivotal role in regulating cell proliferation and tumorigenesis, where its spatiotemporal dynamics, as well as its strength, determine cellular responses. Here, we uncover the ERK activity dynamics in intestinal epithelial cells (IECs) and their association with tumour characteristics. Intravital imaging identifies two distinct modes of ERK activity, sustained and pulse-like activity, in IECs. The sustained and pulse-like activities depend on ErbB2 and EGFR, respectively. Notably, activation of Wnt signalling, the earliest event in intestinal tumorigenesis, augments EGFR signalling and increases the frequency of ERK activity pulses through controlling the expression of EGFR and its regulators, rendering IECs sensitive to EGFR inhibition. Furthermore, the increased pulse frequency is correlated with increased cell proliferation. Thus, ERK activity dynamics are defined by composite inputs from EGFR and ErbB2 signalling in IECs and their alterations might underlie tumour-specific sensitivity to pharmacological EGFR inhibition.

Fig. 1

In vivo imaging of ERK activity dynamics in the mouse small intestine. a Schematic representation of the structure of the FRET biosensor for ERK activity, EKAREV-NLS, and its mechanism of action. Upon activation, ERK phosphorylates the substrate sequence in the biosensor. The WW domain specifically binds to the phosphorylated substrate, which brings CFP and YFP into close proximity. In this “closed” conformation state, excitation energy absorbed by CFP is transferred to YFP without radiation, thereby enabling YFP to emit fluorescence. The biosensor returns from the “closed” to the original “open” conformation by phosphatase-dependent dephosphorylation of the substrate sequence. b Experimental setting of in vivo imaging of the small intestine. The mouse small intestine was exteriorized, fixed on the microscope stage, and observed with an inverted two-photon excitation microscope under inhalation anaesthesia. c The representative FRET/CFP images of the small intestine of EKAREV-NLS mice before and after administration of 0.1 mg kg⁻¹ body weight of TPA. d Bee swarm plots showing the ERK activity (FRET/CFP ratio) in each cell before and after the TPA (n = 50 cells pooled from three crypts). e The representative FRET/CFP images of the small intestine of EKAREV-NLS mice before and after administration of 5 mg kg⁻¹ body weight of a MEK inhibitor (PD0325902). f Bee swarm plots showing the FRET/CFP ratios in each cell before and after MEK inhibitor treatment (n = 50 cells pooled from three crypts). g–j In vivo time-lapse imaging of intestinal crypts of EKAREV-NLS mice. The representative images (g, i) and quantified data from three selected cells (cells 1–3) (h, j) are shown. Note that the ERK activity pulses are spontaneously generated (g, h), or propagated from adjacent cells (i, j). The original time course data of ERK activity used for h and j before smoothing by the moving average are shown in Supplementary Fig. 1b, c. Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U-tests were used for comparison (d, f). *P < 0.05, **P < 0.001, ***P < 0.0001

Fig. 2

Characterization of mouse intestinal organoids expressing the FRET biosensor for ERK activity. a Experimental setting for live imaging of intestinal organoids. Organoids were established from the small intestine of EKAREV-NLS mice, and cultured in media containing EGF, Noggin, and R-spondin1. Imaging was performed within 4 days of the last passage. b A representative FRET/CFP ratio image of an EKAREV-NLS organoid cultured under normal conditions. c–h Time-lapse imaging of ERK activity in the EKAREV-NLS organoids. Organoids were treated with 1 μM TPA (c–e) or 200 nM MEK inhibitor (PD0325901) (f–h) at time point 0. c, f Representative time-lapse images of organoids treated with TPA (c) or a MEK inhibitor (f) are shown. d, g Time courses of the average FRET/CFP values in the organoids (n = 90 (d) and 55 (g) cells). The FRET/CFP values in individual cells were normalized to the mean values before the treatment. e, h Bee swarm plots of the FRET/CFP values in each cell before and after the treatment (n = 90 (e) and 66 (h) cells). The FRET/CFP values in individual cells were normalized to the mean values before the treatment. i Time courses of the average ERK activity (FRET/CFP ratio) in EKAREV-NLS organoids treated with 10 μM, 1 μM, or 100 nM of a BRAF inhibitor (SB590885) (n = 30, 35, and 31 cells, respectively). Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U-tests were used for comparison (e, h). *P < 0.05, **P < 0.001, ***P < 0.0001

