Slap restricts oncogenic Src-family kinase signaling to maintain colonic epithelial homeostasis
- CRBM, Univ. Montpellier, CNRS, France
- Equipe labellisée FRM2023, CRBM, Univ. Montpellier, CNRS, France
- Equipe labellisée LIGUE2020, CRBM, Univ. Montpellier, CNRS, France
- Institut Pasteur, Mouse Genetics Engineering Center, Université Paris Cité, France
- IGF, Univ. Montpellier, CNRS, INSERM, France
- Equipe labellisée LIGUE2022, IGMM, Univ. Montpellier, CNRS, France
- IRCM, Univ. Montpellier, INSERM, France
Figures
Figure 1 with 1 supplement
Murine Slap inactivation induces colonic epithelial hyperplasia.
(A) Constitutive Slap deletion. Top: Hematoxylin and eosin (H&E) staining of transverse colon sections. Crypt thickness was measured and quantified as mean ± SEM from 25 to 40 crypts per mouse (n=6 female mice per group). Middle: Immunohistochemistry (IHC) for the proliferation marker Ki67, with quantification of Ki67-positive colonic epithelial cells (CECs). (mean ± SEM from 20 to 40 crypts per mouse; n=5–6 mice per group). Bottom: RNAscope analysis of Lgr5 to identify colonic stem cells, combined with E-cadherin immunofluorescence to delineate crypt architecture. Inset, higher (×4) magnification highlighting Lgr5 expression. Lgr5 RNA particles were quantified and are shown as mean ± SEM from 20 to 40 crypts per mouse (n=5–6 mice per group). ***p<0.001, ****p<0.0001 Mann–Whitney test. (B) Inducible epithelial-specific Slap deletion (villin-CreERT2). Top: H&E staining of transverse colon sections with quantification of crypt thickness (mean ± SEM from 25 to 40 crypts per mouse, n=6 mice/group). Middle: IHC for Ki67 with quantification of Ki67-positive CECs (mean ± SEM from 20 to 40 crypts per mouse; n=5–6 mice per group). Bottom: RNAscope analysis of Lgr5 combined with E-cadherin immunofluorescence; Inset, higher (×4) magnification highlighting Lgr5 expression. Lgr5 RNA particles were quantified (mean ± SEM from 20 to 40 crypts per mouse, n=5–7 mice per group). Mice were analyzed at 3 months of age, 10 days after tamoxifen induction. ***p<0.001, ****p<0.0001 Mann–Whitney test.
Figure 1—figure supplement 1
Slap inactivation increases goblet cell numbers and SFK activity in the colon.
(A) Left: X-Gal staining of Slap expression. Insert: ×4 magnification highlighting Slap expression at the base of colonic crypts. Right: IF staining for Slap (red), nuclei (blue), and β-catenin (green). (B) Slap inactivation increases goblet cell numbers and SFK activity. Top: Alcian blue staining of goblet cells with quantification. Bottom: IHC for p-SRC and its quantification. (C) Inducible Slap inactivation increases goblet cell numbers. Top: IF staining of Slap (red), nuclei (blue), and β-catenin (green) in the colon after 10 days of tamoxifen treatment. Bottom: Alcian blue staining of goblet cells and quantification.
Figure 2 with 1 supplement
Intestinal Slap inactivation enhances colonic organoid development through SFK activation.
(A) Slap-dependent colonic organoid development. Representative images of colon-derived organoids from Slap f/f and Slap f/f Villin-CreERT2 mice cultured in Matrigel for 2 days. Quantification of organoid number and area is shown as mean ± SEM from 100 to 200 organoids per mouse (n=8 mice per group). ****p<0.0001 Mann–Whitney test. (B) Intestinal Slap deletion increases SFK activity and global protein tyrosine phosphorylation in colonic crypts. Representative immunoblots (right) and quantification (left) of phospho-SFK (pSRC) and total phospho-tyrosine levels in lysates from isolated colonic crypts of the indicated genotypes. Data are presented as mean ± SEM from n=4–5 mice per group. *p<0.05; **p<0.001 t test. (C) Slap-dependent colonic organoid expansion requires SFK activity. Representative images and quantification of colon-derived organoids from Slap f/f and Slap f/f Villin-CreERT2 mice cultured for 2 days in the presence or absence of the SRC-family kinase inhibitor eCF506 (100 nM). Data are shown as mean ± SEM from 100 to 200 organoids per mouse, n=4 mice per group. ***p<0.001, ****p<0.0001 Mann–Whitney test.
