NEDD4 and NEDD4L regulate Wnt signalling and intestinal stem cell priming by degrading LGR5 receptor

Abstract

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post-translational regulation of LGR5. Here, we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apc^min mice with increased numbers of high-grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/β-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression.

Keywords: Lgr5; NEDD4; Wnt; colorectal cancer; intestinal stem cell.

Figure 1. Loss of Nedd4 and Nedd4l increases intestinal crypt proliferation

1. A, B

   Representative images of RNAScope ISH showing Nedd4 and Nedd4l expression in mouse small intestinal tissues derived from normal animal (A) and Apc^min tumours (B). High‐magnification images are shown in the box. Scale bars, 50 µm (A), 100 µm (B).

2. C–N

   Histology and immunostaining of Villin^creERT2 wild‐type (WT) (C, E, G, I, K, M) and Villin^creERT2;Nedd4^fl/fl;Nedd4l^fl/fl DKO (D, F, H, J, L, N) proximal intestine 1‐year post‐tamoxifen induction using the indicated markers (n = 3 per group). Scale bars, 50 µm.

3. O

   Quantitation of Edu⁺ proliferating cells per crypt from (C, D). Each dot represents the average of at least 10 crypts per animal. Data are mean ± standard error. n = 3 per group.

4. P

   Quantitation of crypt length (µm) from (E, F). Each dot represents the average of at least 20 crypts per animal. Data are mean ± standard error. n = 3 per group.

5. Q–T

   Quantitation of Olfm4 (Q), Cyclin D1 (R), Sox9 (S) and lysozyme (T)‐positive cells per crypt. Each dot represents the average of at least 20 crypts per animal. Data are mean ± standard error. n = 3 per group. Error bars represent ± SEM.

Data information: P values were determined using the unpaired two‐sided t‐test (*P < 0.05; **P < 0.01; ns, not significant).

Figure EV1. Short‐term deletion of Nedd4 and/or Nedd4l in the intestine

1. Schematic representation of the crypt–villus axis of the small intestine. mRNA expression of the indicated genes was analysed by qRT–PCR in villi and crypts isolated from wild‐type intestine. Data are presented as fold change normalised to Hprt1 control (n = 4 per group). Error bars represent ± standard error.

2. Representative images of WT, Nedd4 cKO, Nedd4l cKO and DKO proximal intestine collected at 50 dpi stained for H&E, PAS, Cyclin D1, Sox9 and Edu (n = 3 per group). Scale bars, 50 µm.

3. mRNA expression of the indicated genes was analysed by qRT–PCR in crypts from small intestinal organoids isolated from the correspondent WT and DKO mice. Data are presented as fold change normalised to Hprt1 control in triplicate (n = 3 per condition). Error bars represent ± standard error.

Data information: P‐values were determined using the unpaired two‐sided t‐test (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure EV2. Characterisation of in vitro phenotype using small intestinal organoids

1. mRNA expression of the indicated genes was analysed by qRT–PCR in small intestinal organoids isolated from the correspondent WT, Nedd4 cKO, Nedd4l cKO and DKO mice. Data are presented as fold change normalised to Hprt1 control in triplicate (n = 3 per condition). Error bars represent ± standard error. P‐values were determined using the unpaired two‐sided t‐test (*P < 0.05; **P < 0.01).

2. Representative photographs of colony formation assay of organoids derived from WT, Nedd4 cKO, Nedd4l cKO and DKO intestine at 5 dpi. Scale bar, 1,000 µm.

Figure 2. Nedd4/Nedd4l deficiency activates Wnt signalling and promotes growth advantage in intestinal organoids

1. A, B

   mRNA expression of the indicated genes was analysed by qRT–PCR in small intestinal organoids isolated from the correspondent WT, Nedd4 cKO, Nedd4l cKO and DKO mice. Data are presented as fold change normalised to Hprt1 control in triplicate (n = 3 per condition). Error bars represent ± standard error.

2. C

   Immunostaining for proliferating cells after 2‐h EdU incorporation. Scale bars, 100 µm.

3. D

   Quantitation of the organoid formation assay in (Fig EV2B). WT = 9, Nedd4 cKO = 3, Nedd4l cKO = 4 and DKO = 5. Data are mean ± standard error.

4. E

   Representative images of organoids of the indicated genotypes cultured under 5% RSPO (top row) or 1% RSPO (bottom row) conditions at day 3. Asterisks indicate dying organoids. Scale bar, 1,000 µm.

5. F

   Quantitation of dying organoids in (E). Each dot represents the average of dying organoids from three different mice per genotype in the indicated conditions. Data are mean ± standard error.

Data information: P‐values were determined using the unpaired two‐sided t‐test (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3. Loss of Nedd4 and Nedd4l exacerbates Apc^min intestinal tumour phenotype

1. A

   Kaplan–Meier survival analysis of Apc^min control and Apc^min DKO mice. P values were determined using the Mantel–Cox test.

