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

Cigarette smoking is a well-known risk factor inducing the development and progression of various diseases. Nicotine (NIC) is the major constituent of cigarette smoke. However, knowledge of the mechanism underlying the NIC-regulated stem cell functions is limited. In this study, we demonstrate that NIC increases the abundance and proliferative activity of intestinal stem cells (ISCs) in vivo and ex vivo. Moreover, NIC induces Yes-associated protein (YAP)/Transcriptional coactivator with PDZ-binding motif (TAZ) and Notch signaling in ISCs via α7-nicotinic acetylcholine receptor (nAchR) and protein kinase C (PKC) activation; this effect was not detected in Paneth cells. The inhibition of Notch signaling by dibenzazepine (DBZ) nullified the effects of NIC on ISCs. NIC enhances in vivo tumor formation from ISCs after loss of the tumor suppressor gene Apc, DBZ inhibited NIC-induced tumor growth. Hence, this study identifies a NIC-triggered pathway regulating the stemness and tumorigenicity of ISCs and suggests the use of DBZ as a potential therapeutic strategy for treating intestinal tumors.


Introduction
All tissues and organs are generated from stem cells.Abnormal stem cell functions are significantly associated with age-related organ dysfunction and carcinogenesis 1 .Intestinal epithelial turnover is sustained by intestinal stem cells (ISCs) and adjacent Paneth cells.Paneth cells constitute the niche for ISCs that reside at the bottom of the crypts.Most of the ISCs are leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)+ ones that regulate intestinal homeostasis in response to dietary signals 2,3 .In the intestine, Lgr5+ ISCs are possibly the origin of precancerous adenomas 4 .For instance, long-term high-fat diet (HFD)-induced obesity enhances the selfrenewal potential of ISC and promotes in vivo formation of tumors via the induction of peroxisome proliferator-activated receptor delta in ISCs 5 .
Moreover, cigarette smoking has emerged as a potential major risk factor associated with colon cancer, along with metabolic risk factors such as diet and obesity.Previous studies indicate that cigarette smoking is significantly associated with colon cancer and mortality in humans 6 and animal models 7 .
Cigarette smoke contains a wide range of compounds that are harmful to human health; nicotine (NIC) derivatives 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are highly carcinogenic 8,9 , which can induce mutations in tumor suppressive genes like Ras, p53, and Rb 10 .Although NIC itself, the addictive component in cigarette smoke, is generally considered to have a limited ability to initiate cancer, it can stimulate several effects crucial for cancer development independently 11 .However, knowledge of the mechanism underlying the NIC-regulated ISC functions and intestinal tumorigenicity is limited.
In this study, we aimed to demonstrate the effects of NIC treatment on the functions of murine ISCs ex vivo and in vivo.Moreover, we explored the molecular factors and signaling cascades prospectively associated with the regulation of the effects of NIC on ISCs.This study can potentially contribute to the comprehensive knowledge on the pathway by which stem cells respond to NIC, a major component of cigarette smoke, and suggest an effective new strategy for the treatment of smoking-related colon cancer.

NIC treatment enhances the frequency of ISC in the intestine
Histological analyses of the small intestine in C57BL/6 mice treated with 200 μg/ml NIC (which emulates active smoking) revealed that NIC exposure decreased villus length without affecting crypt size (Figure S1A), which was consistent with NIC-induced decrease in the number of differentiated cells, including absorptive enterocytes (Figure S1B) or chromogranin A+ enteroendocrine cells (Figure S1C), in the gut.
We investigated the effect of NIC on the population of proliferative cells in the crypts using Ki67 labeling to mark proliferative stem and progenitor cells in the crypts.NIC exposure increased abundance of Ki67-positive cells in the small and large intestine (Figures 1A and B).Consistently, the number of proliferative olfactomedin-4 (Olfm4)-positive ISCs significantly increased in the small intestine of NIC-treated mice (Figure 1C).Moreover, the number of LgR5+ colonic stem cells (CSCs) was increased in NIC-treated Lgr5-EGFP-IRES-CreERT2 mice expressing EGFP under the control of the LgR5 promoter (Figure 1D).Paneth cells support the proliferation of ISCs.However, we did not observe any changes in the number of Paneth cells in NIC-treated mice (Figure 1E).These results indicate that the self-renewal of ISCs in NIC-treated mice increased with a reciprocal decrease in the number of differentiated cells.

