Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway

  1. Ryosuke Isotani
  2. Masaki Igarashi  Is a corresponding author
  3. Masaomi Miura
  4. Kyoko Naruse
  5. Satoshi Kuranami
  6. Manami Katoh
  7. Seitaro Nomura
  8. Toshimasa Yamauchi  Is a corresponding author
  1. Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Japan
  2. Department of Cardiovascular Medicine, The University of Tokyo Graduate, School of Medicine, Japan
  3. Department of Frontier Cardiovascular Science, The University of Tokyo, Graduate School of Medicine, Japan
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Nicotine (NIC) treatment increases the number of ISCs in the Intestine.

(A and B) Image of Ki67‐positive cells (Red: Ki67, Blue: DAPI) and their quantification at the crypt base of proximal jejunum (A) or colon (B) of NIC-treated and untreated mice (A:3–4 mice per …

Figure 1—figure supplement 1
NIC suppresses the differentiation of ISCs.

(A) Images of the H&E-stained crypt and villus and quantification of their length after or without NIC treatment (3–4 mice per group). (B) The quantification of the number of enterocytes per villus …

Figure 2 with 1 supplement
NIC enhances the formation of intestinal organoids from ISCs.

(A) Crypts from the proximal small intestine were cultured with 10 μM, 1 μM, and 100 nM NIC or cotinine to allow ISCs to form organoid colonies; the control set contained no NIC or cotinine. …

Figure 2—source data 1

The quantification of colonies in organoid assay.

https://cdn.elifesciences.org/articles/95267/elife-95267-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
NIC induces the formation of intestinal organoids from ISCs.

ISCs and Paneth cells were isolated from the small intestine of Lgr5‐EGFP‐IRES‐CreERT2 mice (NIC-treated and untreated); 2×103 cells were co-cultured using medium without 10 μM CHIR99021. …

Figure 2—figure supplement 1—source data 1

The quantification of colonies in organoid assay.

https://cdn.elifesciences.org/articles/95267/elife-95267-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 1 supplement
The effect of NIC is mediated via the α7 subunits of α7-nicotinic acetylcholine receptor (nAChR).

(A) Isolated ISCs were cultured in a medium with or without 1 μM NIC and 10 μM Mecamylamine (3 wells/group). (B) Isolated ISCs were cultured in a medium with or without 1 μM NIC and 3 μM Adiphenine …

Figure 3—figure supplement 1
The distribution of human nAchR subunits in ISCs and Pante cells.

(A) Violin plots for nAchR subunits in ISCs and Panth cells by the analysis of human gut single-cell datasets (Elmentaite et al., 2021). (B) The number of positive cells for each nAchR in ISCs and …

Figure 4 with 1 supplement
NIC induces Hippo-YAP/TAZ and notch signaling in ISCs.

(A and B) Isolated ISCs cultured using a medium supplemented with or without 1 μM NIC combined with either (A) 1 μM H89 dihydrochloride (PKA inhibitor) or (B) 10 nM Gö 6983 (PKC inhibitor; 3 …

Figure 4—figure supplement 1
PI3K/AKT, mTORC1, and p38 /MAPK signaling cascades do not mediate the effect of NIC.

(A) Isolated ISCs cultured using a medium supplemented with or without 1 μM NIC combined with 5 nM Sotrastaurin (PKC inhibitor) (3 wells/group). (B) Immunoblotting analyses of p38, p-p38, p-S6, or …

Inactivation of Hippo-YAP/TAZ and Notch signaling suppresses NIC-induced Colony Formation in mice.

(A) Isolated ISCs were cultured using a medium with or without 1 μM Nicotine and 5 nM K-975 (3 wells/group). (B) Isolated ISCs were cultured using a medium with or without 1 μM Nicotine and 1 μM …

Figure 5—source data 1

The quantification of colonies in organoid assay.

https://cdn.elifesciences.org/articles/95267/elife-95267-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
Dibenzazepine (DBZ) treatment suppresses the NIC-induced expansion of ISCs in vivo.

(A) Schematic representation of the treatment showing daily injection of DBZ (1 mg/kg body weight) for 2 weeks. (B) Immunoblotting analysis of crypt lysate isolated from DBZ- and vehicle-treated …

Figure 6—figure supplement 1
DBZ treatment suppresses the NIC-induced expansion of CSCs in vivo.

(A and B) Immuno-stained Ki67-positive cells (A) (Red, Ki67; Blue, DAPI) and LgR5-GFP-positive cells (B) (Red, Olfm4; blue, DAPI) and quantification of their abundance in the colon of DBZ or …

Figure 6—figure supplement 1—source data 1

The quantification in immunostaining.

https://cdn.elifesciences.org/articles/95267/elife-95267-fig6-figsupp1-data1-v1.xlsx
BZ inhibits intestinal tumor growth by NIC.