2. B

   Representative H&E staining of the small intestines of the indicated genotypes. Scale bar 2,000 µm.

3. C, D

   Total number of adenomas in the small intestine (C) and colon (D) 3 months after induced Nedd4 and/or Nedd4l loss. Data are mean ± standard error. P‐values were determined using the unpaired two‐sided t‐test (*P < 0.05; **P < 0.01).

Figure EV3. Loss of Nedd4 and/or Nedd4l increases Wnt activation in Apc^min adenomas

1. A

   Kaplan–Meier survival analysis of Apc^min control, Apc^min Nedd4 cKO and Apc^min Nedd4l cKO mice. P‐values were determined using the Mantel–Cox test.

2. B

   Quantitation of lysozyme‐positive cells in adenomas from the indicated mice. Each dot represents the average of at least 5 adenomas (with similar size and grade of dysplasia) per animal. Data are mean ± standard error. n = 3 per group.

3. C–N

   Immunostaining of adenoma tissues from Apc^min control (C, G, K), Apc^min Nedd4 cKO (D, H, L), Apc^min Nedd4l cKO (E, L, M) and DKO (F, J, N) mice using the indicated antibodies. Scale bars, 100 µm.

4. O–P

   Quantitation of Sox9‐positive (O) and Edu‐positive proliferating (P) cells per a defined area in the adenoma. Each dot represents the average of at least 5 adenomas (with similar size and grade of dysplasia) per animal. Data are mean ± standard error. n = 3 per group.

Data information: P‐values were determined using the unpaired two‐sided t‐test (*P < 0.05; **P < 0.01).

Figure 4. Inactivation of NEDD4 and NEDD4L increases proliferation and tumour grade in Apc^min mice

1. A, B

   Quantitation of the grades of the adenomas that developed in the small intestine (A) and colon (B) of the mice in (C, D). Data are mean ± standard error. P‐values were determined using the unpaired two‐sided t‐test (*P < 0.05; **P < 0.01).

2. C

   Representative H&E staining from a low‐grade versus a high‐grade tumour in the indicated genotypes. Scale bar, 100 µm.

3. D–O

   Representative images of Lgr5 RNAScope (D–G), lysozyme (H–K) and Edu (L–O) staining in adenoma tissues obtained from the indicated genotypes. High‐magnification images of the boxed areas within the adenomas are shown on the right. Scale bar, 100 µm. Data are representative of at least three mice per group. Adenomas are circled in red.

Figure EV4. NEDD4 and NEDD4L target DVL2 for degradation

1. Cell lysates of HEK293T control (Ctr) and the indicated CRISPR clones were analysed by Western blotting using the indicated antibodies.

2. Relative TOPFlash reporter activities of HEK293T cells with the indicated CRISPR targeting. When indicated, cells were treated with Wnt3a‐conditioned media or Wnt3a supplemented with RSPO. Data represent mean ± standard error of at least three independent experiments. P‐values were determined using the unpaired two‐sided t‐test (*P < 0.05; **P < 0.01).

3. Western blot analysis of HEK293T cells transfected with Flag‐DVL1, Flag‐DVL2 or Flag‐DVL3 with or without MYC‐NEDD4L using the indicated antibodies.

4. HEK293T cells were transfected with constructs expressing Flag‐DVL2, MYC‐NEDD4‐WT, Myc‐NEDD4‐CS mutant, HA‐Ubiquitin or empty vector (EV), as indicated. Cells were treated with MG132, followed by anti‐Flag IP and immunoblotting using the indicated antibodies.

5. Cell lysates of HEK293T control (Ctr) and the indicated CRISPR clones were analysed by Western blotting using the indicated antibodies.

Figure 5. NEDD4 and NEDD4L negatively regulate Wnt signalling pathway

1. A

   Relative TOPFlash reporter activities of HEK293T cells with the indicated CRISPR targeting. Cells were treated with Wnt3a‐conditioned media.

2. B

   Western blot analysis of HEK293T cells transfected with Flag‐DVL1, Flag‐DVL2 or Flag‐DVL3 with or without Myc‐NEDD4 using the indicated antibodies.

3. C

   Relative TOPFlash reporter activity upon ectopic expression of the indicated plasmids in HEK293T cells treated with Wnt3a and RSPO.

4. D

   Relative TOPFlash reporter activities of HEK293T cells with CRISPR deletion of DVL2 upon ectopic expression of the indicated plasmids. Cells were treated with Wnt3a and RSPO.

5. E–G

   Relative TOPFlash reporter activities of HCT116 (E), APC4 (Novellasdemunt et al, 2017) (F) and DLD1 (G) cells transfected with the indicated plasmids.