NIC treatment enhances the formation of intestinal organoids from ISCs
Our ISC proliferation analysis using intestinal crypts of wild-type mice revealed that a range of nicotine concentrations (100nM, 1 µM, and 10 µM) promoted the organoid formation from crypts from the small intestine (Figure 2A), which was consistent with the in vivo data (Figures 1A and C).
However, the same dose of cotinine, a minor tobacco alkaloid, and a major metabolite of NIC 12 did not exhibit similar effects (Figure 2A).Furthermore, the addition of 1 µM NIC promoted the organoid formation from colonic crypts (Figure 2B).Next, to address how ISCs and Paneth cells interact functionally, we isolated Lgr5-positive ISCs and Paneth cells from control or NICtreated Lgr5-EGFP-IRES-CreERT2 mice as described previously 2 .ISCs and Paneth cells were co-cultured in the culture media containing glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021, which induces β-catenin and thus stimulates organoid formation 2,13 .Lgr5-positive ISCs isolated from NIC mice formed more organoid colonies than those isolated from control mice when cultured with or without Paneth cells (Figure 2C).Consistently, the addition of 1 µM NIC to control ISCs stimulated the organoid colony formation (Figure 2D).However, Paneth cells exhibited no significant difference in function (organoid formation) between control and NIC-treated groups, with or without CHIR99021 (Figures 2C and S2A).Therefore, this ex vivo assay for ISC function and ISC-Paneth cell interaction demonstrated that NIC directly stimulates ISC proliferation without affecting the supportive function of Paneth cell for ISC.

The 7 subunits of nAChR control the effects of NIC on ISC proliferation
We investigated the pathway underlying the NIC-regulated proliferation of ISCs.
NIC interacts with nicotinic acetylcholine receptors (nAchRs), which are heterodimers of nine types of  subunits (α2-α10) and three types of β subunits (β2-β4) 14 .We validated the significance of nAChR signal transduction using ISCs, isolated wild-type mice cultured in the presence of NIC, and the nonselective nAChR antagonist Mecamylamine, which indicated that Mecamylamine treatment completely abolishes the NIC-mediated formation of ISC-derived organoids (Figure 3A).We further explored the nAChR subtypes.
Considering that NIC has a high affinity for the nAChR comprised of α4 and β2 subunits 15 , we cultured ISCs in the presence of NIC and Adiphenine, a noncompetitive inhibitor of nAChR (α1, α3β4, α4β2, and α4β4).However, Adiphenine exhibited no effect on the NIC-induced organoid formation from ISCs, indicating that the effect of NIC was not mediated by these nAChRs (Figure 3B).As some cancer stem cells are known to express 7-nAChR 16,17 , we further analyzed the role of 7-nAChR.The existence of 7-nAChR in ISCs was detected by immunoblotting and RT-PCR, respectively (Figures 3C and D).
The higher expression of α7 and α8 subunits in both ISCs and Paneth cells were observed compared with other nAChR subunits.However, there were not the significant difference in expression of α7 or α8 subunit between ISCs and Paneth cells (Figure 3C).To investigate the distribution of nAChRs subunits in human intestine, we analyzed scRNA-seq datasets of the human intestinal epithelium 18 .In consistent with mouse data (Figure 3C), the expression of human α7 subunit (CHRNA7) is higher than that of other subunits in both ISCs and Paneth cells (Figures S3A and B), although the predominance of the expression in ISCs or Paneth cells was not clear (Figures S3A and B).
Interestingly, NIC treatment significantly upregulated 7-nAChR in ISCs rather than Paneth cells (Figures 3C and D).Importantly NIC treatment did not induce other nAChR subunits in ISCs (Figure 3C).Moreover, addition of the 7-selective nAChR agonist PNU282987 increased organoid colony formation from ISCs (Figure 3E), which was consistent with the effects of NIC.Furthermore, α-Bungarotoxin, an 7-selective nAChR antagonist, completely inhibited the NIC-induced increase in organoid formation (Figure 3F).These outcomes indicate the effect of nicotine is mediated via the 7 subunits of nAChR.