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Figure 2—source data 1
PDF file containing original western blots for Figure 2, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/110324/elife-110324-fig2-data1-v1.pdf
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Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2.
- https://cdn.elifesciences.org/articles/110324/elife-110324-fig2-data2-v1.zip
Figure 2—figure supplement 1
EPH-dependent phosphotyrosine accumulation and SFK activation in Slap-deficient colonic organoids.
pTyr and activated SFKs (pSRC) levels in WT and Slap-deficient colonic organoids under the indicated conditions (EPHi and SRCi: 100 nM for 3 hrs); top: representative example; bottom: quantification (mean ± SEM n=3; ns: p>0.05; *p<0.05, **p<0.01, ****p<0.0001).
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Figure 2—figure supplement 1—source data 1
PDF file containing original western blots for Figure 2–figure supplement 1, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/110324/elife-110324-fig2-figsupp1-data1-v1.pdf
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Figure 2—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 2–figure supplement 1.
- https://cdn.elifesciences.org/articles/110324/elife-110324-fig2-figsupp1-data2-v1.zip
Figure 3 with 1 supplement
Inducible Slap intestinal inactivation drives colon tumorigenesis and promotes SFK-dependent tumoroid growth.
(A) Experimental workflow of AOM/DSS-induced colon tumorigenesis. Mice received tamoxifen, followed 10 days later by a single azoxymethane (AOM) injection to induce mutagenesis and one cycle of dextran sodium sulfate (DSS) administered for 7 consecutive days. A second AOM injection was then performed, followed by two additional DSS cycles (7 days each). Mice were sacrificed at day 80. (B) Epithelial Slap loss accelerates colon tumor development. Left, representative H&E-stained sections showing transformed regions. Right, quantification of epithelial lesion area (mean ± SEM; n=11–15 mice per group). Statistical analysis is shown (Mann–Whitney test). (C) Slap depletion increases SFK activation in colonic tumors. Left, representative immunohistochemistry for phosphorylated SFK (pSRC) in transformed epithelium. Right, quantification of p-SFK–positive cells per mm² (mean ± SEM; n=7–9 mice per group; ****p<0.0001, Mann–Whitney test). (D) Tumoroids derived from Slap f/f and Slap f/f Villin-CreERT2 mice were cultured in 3D Matrigel in the presence or absence of SRC inhibitor (SRCi) and imaged at day 3. Tumoroid area (µm²) was quantified following SRCi treatment (mean ± SEM; 150–250 organoids per mouse, n=4 mice per group; ns: p>0.05; ****p<0.0001, Mann–Whitney test).
Figure 3—figure supplement 1
SLAP suppresses human CRC stem cell properties.
(A) SLAP overexpression reduces ALDH activity in SLAP-low HT29 and SW620 cells (representative FACS plots and quantification; mean ± SEM; n=3; **p<0.01, ****p<0.0001; unpaired t-test). (B) SLAP overexpression decreases tumoroid formation in HT29 and SW620 cells (representative images and quantification; 25–50 organoids/condition; mean ± SEM; n=3; ****p<0.0001; Mann–Whitney).
Figure 4 with 1 supplement
Slap targets EphB2 to restrict SFK proliferative signaling in CEC.
(A) Intestinal Slap deletion increases EphB2 expression, EphB2 tyrosine phosphorylation, and its association with active SRC (pSRC) in colonic crypts. Left, quantification; right, representative immunoblots of EphB2 levels, EphB2 tyrosine phosphorylation, and associated pSRC following EphB2 immunoprecipitation from isolated colonic crypt lysates of the indicated mouse strains. Mean ± SEM; n=3–5 mice per group; *p<0.05, Student’s t-test. (B) IHC analysis of EphB2 expression in colonic crypts from control and Slap-deficient mice. Left, representative images; right, quantification (mean ± SEM; n=3 mice per group; ****p<0.0001, Mann–Whitney test). (C) SLAP–EPHB2 interaction in HEK293T cells. Co-immunoprecipitation of the indicated SLAP-FLAG constructs (wild-type, WT; or SH3*SH2* mutant, SLAP mut) and EPHB2-MYC constructs (wild-type, WT; or Y596F/Y602F mutant, YF) transfected into HEK293T cells. Expression levels of SLAP and EPHB2 and relative quantification of SLAP-EPHB2 interaction are shown (representative example of three independent experiments). (C) Slap-dependent colon organoid expansion requires EphB2 activity. Representative images and quantification of colon-derived organoids from Slap f/f and Slap f/f Villin-CreERT2 mice treated with the indicated EPH inhibitors (EPHB2i: 200 nM, pan-EPH2i: 100 nM, or DMSO control) and analyzed at day 2 (mean ± SEM; 150–200 organoids per mouse; n=4 mice per group; ns: p>0.05; ****p<0.0001, Mann–Whitney test).