Fig. 3

Spontaneous pulse-like ERK activation and propagation of ERK activity in intestinal organoids. a–d Time-lapse imaging of the EKAREV-NLS organoids was performed for 90 min at 1.5 min intervals (n = 71 cells). a Representative time-lapse images of ERK activity (FRET/CFP ratio) in an organoid showing pulse-like ERK activation. b Heat map showing the time course of ERK activity (FRET/CFP ratio) in each cell. c Time courses of ERK activity in three representative cells. The original data before smoothing by the moving average are shown in Supplementary Fig. 2c. d Temporal autocorrelation coefficients of ERK activity in the three cells shown in c. e, f Time-lapse imaging of the EKAREV-NLS organoids cultured under EGF-starved conditions for 24 h. e Bee swarm plots of ERK activity in organoids cultured under the normal condition (ENR) or EGF-starved condition (NR) (n = 193 and 148 cells pooled from three organoids). f Time course of the average ERK activity in the control or EGF-starved organoids after stimulation with EGF. The organoids were cultured under the normal (ENR) or EGF-starved condition (NR), and then stimulated with 50 ng ml⁻¹ of EGF at time point 0 (ENR: n = 129, NR: n = 59 cells). g Representative time-lapse images of an organoid cultured under the NR condition showing propagation of ERK activity. ERK activity (FRET/CFP ratio) is shown in golden pseudo colour mode. h Time courses of ERK activity in the three representative cells marked in g. i, j Representative time-lapse images of organoids treated with an EGFR inhibitor (PD153035, 1 μM) (i) or an MMP inhibitor (marimastat, 100 μM) (j). Organoids were cultured in NR media for 24 h, and subsequently treated with either inhibitor. ERK activity is shown in the golden pseudo colour mode. Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U-test was used for comparison (e). *P < 0.05, **P < 0.001, ***P < 0.0001, n.s., not significant

Fig. 4

EGFR and ErbB2 generate two distinct modes of ERK activity in intestinal organoids. a, b EKAREV-NLS organoids were treated with EGFRi (PD153035) (1 μM), and/or ErbB2i (CP-724714) (10 μM), at time point 0. a Time courses of average ERK activity under each condition (EGFRi: n = 70, ErbB2i: n = 62, EGFRi + ErbB2i: n = 57 cells). b Quantification of ERK activity before and after treatment with EGFRi or ErbB2i (EGFRi: n = 113, ErbB2i: n = 147 cells, pooled from two organoids). c Time course of average ERK activity in organoids that were cultured under EGF-starved condition (NR) for 24 h, treated with either EGFRi or ErbB2i for 60 min, and then stimulated with 50 ng ml⁻¹ of EGF (EGFRi: n = 48, ErbB2i: n = 32 cells). d Quantification of ERK activity in organoids infected with a control lentivirus (control) or lentiviruses expressing either a dominant negative form of EGFR (dnEGFR) or that of ErbB2 (dnErbB2) (Control: n = 134, dnEGFR: n = 129, dnErbB2: n = 151 cells, pooled from five organoids cultured in the ENR medium). e, f Quantification of ERK activity pulses in EKAREV-NLS organoids cultured in the ENR medium and treated with EGFRi and/or ErbB2i (−/−: n = 71, EGFRi/−: n = 49, −/ErbB2i: n = 36, EGFRi/ ErbB2i: n = 53 cells). Organoids were treated with indicated inhibitors and imaged for 90 min. ERK activity data were smoothened by 6-min moving average, and fitted to flat lines or multi-peak functions. e The proportion of cells exhibiting the pulse-like ERK activation (ERK-pulse⁺) under each condition. f Frequencies of ERK activity pulses under each condition. Duration (g) and frequencies (h) of ERK activity pulses in organoids cultured in the ENR medium and treated with 0, 10, 1, or 0.1 μM of a BRAF inhibitor (SB590885) for 90 min (n = 55, 52, 29, and 65 cells, respectively). Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U-test (b) and Steel–Dwass test (d, f–h) were used for comparison. *P < 0.05, ** P < 0.001, ***P < 0.0001, n.s., not significant