NIC induces a Hippo-YAP/TAZ and notch signaling in ISCs
As NIC can activate protein kinase C (PKC) or cAMP-dependent protein kinase A (PKA) via α7-nAChR activation 17,19 , we cultured isolated ISCs in the presence of NIC combined with Go6983, a pan-PKC inhibitor, or H89, a selective PKA inhibitor.H89 did not suppress NIC-induced organoid formation, however, Go6983 completely abolished this effect of NIC (Figures 4A and B).
Additionally, we cultured ISCs with NIC and Sotrastaurin, another pan-PKC inhibitor inactive to PKCζ, the loss of which is reported to increase ISC activity both in vivo and in vitro 20 .The observed results with Go 6983 was reproduced by Sotrastaurin treatment (Figures S4A), demonstrating other PKCs than PKCζ mediates the effect of NIC.Consistently, the PKC activator Ingenol-3-angelate stimulated the formation of organoid colonies from ISCs (Figures 5E and 5F).Next, to investigate the possible downstream signaling of 7-nAChR and PKC associated with NIC-induced renewal of ISCs, we explored PI3K/AKT signaling 21 , p38/ mitogen-activated protein kinase (MAPK) signaling 22 , and mTORC1 signaling 2,23 , each of which plays a crucial role in the expansion of ISCs.Results indicate that NIC does not induce these cascades and these were not required for the effects of NIC on ISCs (Figures S4 B-E).YAP/TAZ, the downstream effectors of the Hippo signaling pathway, and Notch receptor 1 (Notch1)/Delta-Like protein 1(Dll1), the main components of Notch signaling are reported to be upregulated in NIC-treated organoids 24 .
Both the Hippo-YAP/TAZ and Notch signaling pathways regulate intestinal homeostasis via the control of ISC function 25,26 and Notch signaling is positively regulated by YAP/TAZ in the intestine 27 .Hence, we examined Hippo-YAP/TAZ and Notch signaling.YAP1 and TAZ expression was significantly induced in the crypts of NIC-treated mice (Figure 4C).YAP1 and TAZ also upregulated at mRNA level in ISCs from NIC mice (Figure 4D).Moreover, activation of Notch signaling in crypts of NIC-treated mice was confirmed through immunoblotting assay (Figure 4E).The expression of genes, including Jagged1, HeyL, Hes1, Hes5, and Dll1, involved in Notch signaling, significantly increased in ISCs obtained from NIC-treated mice (Figure 4D).Notably, Hippo-YAP/TAZ and Notch signaling were not significantly activated in Paneth cells, indicating that NIC affects α7-nAchR of ISCs rather than Paneth cells.
Collectively, these results suggest that NIC induces a Hippo-YAP/TAZ and Notch signal pathway in ISCs via activation of α7-nAchR and PKC.

Inactivation of Hippo-YAP/TAZ and Notch signaling suppresses the NICinduced colony formation
To further explore the role of the Hippo-YAP/TAZ and Notch signaling in NIC-induced ISC expansion, ISCs were cultured with NIC in the presence of either K-975, a specific inhibitor of transcriptional enhanced associate domain (TEAD), which binds to its transcriptional co-activators YAP or TAZ and forms a transcription complex 28 , or -secretase inhibitor MK-0752 that inhibits the cleavage of Notch into its active signaling effector, Notch intracellular domain (NICD) 29 .Treatment with K-975 and MK-0752 completely abolished the NICinduced increased organoid formation (Figures 5A and B).Furthermore, K-975 or MK-0752 prevented the increase in the organoid formation in ISCs treated with PNU 298987 or Ingenol-3-angelate (Figures 5C-F), suggesting that YAP/Notch signaling acts downstream of α7-nAchR or PKC leading to the response of ISC to NIC (Figure 5G).