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Figure 4—source data 1
PDF file containing original western blots for Figure 4, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/110324/elife-110324-fig4-data1-v1.pdf
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4.
- https://cdn.elifesciences.org/articles/110324/elife-110324-fig4-data2-v1.zip
Figure 4—figure supplement 1
SLAP interacts with EPHB2 and reduces its levels in HT29 cells.
(A) EPHB2 expression and association with SLAP in CRC cells (transcript and protein; mean ± SEM; n=3–5; ns >0.05; *p<0.05; t-test). (B) EPHB2i (400 nM) reduces EPHB2 activity (i.e. pTyr level) in HEK293T cells. (C) Model of SLAP regulation of SFK signaling in CSCs to maintain colonic epithelial homeostasis by limiting RTK activity.
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Figure 4—figure supplement 1—source data 1
PDF file containing original western blots for Figure 4—figure supplement 1, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/110324/elife-110324-fig4-figsupp1-data1-v1.pdf
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Figure 4—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/110324/elife-110324-fig4-figsupp1-data2-v1.zip
Figure 5 with 1 supplement
SLAP suppresses EPHB2-dependent tumoroid development of CRC cells and is associated with improved prognosis in MSS CRC.
(A) SLAP expression in SW620 CRC cells transduced with empty vector (mock), wild-type SLAP, or an EPHB2-binding-defective SLAP mutant (SLAP Mut). (B) Representative tumoroid images from SW620 cells expressing mock, SLAP, or SLAP m and treated with indicated EPHB2 inhibitors (EPHB2i: 200 nM, pan-EPH2i: 100 nM, or DMSO control). (C) Quantification of tumoroid size from (B) (mean ± SEM; 150–200 tumoroids; n=3; ns: p>0.05; **p<0.001; ***p<0.0005; ****p<0.0001, Mann–Whitney test). (D) Disease-free relapse analysis of TCGA MSS and MSI CRC patients stratified according to combined EPHB2, SLAP, and SLA2 expression. High SLAP/SLA2 and EPHB2 co-expression is associated with improved prognosis in MSS patients.
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Figure 5—source data 1
PDF file containing original western blots for Figure 5, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/110324/elife-110324-fig5-data1-v1.pdf
-
Figure 5—source data 2
Original files for western blot analysis displayed in Figure 5.
- https://cdn.elifesciences.org/articles/110324/elife-110324-fig5-data2-v1.zip
Figure 5—figure supplement 1
Contribution of SLAP expression to the regulation of EPHB2-dependent CSC signaling in CRC patients.