Fig. 5

Adenoma-derived organoids exhibit increased dependence on EGFR signalling. a, b Organoids were generated from the normal epithelium or adenomas of the Apc^Δ716 mouse small intestine, and infected with lentiviruses expressing a FRET biosensor for ERK activity (EKAREV-NLS). a Representative images of ERK activity (FRET/CFP ratio) in organoids derived from the normal epithelium and adenomas. b Bee swarm plots of ERK activity in the normal epithelium-derived and adenoma-derived organoids (Normal: n = 61, Adenoma: n = 99 cells, from more than three organoids). c–f Adenoma-derived organoids expressing EKAREV-NLS were treated with 1 μM of an EGFR inhibitor (EGFRi), PD153035, and/or 10 μM of an ErbB2 inhibitor (ErbB2i), CP-724714. c Representative images of adenoma-derived organoids before and after treatment with EGFRi or ErbB2i. d Bee swarm plots of ERK activity before and after inhibitor treatment (EGFRi: n = 120, ErbB2i: n = 119, EGFRi + ErbB2i: n = 109 cells, pooled from three organoids). e, f Quantification of ERK activity pulses in adenoma-derived organoids. Organoids were treated with EGFR and/or ErbB2 inhibitors, and then imaged for 90 min. ERK activity data from each cell were processed as described for Fig. 4e (−/−: n = 40, EGFRi/−: n = 45, −/ErbB2i: n = 32, EGFRi/ErbB2i: n = 29 cells). Frequencies of the pulse-like ERK activity (e) and the proportion of cells with pulse-like ERK activity (ERK-pulse⁺) (f) under each condition. Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U-test (b, d) and Steel–Dwass test (e) were used for comparison. *P < 0.05, **P < 0.001, ***P < 0.0001, n.s., not significant

Fig. 6

Pharmacological activation of Wnt signalling promotes cell proliferation by augmenting EGFR signalling. a Representative images and b quantification of ERK activity in organoids treated with or without CHIR99021 (5 μM) for 24 h (n = 210 and 213 cells (from the left), from three organoids). c Time courses of ERK activity in organoids treated with CHIR99021 for 24 h and then treated with EGFRi (PD153035), and/or ErbB2i (CP-724714) (n = 45, 29, and 29 cells from the left). d Quantification of ERK activity before (Pre) and after (Post) EGFRi treatment (bottom) (n = 30, 44, and 53 cells from the left). Organoids cultured in ENR, ENR+CHIR, or ENR+CHIR99021 for 24 h and then in ENR for another 24 h (Re-ENR) were treated with EGFRi (top). e, f Quantification of ERK activity pulses in organoids treated with CHIR99021 for 24 h and then with EGFRi and/or ErbB2i for 90 min during imaging. The proportion of cells with ERK activity pulses (ERK-pulse⁺) (e), and the frequency of the pulses (f) under each condition (n = 36, 47, 47, and 25 cells from the left). g Schematic structure of Fucci2a (top), cell cycle diagram marked with corresponding fluorescence (bottom left), and the representative image of organoid expressing Fucci2a (bottom right). h, i Proportion of cells in the S/G2/M phases in the Fucci2a organoids. h Organoids were cultured for 24 h in ENR, EGF-deficient medium (NR), or ENR supplemented with EGFRi ErbB2i, and/or a MEK inhibitor (MEKi) (n = 158, 264, 400, 359, 423, and 243 cells (from the left), from more than four organoids). i Organoids treated with CHIR99021 for 24 h were subsequently treated with EGFRi, and/or ErbB2i for another 24 h (n = 156, 1062, 461, 220, and 234 cells (from the left), from more than four organoids). Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U-test (b, d), Steel–Dwass test (f), and χ² test with BH procedure (h, i) were used for comparison.*P < 0.05, **P < 0.001, ***P < 0.0001, n.s., not significant