DBZ treatment suppresses the expansion of ISCs by NIC in vivo
To validate the significance of Notch signaling in the ISC expansion, NIC-treated mice were subjected to daily IP injection of -secretase inhibitor DBZ (1 mg/kg body weight) for 2 weeks (Figure 6A).We confirmed that DBZ treatment significantly downregulated Hes5 protein expression in the crypts of NIC-treated mice (Figure 6B).Notably, DBZ suppressed the expression of YAP and TAZ in NIC-treated mice (Figure 6B).In the intestine, YAP/TAZ regulates Notch signaling 27 and Notch activation can activate YAP/TAZ 30 .Consistent with previous reports, our results demonstrate that Notch inhibitor suppresses YAP/TAZ and Notch activities, indicating a positive feedback loop between Hippo-YAP/TAZ and Notch signaling in ISCs of NIC mice (Figure 5G).Ki67 labeling and immunostaining for the ISC marker Olfm4 elucidated the effect of DBZ treatment on the frequency of ISCs in control and NIC-treated mice.DBZ treatment did not alter the population of ISCs in the control mice but significantly suppressed the expansion of Ki67+ and Olfm4+ cells in NIC-treated mice (Figures 6C and D).Moreover, DBZ suppressed the expansion of Ki67+ cells and CSCs in the colon of NIC-treated mice (Figures S4A and B).These outcomes demonstrate that the Hippo-YAP/TAZ and Notch signaling pathways are crucial for ISC expansion in NIC-treated mice analyzed in vivo.

DBZ inhibits intestinal tumor growth by NIC
As ISCs are the potential origin of tumors 4 , we hypothesized that NICinduced ISC expansion can promote tumor formation in a tumor-initiating background, such as Apc loss.We crossed stem cell-specific Lgr5-EGFP-IRES-creERT2 knock-in mice with Apc flox/flox mice (Lgr5Cre ER Apc fl/fl mice); in the resulting mice, the transformation of Lgr5-GFP positive stem cells efficiently drives adenoma formation throughout the intestine after Apc loss induced by Cre activation using tamoxifen (Figure 7A).
To test whether NIC promotes tumor formation via ISC expansion, tumor formation was induced in NIC-treated Lgr5Cre ER Apc fl/f (NIC-Lgr5Cre ER Apc fl/fl ) mice, and entire intestines, isolated from them, were examined for polyps (Figure 7A).A marked increase in the abundance of polyps was detected throughout the intestine of NIC-treated Lgr5Cre ER Apc fl/fl mice; moreover, these polyps were significantly larger than that in the control mice (Figure 7B).Consistently, the area of β-catenin positive adenomatous lesions throughout the entire intestine significantly increased in NIC-Lgr5Cre ER Apc fl/fl mice (Figure 7C).These results indicated that NIC increased the overall polyp burden in the intestines of Lgr5Cre ER Apc fl/fl mice by increasing their size and number.
Finally, to validate whether DBZ can suppress NIC-induced intestinal adenomas, we treated NIC-Lgr5Cre ER Apc fl/fl mice with DBZ four weeks after the induction of Apc loss (Figure 7D).Notably, this treatment effectively reduced the abundance of polyps and β-catenin positive adenomatous lesions in NIC-Lgr5Cre ER Apc fl/fl , indicating the efficiency of DBZ for the prevention and treatment of NIC-induced intestinal tumors (Figures 7E and F).Treatment of organoids with NIC has been reported to enhance cell growth and expression of marker genes of stem cells 24 .Moreover, mRNA analysis of NIC-treated organoids showed that the expression of YAP1/TAZ and Notch1/Dll1 was upregulated after treatment with NIC 31 .This previous report predicted that NIC activates the Hippo and Notch signaling pathway in Paneth cells rather than ISCs, because the α2β4-nAChR was mainly expressed in Paneth cells 31 .However, the hypothesis was not fully testified in the report.In contrast, our analysis validates that NIC affects ISC rather than Panth cells via Hippo-YAP/TAZ and Notch signaling activated though nAchRa7 by mRNA and protein analyses of sorted ISCs or Paneth cells and ex vivo functional assays.

