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

Reviewer #1 (Public review):

Summary:

In their manuscript, authors Isotani et al used in vivo and ex vivo models to show that nicotine could promote stemness and tumorigenicity in murine model. The authors further provided data supporting that the effects of nicotine on stem cell proliferation and tumor initiation were mediated by the Hippo-YAP/TAZ and Notch signal pathway.

Strengths and weaknesses:

The major strength of this study is the using a set of tools, including Lgr5 reporter mice (Lgr5-EGFP-IRES-CreERT2 mice), stem cell-specific Apc knockout mice (Lgr5CreER Apcfl/fl mice), organoids derived from these mice and chemical compounds (agonists and antagonists) to demonstrate nicotine affects stem cells rather than Paneth cells, leading to increased intestinal stemness and tumorigenicity. Whereas, all models are restricted to mice, lacking analysis of human samples or human intestinal organoids to prove the human relevant of these findings. Although the revised manuscript has significantly improved in the quality of pictures, there seems to be still a discrepancy in Figure 2A: quantification result suggested that NIC (1um) treatment increased the number of colonies from 300 to around 450 (1.5 folds), whereas representative picture shown that the difference was 3 to 12 living organoids (4 folds).

Overall, the presented results could support their conclusions. A previous study reported that nicotine acts through the α2β4 nAChR to enhance Wnt production by Paneth cells, which subsequently affects ISCs. In contrast, this manuscript demonstrated that nicotine directly promotes ISCs through α7-nAChR, independent of Paneth cells. Therefore, this manuscript offers novel insights into the mechanism of nicotine's effects on the mouse intestine.

Reviewer #2 (Public review):

Summary:

The manuscript by Isotani et al characterizes the hyperproliferation of intestinal stem cells (ISCs) induced by nicotine treatment in vivo. Employing a range of small molecule inhibitors, the authors systematically investigated potential receptors and downstream pathways associated with nicotine-induced phenotypes through in vitro organoid experiments. Notably, the study specifically highlights a signaling cascade involving α7-nAChR/PKC/YAP/TAZ/Notch as a key driver of nicotine-induced stem cell hyperproliferation. Utilizing a Lgr5CreER Apcfl/fl mouse model, the authors extend their findings to propose a potential role of nicotine in stem cell tumorgenesis. The study posits that Notch signaling is essential during this process.

Strengths and Weaknesses:

One noteworthy research highlight in this study is the indication, as shown in Figure 2 and S2, that the trophic effect of nicotine on ISC expansion is independent of Paneth cells. In the Discussion section, the authors propose that this independence may be attributed to distinct expression patterns of nAChRs in different cell types. To further substantiate these findings, the authors provided qPCR analysis of nAchRs in ISCs and Paneth cells from isolated whole small intestine, indicating that α7-nAChR uniquely responds to nicotine treatment among various nAChRs. And the authors further strengthen the clinical relevance of the study by exploring human scRNA-seq dataset, in which α7-nAChR is indeed also expressed in human ISCs and Paneth cells.

As shown in the same result section, the effect of nicotine on ISC organoid formation appears to be independent of CHIR99021, a Wnt activator. In the Lgr5CreER Apcfl/fl mouse model, it is known that APC loss results in a constitutive stabilization of β-catenin, thus the hyperproliferation of ISCs by nicotine treatment in this mouse model is likely beyond Wnt activation. The authors have included such discussion.

In Figure 4, the authors investigate ISC organoid formation with a pan-PKC inhibitor, revealing that PKC inhibition blocks nicotine-induced ISC expansion. It's noteworthy that PKC inhibitors have historically been used successfully to isolate and maintain stem cells by promoting self-renewal. Therefore, it is surprising to observe no or reversal effect on ISCs in this context. The authors have now included an additional PKC inhibitor Sotrastaurin to confirm the role of PKC in nicotine-induced ISC expansion.

Overall, the manuscript has provided sufficient experimental evidence to address my concerns and also significantly enhanced its quality.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

In their manuscript, "Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway", authors Isotani et al claimed that this study identifies a NIC-triggered pathway regulating the stemness and tumorigenicity of ISCs and suggest the use of DBZ as a potential therapeutic strategy for treating intestinal tumors. However, the presented data do not support the primary claims.

Weaknesses:

My main reservation is that the quality of the results presented in the manuscript may not fully substantiate their conclusions. For instance, in Figure 2 A and B, it is challenging to discern a healthy organoid. This is significant, as the entirety of Figure 2 and several panels in Figures 3 - 5 are based on these organoid assays. Additionally, there seems to be a discrepancy in the quality of results from the western blot, as the lanes of actin do not align with other proteins (Figure 6B).