(A) Negative correlation between SLAP expression and curated CSC/progenitor gene signature (15 genes including LGR5, ASCL2, OLFM4, BMI1, PROM1, CD44, MKI67, EPCAM, SOX9, SMOC2, TNFRSF19, AXIN2, CYP26A1, ITGA6, PCNA) in TCGA COAD samples (R = −0.2, p=0.0011). (B) EPHB2, or SLA and the SLAP-related gene SLA2 expression do not correlate with disease-free survival in TCGA MSS COAD samples.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Slap tm1a mice | KOMP Repository | Slaptm1a (KOMP); RRID:MMRRC_075158-UCD | C57BL/6 N background; LacZ reporter; FRT- and loxP-modified allele |
| Strain, strain background (Mus musculus) | Slap -/- mice | This study | Functional gene inactivation by LacZ cassette insertion | |
| Strain, strain background (Mus musculus) | Slap f/fl mice | This study | Generated after FlpO-mediated recombination | |
| Strain, strain background (Mus musculus) | Tg(CAG-flpo)1Afst | Kranz A et al., 2010 | MGI:4453967; RRID:MMRRC_036512-UCD | Flp deleter mouse in C57Bl/6 based on Flpo recombinase |
| Strain, strain background (Mus musculus) | Tg(Vil1-cre/ERT2)23Syr | el Marjou et al., 2004 | MGI:3053826; RRID:MGI:3053826 | Tamoxifen-inducible Cre expression in intestinal epithelial cells |
| Strain, strain background (Mus musculus) | Slap f/f Villin CreERT2 mice | This study | Conditional epithelial SLA knockout induced by tamoxifen | |
| Cell line (human) | HT29 | ATCC | HTB-38; RRID:CVCL_0320 | Human colorectal adenocarcinoma |
| Cell line (human) | SW620 | ATCC | CCL-227; RRID:CVCL_0547 | Human colorectal adenocarcinoma |
| Cell line (human) | HEK293T | ATCC | CRL-3216, RRID:CVCL_0063 | Human embryonic kidney cells used for transient transfection |
| Transfected construct (M. musculus) | SLAP-FLAG (WT) | Naudin et al., 2014 | FLAG-tagged wild-type SLAP expression plasmid | |
| Transfected construct (M. musculus) | SLAP-FLAG (SH3/SH2 mutant) | Naudin et al., 2014 | FLAG-tagged SH3/SH2-deficient SLAP mutant expression plasmid | |
| Transfected construct (human) | EPHB2-myc | Narayanan et al., 2023 | Myc-tagged human EPHB2 expression plasmid | |
| Transfected construct (human) | EPHB2 Y596F/Y602F mutant | This study | Myc-tagged human EPHB2 YF mutant expression plasmid | |
| Sequence-based reagent | Mm-LGR5-C2 | Bio-Techne | Catalog # 312178 | RNAscope probe for detection of mouse LGR5 |
| Antibody | Anti-Myc (mouse monoclonal) | Cell Signaling Technology | sc-2276S; RRID:AB_331783 | WB (1:1000), IP (1:250) |
| Antibody | Anti-EPHB2 (rabbit monoclonal) | Cell Signaling Technology | sc-83029 | WB (1:1000), IP (1:50) |
| Antibody | Anti-EPHB2 (goat polyclonal) | R&D Systems | Cat#AF467; RRID:AB_355375 | IHC (1:100) |
| Antibody | Anti-FLAG M2 (mouse monoclonal) | Sigma-Aldrich | Clone M2; RRID:AB_262044 | WB (1:1000), IP (1:250) |
| Antibody | Anti-HA (mouse monoclonal) | Invitrogen | Cat#26183; RRID:AB_10978021 | WB (1:1000) |
| Antibody | Anti-β-actin (mouse monoclonal) | Sigma-Aldrich | Cat#A2228; RRID:AB_476697 | WB (1:2000), (loading control) |
| Antibody | Anti-phosphotyrosine (4G10) (mouse monoclonal) | Gift from P. Mangeat | Clone 4G10; RRID:AB_2891016 | WB (1:1000) |
| Antibody | Anti-SLAP (goat polyclonal) | Santa Cruz Biotechnology | sc-1215 | WB (1:1000) |
| Antibody | Anti-phospho-SRC (rabbit polyclonal) | Invitrogen | PA5-97366; RRID:AB_2809160 | WB (1:1000) |
| Antibody | Anti-phospho-SRC (rabbit polyclonal) | Cell Signaling Technology | Cat#2101; RRID:AB_331697 | IHC (1:100) |
| Antibody | Alexa Fluor 488-conjugated secondary antibody (donkey polyclonal) | Jackson ImmunoResearch | Cat#711-545-152; RRID:AB_2340621 | Fluorescence IHC (1:500) |
| Antibody | Cy3-conjugated secondary antibody (donkey polyclonal) | Jackson ImmunoResearch | Cat#711-165-152; RRID:AB_2307443 | Fluorescence IHC (1:500) |