Discussion
The crosstalk between Hippo, Notch, and Wnt signaling regulates mammalian intestinal homeostasis 30,32,33 .Activated YAP/TAZ, which act downstream of Hippo signaling, translocate to the nucleus and induce the gene expression of Notch receptors and Notch ligands, and thus, Notch is a mediator of YAP1-induced ISC expansion 27,30 .Moreover, YAP/TAZ and Notch signaling congruently induces nuclear translocation of YAP/TAZ and NICD, regulating the expression of common target genes 30 .Furthermore, YAP with TEADs enhances Jagged1-Notch1 signaling; Notch1 promotes YAP stability through inhibited β-TrCP-mediated degradation and formation of a YAP1-jagged-1/Notch1 positive feedback loop in breast cancer cells 34 .Similarly, we propose a YAP/TAZ-Notch positive feedback loop in ISCs of NIC-treated mice (Figure 5G).This model is consistent with DBZ-induced suppression of Hippo-YAP/TAZ and Notch signaling in ISCs from NIC-treated mice (Figure 6B).As NIC effectively expands the ISC population through the upregulated 7-nAChR and the formation of the YAP/TAZ-Notch loop, we speculate these factors to be good therapeutic targets for treating NIC-induced colon cancer.
In the intestine, YAP activation enhances the expression of both βcatenin and transcriptional targets of Wnt signaling 35 .Moreover, Notch signaling concomitantly regulates intestinal cell proliferation via Wnt signaling 36 .NIC is supposed to activate Wnt signaling via Hippo-YAP/TAZ and Notch signaling.In consistent with this notion.Similarly, the expression of target proteins (Sox9, TCF4 and, C-myc) of the Wnt/β-catenin pathway as well as cell-cycle regulatory proteins, such as cyclin B, and cyclin E, was up-regulated in crypts obtained from NIC-treated mice in our experiment (data not shown).However, the effect of NIC on ISC organoid formation appears to be independent of CHIR99021, a Wnt activator (Figure 2).Moreover, In the Lgr5Cre ER Apc fl/fl mouse model, APC loss results in a constitutive stabilization of β-catenin, thus the hyperproliferation of ISCs by NIC treatment in this mouse model is likely beyond Wnt activation (Figure 7).The downstream pathway of Hippo-YAP/TAZ and Notch signaling remains to be determined.
Our study demonstrated that NIC increased the expression of YAP/TAZ and Notch target genes, as well as the ISC population.Our ex vivo organoid assay revealed the role of Hippo-YAP/TAZ and Notch signaling in NIC-induced ISC expansion.However, the mechanism underlying NIC-activated Hippo-YAP/TAZ and Notch signaling also remains unclear.NIC administration can induce the nuclear translocation and activation of YAP in Esophageal Squamous Cell Cancer 37 .The nAChRs physically interact with YAP, leading to the upregulation and nuclear translocation of YAP1.This process is considered to be mediated by PKC activation because PKC-specific inhibitors block NIC-induced YAP activation 37 .Moreover, PKC activity induces the translocation of ADAM-10, which is implicated in the cleavage of Notch receptors 38 , to the cell membrane of glioblastoma 39 .Hence, we propose the possibility of NIC-induced activation of YAP and ADAM via PKC, leading to Hippo-YAP/TAZ and Notch activation and ISC expansion.
The lifetime risk of various cancers is strongly correlated with frequency of stem cell divisions that maintain tissue homeostasis 40 .However, the risk of cancer associated with stem cell divisions is heavily influenced by both extrinsic and intrinsic factors 41 .Extrinsic factors, such as NIC, may increase the risk of cancer by promoting stem cell division.Our data indicated that NIC increased the abundance and proliferation of ISCs, which may partly explain the increased rate of intestinal tumors in smokers.
In conclusion, we demonstrated that NIC enhances the ISC population via 7-nAChR as well as Hippo-YAP/TAZ and Notch signaling.These findings revealed the pivotal role of NIC as a stimulant of the cancer stem cell proliferation in intestinal tumors, and thus, explains colon cancer development in cigarette smokers.The development of drugs that can block the 7-nAChR, Hippo-YAP/TAZ, and Notch signaling may provide a new therapeutic strategy for treating colon cancers.