Fig. 7

EGFR signalling is augmented in adenomas and CHIR99021-treated mouse intestine in vivo. a Bee swarm plots of ERK activity in the vehicle- (Control) or CHIR99021-treated (CHIR) mouse intestinal epithelium before (Pre) and after (Post) treatment with an EGFR inhibitor, erlotinib. Mice were injected with vehicle or 20 mg kg⁻¹ body weight of CHIR99021 for 3 days and then treated with 100 mg kg⁻¹ body weight erlotinib for 30 min (Control: n = 48, CHIR: n = 61 cells from three crypts). b, c Immunofluorescence staining of the small intestine in the mice injected with vehicle (Control) or CHIR99021 for 3 days using anti-EGFR and anti-E-cadherin antibodies. b The ratio of EGFR and E-cadherin staining intensities is shown in the IMD mode according to the colour scale. Counterstaining was performed with Hoechst. Note that strong staining of stromal cells results from non-specific binding of the secondary antibodies used here (anti-mouse IgG). c Quantification of the EGFR/E-cadherin staining intensity ratio in each cell under vehicle or CHIR99021-treated condition (n = 50 cells). d Immunofluorescence staining of an adenoma and the normal small intestine of Apc^Δ716 mice with anti-EGFR and anti-E-cadherin antibodies. EGFR/E-cadherin staining intensity ratio is shown in the IMD mode. e Quantification of the EGFR/E-cadherin ratio in each cell located in the indicated regions (n = 50 cells). f Immunofluorescence staining of normal and adenoma-derived organoids with anti-EGFR and anti-ErbB2 antibodies. EGFR/ErbB2 staining intensity ratio is shown in the IMD mode. g Quantification of the EGFR/ErbB2 ratio in each cell in the organoids (n = 40 cells pooled from at least two organoids). h, i EdU staining of the small intestine of CHIR99021- and/or erlotinib-treated mice. Mice were injected with vehicle, 20 mg kg⁻¹ body weight of CHIR99021, and/or 100 mg kg⁻¹ body weight of erlotinib for 3 days. i Quantification of EdU⁺ cells per crypt section (−/−: n = 46, CHIR/–: n = 42, −/Erlotinib: n = 54, and CHIR/Erlotinib: n = 45 crypts from three mice). Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U-test (a, c, g), and Steel–Dwass test (e, i) were used for comparison.*P < 0.05, **P < 0.001, ***P < 0.0001

Fig. 8

Wnt signalling activation affects EGFR–ERK signalling dynamics through regulating expression of multiple molecules. a A volcano plot depicting the fold changes in gene expression levels between normal (ENR) and CHIR99021-treated organoids (ENR + CHIR), and statistical significance of the changes. b–e Enrichment plots from gene set enrichment analysis (GSEA). GESA plots for genes downregulated after Apc knockout (b), genes upregulated after Apc knockout through Myc (c), genes downregulated in colorectal adenoma (d), and genes upregulated in colorectal adenoma (e) are shown. f, g RT-PCR analysis revealed that Egfl6, Flna, and Troy were upregulated, and that Lrig3 was downregulated in both CHIR99021-treated and adenoma-derived organoids. The relative mRNA levels of indicated genes in normal (ENR) versus CHIR99021-treated organoids (ENR + CHIR) (f), and those in normal versus adenoma-derived organoids (g) are shown (n = 3 samples containing more than ten organoids). h, i Adenoma-derived organoids expressing the ERK biosensor were infected with lentiviruses expressing control vector (control), Lrig3 (Lrig3), or shRNAs for Egfl6 (shEgfl6), Flna (shFlna), or Troy (shTroy). h Bee swarm plots of ERK activity in organoids before (Pre) and after (Post) EGFR inhibitor treatment under each condition (n = 80 cells pooled from two organoids). i The frequency of ERK activity pulses under each condition. Time-lapse imaging was performed for 90 min (n = 50 cells). j Schematic representation of ERK activity dynamics generated by kinase activity of EGFR and ErbB2 in the normal and Wnt signalling-activated intestinal epithelia. Red lines represent mean. Error bars represent s.e.m. Welch’s t test (f, g), Mann–Whitney U-test (h), and Steel–Dwass test (i) were used for comparison.*P < 0.05, **P < 0.001, ***P < 0.0001