| Peptide, recombinant protein | Recombinant human R-spondin-1 | PeproTech | Cat#78213 | Organoid culture supplement |
| Peptide, recombinant protein | Recombinant human EGF | Bio-Techne | 236-EG-200 | Organoid culture supplement |
| Peptide, recombinant protein | Recombinant mouse Noggin | StemCell Technologies | Cat#78061 | Organoid culture supplement |
| Peptide, recombinant protein | Recombinant mouse WNT3A | Thermo Fisher Scientific | Cat#315–20 | Organoid culture supplement |
| Commercial assay or kit | ALDEFLUOR Kit | StemCell Technologies | Cat#01700 | ALDH activity assay |
| Commercial assay or kit | RNeasy Plus Mini Kit | Qiagen | Cat#74134 | RNA extraction |
| Commercial assay or kit | SuperScript VILO cDNA Synthesis Kit | Thermo Fisher Scientific | Cat#11754050 | Reverse transcription |
| Commercial assay or kit | RNAscope Multiplex Fluorescent Reagent Kit | Bio-Techne | Cat#323100 | RNA in situ hybridization assay for FFPE tissues |
| Commercial assay or kit | QuickChange Site-Directed Mutagenesis Kit | Agilent | Cat#200519 | Site-directed mutagenesis |
| Commercial assay or kit | Tumor Dissociation Kit, Mouse | Miltenyi Biotec | Cat#130-096-730 | Tumor dissociation |
| Commercial assay or kit | BIOXALL | Vector Laboratories | SP-6000–100 | Endogenous Peroxidase and Alkaline Phosphatase Blocking Solution |
| Commercial assay or kit | DAB | Vector Laboratories | SK-4100 | Chromogenic detection of HRP activity |
| Chemical compound, drug | Tamoxifen | Merck | Cat#5648–1 G | CreERT2 activation |
| Chemical compound, drug | Azoxymethane (AOM) | Sigma-Aldrich | Cat#A5486 | Colon carcinogenesis model induction |
| Chemical compound, drug | Dextran sodium sulfate (DSS) | TdB Labs AB | DB001 | Colitis induction |
| Chemical compound, drug | X-gal | Sigma-Aldrich | Cat#71077–3 | β-Galactosidase staining |
| Chemical compound, drug | eCF506 | MedChemExpress | Cat#HY-112096 | SRC inhibitor |
| Chemical compound, drug | ALW-II-49–7 | MedChemExpress | Cat#HY-18833 | EPHB2 inhibitor |
| Chemical compound, drug | ALW-II-41–27 | MedChemExpress | Cat#HY-18007 | Pan-Eph inhibitor |
| Chemical compound, drug | Y-27632 dihydrochloride | Sigma-Aldrich | Cat#Y0503 | Rock inhibitor |
| Chemical compound, drug | CHIR-99021 | Tebu-Bio | Cat#10–1279 | GSK3 inhibitor |
| Chemical compound, drug | N-acetyl-L-cysteine | Sigma-Aldrich | Cat#A9165 | Organoid culture supplement |
| Other | Matrigel GFR phenol-red free | Corning | REF 356231 | Basement Membrane Matrix for organoids and tumoroids |
| Other | Advanced DMEM/F12 | Gibco | Cat#12634010 | Basal culture medium |
| Other | B27 Supplement | Gibco | Cat#17504044 | Serum-free supplement |
| Other | N2 Supplement | Gibco | Cat#17502048 | Serum-free supplement |
| Other | GlutaMAX | Gibco | Cat#35050061 | Cell culture supplement |
| Software, algorithm | GraphPad Prism | GraphPad Software | Version 9.3.1 | Statistical analysis |
| Software, algorithm | ImageJ | NIH | RRID:SCR_003070 | Image processing and quantification |
| Software, algorithm | NovoExpress | Agilent | Flow cytometry analysis | |
| Software, algorithm | ZEN | Zeiss | Microscope image acquisition | |
| Software, algorithm | NDP.view2 | Hamamatsu | U12388-01 | Image viewing |
| Software, algorithm | QuPath | Bankhead et al., 2017 | RRID:SCR_018257 | IHC quantification |
| Software, algorithm | GEPIA2 | Tang et al., 2019 | http://gepia2.cancer-pku.cn | TCGA gene expression and survival analyses |
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Slap restricts oncogenic Src-family kinase signaling to maintain colonic epithelial homeostasis
eLife 15:RP110324.
https://doi.org/10.7554/eLife.110324.3