Nicotine treatment
Mice were administered with 200 μg/ml NIC (Nicotine hemisulfate salt, Sigma-Aldrich, Saint Louis, MO) through drinking water for more than 8 weeks.
Water bottles were replaced every alternate day.

Tamoxifen treatment
Recombination by Lgr5Cre ER in Lgr5Cre ER Apc fl/fl :tdTomato mice was induced with a single dose of tamoxifen (30 mg kg −1 body weight) suspended in corn oil, administered through the intraperitoneal injection.LgR5 Cre ER -induced mice were analyzed 4 weeks after induction.

DBZ treatment
The mice were intraperitoneally injected with DBZ (1 mg/kg body weight; Cayman Chemical, Ann Arbor, MI) or DMSO (in PBS) for 2 or 4 weeks.

Crypt Isolation and Culture
Crypts were isolated as described previously 2 , 23 .For this purpose, the proximal half of the small intestine was isolated, opened longitudinally, and washed with cold PBS.After washing the intestine with cold PBS, it was cut into small (5 mm long) pieces with scissors and washed using cold PBS.

Flow cytometry
ISC and Paneth cells were isolated from dissociated intestinal crypts using flow cytometry as described previously 2,23 .The crypts were centrifuged for 5 min at 300 × g at 4 °C and the pellets were gently resuspended in 800 μl TrypLE Express (Thermo Fisher Scientific) supplemented with 200 μl PBS followed by incubation in a water bath at 32 °C for 1.5 min; after incubation, the samples were placed on ice.Next, 12 mL of cold minimum essential medium (MEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was added, and the samples were gently triturated twice.After centrifugation for 5 min at 200 × g at 4 °C, the pellets were resuspended and incubated for 15 min on ice in 0.5 ml MEM containing CD24-APC antibody (1:500, 101814, Biolegend, San Diego, CA).After centrifugation for 5 min at 200 × g at 4 °C, the pellets were resuspended in MEM containing 1.5 µM propidium iodide (PI) (Fujifilm Wako Pure Chemical Corporation).The samples were filtered through a 40 µm mesh (Corning) and immediately sorted using a BD FACS Aria III Cell Sorter (BD Life Sciences, San Jose, CA).ISCs were isolated as Lgr5-EGFP hi CD24 low PI -and Paneth cells were isolated as CD24 hi SideScatter hi Lgr5-EGFP − PI − .

Colonic crypt isolation and culture
Colonic crypts were isolated from the large intestine as described previously with a few modifications23.For this purpose, a 5-7 cm part of the proximal large intestine was isolated, opened longitudinally, and washed with cold PBS.After washing with cold PBS, the intestine was cut into small (5 mm long) pieces with scissors, placed in cold 5 mM EDTA-PBS, and gently rocked for 15 min at 4 °C.After removal of EDTA-PBS, pieces of the intestine were incubated in DMEM/F12 containing 500 U/ml Collagenase Type IV (Worthington Biochemical Corporation, Lakewood, NJ) for 30 min at 37 °C using a water bath.Subsequently, pieces of the intestine were pipetted up and down in cold PBS until most of the crypts were released.The crypt fraction was obtained by passing the suspension through a 70 μm cell strainer followed by centrifuging at 250 × g for 5 min.Isolated crypts were collected in a crypt culture medium, counted, and embedded in Matrigel.A total of 1,000 crypts were plated per well of a 48-well plate and cultured using a colonic crypt culture medium (DMEM/F12 supplemented by 1xN2, 1xB27, 1 mM N-Acetyl-L-cysteine, 50 ng/ml EGF, 100 ng/ml Noggin, 500 ng/ml R-spondin, 2 mM Valproic acid [FUJIFILM Wako Pure Chemical Corporation], and 10 μM CHIR99021).The medium was replaced in 2 days.
Nicotine hemisulphate salt was added to the crypt culture medium as needed for experiments.The number of alive spherical organoids formed from the crypts was measured under microscope 5 days after plating them.