(A) Schematic representation of Apcflox/flox; Lgr5-EGFP-IRES-CreERT2 (Lgr5CreERT2 Apcfl/fl)tumor initiation. Mice were treated with control or NIC more than 8 weeks before a single Tamoxifen …

Figure 7—source data 1

The quantification of polyps and β-catenin positive lesions.

https://cdn.elifesciences.org/articles/95267/elife-95267-fig7-data1-v1.xlsx

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)Lgr5-EGFP-IRES-CreERT2 miceJackson Laboratory#008875
Strain, strain background (Mus musculus)Apc CKO miceNational Cancer Institute#01XAA
Strain, strain background (Mus musculus)Rosa26-CAG-lsl-tdTomato miceJackson Laboratory#007909
Antibodyrabbit monoclonal anti-Ki67Cell Signaling Technology#12202IHC (1:200)
Antibodyrabbit monoclonal anti-Olfm4Cell Signaling Technology#39141IHC (1:400)
Antibodymouse monoclonal anti-GFPSanta Cruz Biotechnology#sc-9996IHC (1:50)
Antibodyrabbit polyclonal anti-LysozymeThermo Fisher Schientific#PA5-16668IHC (1:50)
Antibodymouse monoclonal anti-chromogranin ASanta Cruz Biotechnology#sc-393941IHC (1:50)
Antibodyrabbit polyclonal anti-β-cateninCell Signaling Technology#9562IHC (1:200)
Antibodygoat polyclonal anti-mouse IgG H&L (Alexa Fluor 488)Abcam#ab150113IHC (1:200)
Antibodymouse monoclonal anti-β-actinSanta Cruz#sc-47778WB (1:200)
Antibodymouse monoclonal anti-YAPSanta Cruz#sc-101199WB (1:200)
Antibodymouse monoclonal anti-TAZSanta Cruz#sc-293183WB (1:200)
Antibodymouse monoclonal anti-α7-AchRSanta Cruz#sc-58607WB (1:200)
Antibodymouse monoclonal anti-Notch1Santa Cruz#sc-376403WB (1:100)
Antibodymouse monoclonal anti-Jagged1Santa Cruz#sc-390177WB (1:100)
Antibodymouse monoclonal anti-Jagged2Santa Cruz#sc-515725WB (1:100)
Antibodymouse monoclonal anti-Hes5Santa Cruz#sc-293445WB (1:200)
Antibodymouse monoclonal anti-p38Santa Cruz#sc-81621WB (1:200)
Antibodymouse monoclonal anti-phospho-p38Santa Cruz#sc-166182WB (1:100)
Antibodyrabbit monoclonal anti-S6Cell Signaling#2217WB (1:1000)
Antibodyrabbit monoclonal anti-phospho-S6 Ser235/236Cell Signaling#4858WB (1:2000)
Antibodyanti-Mouse IgG, HRP-Linked Whole Ab SheepCytivaNA931WB (1:5000)
Antibodyanti-Rabbit IgG, HRP-Linked Whole Ab DonkeyCytivaNA934WB (1:5000)
Antibodyrat monoclonal APC-conjugated anti-mouse CD24 AntibodyBiolegend#101814FCY (1:500)
Commercial assay or kitTSA Plus Cyanine 3 SystemAkoya Biosciences#NEL744001KT
Chemical compound, drugMecamylamineCayman Chemical#14602
Chemical compound, drugAdiphenine hydrochlorideMedChemExpress#HY-B0379A
Chemical compound, drugPNU282987MedChemExpress and Cayman Chemical#17424
#HY-12560A
Chemical compound, drugα-BungarotoxinR&D#2133/1
Chemical compound, drugH-89 dihydrochlorideMedChemExpress#HY-15979A
Chemical compound, drugGö 6983Cayman Chemical#13311
Chemical compound, drugSotrastaurinMedChemExpress#HY-10343
Chemical compound, drugK-975MedChemExpress#HY-138565
Chemical compound, drugMK-0752MedChemExpress#HY-10974
Chemical compound, drugIngenol-3-angelateCayman Chemical#16207
Chemical compound, drugAKT Inhibitor VIIIMedChemExpress#HY-10355
Chemical compound, drugSB203580Tokyo Chemical Industry#F0864
Chemical compound, drugRapamycinLKT Laboratories, Inc#R0161
Chemical compound, drugCollagenase Type IVWorthington Biochemical Corporation#CLS4
Chemical compound, drugValproic acid sodium saltFUJIFILM Wako Pure Chemical Corporation#2815/100
Chemical compound, drug[-]-CotinineSigma-Aldrich#C-016
Chemical compound, drugNicotine hemisulfate saltSigma-Aldrich#N1019
Chemical compound, drugDBZ (Dibenzazepine)Cayman Chemical#14627
Chemical compound, drugTamoxifenCayman Chemical#13258
Chemical compound, drugMounting medium With DAPI Aqueous FluoroshieldAbcam#ab104139
Chemical compound, drugCan Get Signal Immunoreaction Enhancer SolutionTOYOBONKB-101

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