Data information: Data represent mean ± standard error of at least three independent experiments. P‐values were determined using the unpaired two‐sided t‐test (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure EV5. NEDD4 and NEDD4L selectively degrade Lgr4 and Lgr5 but not FZD receptors of Wnt signalling pathway

1. A

   HEK293T cells were transfected with empty vector (EV), LGR4‐Flag, MYC‐NEDD4 WT or C854S (CS) mutant, MYC‐NEDD4L wild‐type (WT) or C962A (CA) mutant, followed by Western blot analysis of the indicated antibodies.

2. B, C

   HEK293T cells were transfected with V5‐FZD4 (B) or V5‐FZD5 (C) with or without MYC‐NEDD4 or MYC‐NEDD4L or empty vector (EV) as control. Lysates were subjected to Western blotting using the indicated antibodies.

3. D

   Subcellular localisation of SNAP‐FZD5 in HEK293T cells co‐expressed with the indicated plasmids. Surface SNAP‐FZD5 was labelled with SNAP Alexa‐488 for 10 min. Scale bars, 10 µm.

4. E

   Quantitation of fluorescent intensity in total and surface LGR5 and FZD5 with the indicated transfections of NEDD4 and NEDD4L. Data are presented as percentage of fluorescence intensity compared to the EV control in triplicate for LGR5 and duplicate for FZD5. Error bars represent ± SEM. P‐values were determined using the unpaired two‐sided t‐test (*P < 0.05; ***P < 0.001).

5. F

   HEK293T cells WT, NEDD4 CRISPR or NEDD4L CRISPR mutants were transfected with LGR5‐Flag. Twenty‐four hours later, cells were treated with cycloheximide (Chx) (50 µg/µl) and were collected at different time points as indicated. Cell lysates were subjected to Western blot analysis using the indicated antibodies. Quantitation of the blots is shown at the bottom. Data represent mean ± standard error of at least three independent experiments. P‐values were determined using the unpaired two‐sided t‐test compared between Nedd4 and WT (indicated by *) or Nedd4l and WT (indicated by #) at the same time point *P < 0.05; ^## P < 0.01).

6. G, H

   HEK293T cells were transfected with constructs expressing LGR5‐Flag, HA‐Ubiquitin, EV or the indicated NEDD4 (G) or NEDD4L (H) plasmids. Cells were treated with Bafilomycin A1 followed by anti‐Flag IP and Western blot analysis using the indicated antibodies.

Figure 6. NEDD4 and NEDD4L target LGR5 receptor for lysosomal and proteasomal degradation

1. A

   HEK293T cells were transfected with empty vector (EV), LGR5‐Flag, MYC‐NEDD4 WT or C854S (CS) mutant, MYC‐NEDD4L wild‐type (WT) or C962A (CA) mutant, followed by Western blot analysis of the indicated antibodies. Black triangle indicates the mature glycosylated form of LGR5, while white triangle indicates the immature unprocessed form of LGR5.

2. B

   Subcellular localisation of SNAP‐Lgr5 in the absence or presence of Myc‐NEDD4‐WT, catalytically inactive HA‐NEDD4‐CS, Myc‐NEDD4L‐WT or catalytically inactive HA‐NEDD4L‐CA. Surface SNAP‐Lgr5 was labelled with SNAP Alexa‐488 for 10 min. Scale bar, 10 µm.

3. C, D

   HEK293T cells were transfected with constructs expressing LGR5‐Flag, HA‐Ubiquitin, EV, or the indicated NEDD4 (C) or NEDD4L (D) plasmids. Cells were treated with MG132 followed by anti‐Flag IP under denaturing conditions and Western blot analysis using the indicated antibodies.

4. E

   HEK293T control, NEDD4 or NEDD4L CRISPR‐mediated mutant cells were transfected with LGR5‐Flag and HA‐Ubiquitin. Cells were pre‐treated with MG132 followed by anti‐Flag IP and Western blot analysis using the indicated antibodies.

5. F

   Relative TOPFlash reporter activities of HEK293T cells and NEDD4 and NEDD4L CRISPR cell lines with the indicated siRNA constructs. Cells were treated with Wnt3a supplemented with RSPO‐conditioned media. Data represent mean ± standard error of at least three independent experiments. P‐values were determined using the unpaired two‐sided t‐test (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 7. Loss of Nedd4 and Nedd4l enhances ISC expansion upon RSPO stimulation

1. A

   Experimental design of RSPO3 stimulation model.

2. B, C

   Representative images of EdU (B) and Olfm4 (C) staining in intestinal tissues obtained from the indicated genotypes. Scale bar, 50 µm.

3. D

   Model of proposed mechanism of NEDD4/NEDD4L‐mediated regulation of Wnt pathway. NEDD4 and NEDD4L target two Wnt pathway components: (i) LGR4/5 receptor for lysosomal and proteasomal degradation, and (ii) DVL2 for proteasomal degradation.