Investigation of intestinal polyps
The entire intestine of Lgr5Cre ER Apc fl/fl :tdTomato mice, in which adenomatous polyps were labeled with tdTomato using tamoxifen injection, was promptly excised and cut with the mucosal side up, washed with ice-cold PBS, pinned open on a dissection tray, and fixed using 10% neutral buffered formalin (FUJIFILM Wako Pure Chemical Corporation).The fixed intestine was then photographed, polyps were counted, their diameters were measured using a caliper, and the surface of the polyps was estimated.For the histopathological assay, the entire intestine was fixed on a dry board and gently rolled to form a Swiss roll, which was further used in the immunohistological test.
Our data propose a model in which NIC enhances the self-renewal of ISCs via activated Hippo-YAP/TAZ and Notch signaling (Figure.5G).Our analyses revealed an increase in the number of ISCs in NIC-treated mice.In contrast to ISCs, Paneth cells showed no increase in cell numbers in NICtreated mice.Similarly, we assessed the functional ability of ISCs and Paneth cells in terms of ex vivo organoid formation, which indicated a NIC-induced gain-of-function in ISCs but not in Paneth cells.Hence, we propose that the increase of ISC activity induced by NIC are not dependent on Paneth cells in functional assays.Furthermore, we traced the signaling pathway in ISCs and detected that ISCs respond to NIC via α7-nAchR, PKC activation, and stimulation of Hippo-YAP/TAZ and Notch signaling (Figure 5G).Consistent with this model, DBZ treatment inhibited Hippo-YAP/TAZ and Notch signaling in mice and prevented NIC-induced ISC expansion and transformation through Apc loss.

Figure 1 .
Figure 1.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 NICtreated and untreated mice (A:3-4 mice per group, B: 3 mice per group).(C) Olfm4 staining image (Red: Olfm4, Blue: DAPI) and the quantification of Olfm4positive cells at the crypt base of proximal jejunum with or without NIC treatment (3-4 mice per group).(D) GFP staining image (Green: GFP, Blue: DAPI) and the quantification of LgR5-GFP-positive cells at the crypt base of the colon in NIC or control-treated Lgr5-EGFP-IRES-CreERT2 mice (3 mice per group).(E) Lysozyme staining image (Red: Lysozyme, Blue: DAPI) and the quantification of Lysozyme-positive Paneth cells with or without NIC treatment (3-4 mice per group).Original magnifications: 400× (A-E).Scale bar: 50 µm (A-E).Values represent the mean ± standard error of the mean (SEM).Significant differences are denoted by p values (Student's t-test).See also Figure S1.

Figure 2 .
Figure 2. 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.Representative images of the organoids and the quantification of organoids number at day 5 (3 wells/ group) (yellow arrow marks organoids and red arrow indicates aborted crypts).(B) Colonic crypts were cultured with or without 1 μM NIC to allow CSCs to form organoid colonies.Representative images of the organoids and the quantification of organoids number at day 5 are shown (3 wells/ group) (yellow arrow marks organoids and red arrow indicates aborted crypts).(C) ISCs and Paneth cells were isolated from the small intestine of Lgr5-EGFP-IRES-CreERT2 mice treated with or without NIC; 2 × 10 3 cells each were co-cultured in the medium containing 10 μM CHIR99021.Representative images of the organoids and the frequency of organoids at day 5 (3 wells/ group).(D) ISCs isolated from the

Figure 3 .
Figure 3.The effect of NIC is mediated via the 7 subunits of α7nicotinic 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 hydrochloride (3 wells/group).(C) In ISCs and Paneth cells isolated from control and NIC mice, mRNA levels of nAchR subunits were analyzed using quantitative real-time PCR (n = 5 per group) (C: control, N: NIC).(D) ISC or Paneth cell lysates prepared from control and NIC mice were immunoblotted with antibodies against α7-nAchR and β-actin.(E) Isolated ISCs were cultured in a medium supplemented with or without 10 μM PNU 282987 (3 wells/group).(F) Isolated ISCs were cultured in a medium with or without 1 μM NIC and 1 μM Bungarotoxin (3 wells/group).Values represent the mean ± SEM.Significant differences are denoted by p values (Student's t-test).See also Figure S3.

Figure 4 .
Figure 4. 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 (PKA inhibitor) or (B) 10 nM Go6983 (PKC inhibitor) (3 wells/group).(C) Crypt lysates isolated from control and NIC-treated mice were immunoblotted using antibodies against YAP, TAZ, and β-actin.(D) In ISCs or Paneth cells (n = 5 per group) isolated from control or NIC mice, mRNA levels of genes associated with Hippo-YAP/TAZ and Notch

Figure 5 .
Figure 5. 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 MK-0752 (3 wells/group).(C) Isolated ISCs were cultured using a medium with or without 10 μM PNU282987 and 5 nM K-975 (3 wells/group).(D) Isolated ISCs were cultured using a medium with or without 10 μM PNU282987 and 1 μM MK-0752 (3 wells/group).(E) Isolated ISCs were cultured in a medium with or without 1 nM Ingenol-3angelate and 5 nM K-975 (3 wells/group).(F) Isolated ISCs were cultured in a medium with or without 1 nM Ingenol-3-angelate and 1 μM MK-0752 (3 wells/group).(G) Schematic model of NIC-associated signaling pathway in ISCs.The model traces a signaling cascade via α7-nAchR, PKC, Hippo-YAP/TAZ and Notch signaling in ISCs.Values represent the mean ± SEM.Significant differences are denoted by p values (Student's t-test).

Figure 6 .
Figure 6.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 mice in control and NIC-treatment groups using Hes5, YAP, TAZ, and β-actin antibodies.(C and D) Immunostained Ki67-positive and (C) (Red, Ki67; Blue, DAPI) Olfm4 positive cells (D) (Red, Olfm4; blue, DAPI) and their quantification in the proximal jejunum of DBZ-or vehicle-treated mice (NIC-treated and untreated) (n = 3 per

Figure 7 .
Figure 7. DBZ inhibits intestinal tumor growth by NIC (A) Schematic representation of Apc flox/flox ; Lgr5-eGFP-IRES-CreERT2 (Lgr5Cre ER Apc fl/fl ) tumor initiation.Mice were treated with control or NIC more than 8 weeks before a single Tamoxifen injection (30 mg/kg body weight), continued for 4 weeks before tissue collection.(B) Macroscopic quantification of the number and area of polyps in the entire intestine of control or NIC-treated Lgr5Cre ER Apc fl/fl mice.(C) Representative images (Red: β-catenin, Blue: DAPI) and the quantification of the number of β-catenin positive adenomatous lesions in the entire intestine of control or NIC-treated Lgr5Cre ER Apc fl/fl mice.(D) Schematic presentation of Lgr5Cre ER Apc fl/fl tumor initiation.Control or NICtreated Lgr5Cre ER Apc fl/fl mice were subjected to a single Tamoxifen injection (30 mg/kg body weight), followed by daily DBZ or vehicle injections continued for 4 weeks before tissue collection.(E) Macroscopic quantification of the number of polyps in the entire intestine of DBZ or vehicle-treated Lgr5Cre ER Apc fl/fl mice (NIC-treated and untreated).(F) Representative images (Red: βcatenin, Blue: DAPI) and the quantification of the number of β-catenin positive adenomatous lesions in the entire intestine of DBZ or vehicle-treated Lgr5Cre ER Apc fl/fl mice (NIC-treated and untreated).Original magnifications: 200× (C, and F).Scale bar: 50 µm (C, and F).Values represent the mean ± SEM.Significant differences are denoted by p values (Student's t-test).
Lgr5-EGFP-IRES-CreERT2 mice were purchased from Jackson Laboratories (Bar Harbor, ME).Apc flox mice were obtained from the National Cancer Institute.Lgr5-EGFP-IRES-CreERT2 mice were crossed with Apc flox/flox mice (Lgr5Cre ER Apc fl/fl ).Lgr5Cre ER Apc fl/fl :tdTomato mice were generated by crossing Lgr5Cre ER Apc fl/fl mice with Rosa26-CAG-lsl-tdTomato mice, provided by the National Cancer Institute (Bethesda, MD).All lines were maintained in the C57BL/6 background.Mice were housed in a controlled environment maintaining 12 hr:12 hr light:dark cycle at 25 ± 1 °C.All animal procedures were performed following the guidelines of the Animal Care Committee of the University of Tokyo.