We directly count organoids under microscopy as described previously (Igarashi M et.al., Cell.2016 Igarashi M et.al., Aging Cell.2019). When we count the number of organoids, we exactly can discern which are alive or dead organoids under microscope. Hence, we will detail the method and show which are alive or dead organoids using arrows in our revised version (Figure2A and B).

Moreover, as reviewer1 pointed out, the number of organoids originated from intestinal or colonic crypts can be affected by dead organoids as in Figure2A and 2B. However, almost all colonies from isolated intestinal stem cells (ISCs) (Figure 2C and D) are alive, so the number of colonies are less affected by dead colonies in those experiments using isolated ISCs. Since all organoid data in Figure 3-5 are based on the same method as that of Figure2C and D, the data quality of Figures 3-5 cannot be affected by dead colonies.

Finally, to improve data quality of Figure6B, we repeated this experiments and replaced it by new figures.

Reviewer #2 (Public Review):

Summary:

The manuscript by Isotani et al characterizes the hyperproliferation of intestinal stem cells (ISCs) induced by nicotine treatment in vivo. Employing a range of small molecule inhibitors, the authors systematically investigated potential receptors and downstream pathways associated with nicotine-induced phenotypes through in vitro organoid experiments. Notably, the study specifically highlights a signaling cascade involving α7-nAChR/PKC/YAP/TAZ/Notch as a key driver of nicotine-induced stem cell hyperproliferation. Utilizing a Lgr5CreER Apcfl/fl mouse model, the authors extend their findings to propose a potential role of nicotine in stem cell tumorgenesis. The study posits that Notch signaling is essential during this process.

Strengths and Weaknesses:

One noteworthy research highlight in this study is the indication, as shown in Figure 2 and S2, that the trophic effect of nicotine on ISC expansion is independent of Paneth cells. In the Discussion section, the authors propose that this independence may be attributed to distinct expression patterns of nAChRs in different cell types. To further substantiate these findings, it is suggested that the authors perform tissue staining of various nAChRs in the small intestine and colon. This additional analysis would provide more conclusive evidence regarding how stem cells uniquely respond to nicotine. It is also recommended to present the staining of α7-nAChR from different intestinal regions. This will provide insights into the primary target sites of nicotine in the gut tract. Additionally, it is recommended that the authors consider rephrasing the conclusion in this section (lines 123-124). The current statement implies that nicotine does not affect Paneth cells, which may be inaccurate based on the suggestion in line 275 that nicotine might influence Paneth cells through α2β4-nAChR. Providing a more nuanced conclusion would better reflect the complexity of nicotine's potential impact on Paneth cells.

It was difficult to obtain nAchRs antibodies usable in immunostaining. Hence, we instead performed qPCR of nAchRs in ISCs and Paneth cells from isolated whole small intestine (new Figure3C), although we cannot know the difference of the nAchRs expression in different intestinal regions by this method. Although the comparatively high expression was observed in α7-nAChR and α8nAChR in both ISCs and Paneth cells, the significant difference between ISCs and Paneth cells were not observed (Figure3C).

Interestingly, nicotine up-regulated only the expression of α7-nAChR in ISCs, suggesting the specifical response of α7-nAChR to nicotine (Figures 3C and D). We paraphrased the conclusion of the paragraph according to reviewer’s suggestion.

As shown in the same result section, the effect of nicotine on ISC organoid formation appears to be independent of CHIR99021, a Wnt activator. Despite this, the authors suggest a potential involvement of Wnt/β-catenin activation downstream of nicotine in Figure 4F. In the Lgr5CreER Apcfl/fl mouse model, it is known that APC loss results in a constitutive stabilization of β-catenin, thus the hyperproliferation of ISCs by nicotine treatment in this mouse model is likely beyond Wnt activation. Therefore, it is recommended that the authors reconsider the inclusion of Wnt/β-catenin as a crucial signaling pathway downstream of nicotine, given the experimental evidence provided in this study.

We appreciate for this important suggestion. Certainly, Wnt/β-catenin was activated in Nicotine treated ISCs. However, as reviewer points out, the hyperproliferation of ISCs by nicotine treatment is likely beyond Wnt activation. According to the reviewer’s suggestion, we removed Wnt/β-catenin as a crucial signaling pathway downstream of nicotine (Figure 5G).

In Figure 4, the authors investigate ISC organoid formation with a panPKC inhibitor, revealing that PKC inhibition blocks nicotine-induced ISC expansion. It's noteworthy that PKC inhibitors have historically been used successfully to isolate and maintain stem cells by promoting self-renewal. Therefore, it is surprising to observe no effect or reversal effect on ISCs in this context. A previous study demonstrated that the loss of PKCζ leads to increased ISC activity both in vivo and in vitro (DOI: 10.1016/j.celrep.2015.01.007). Additionally, to strengthen this aspect of the study, it would be beneficial for the authors to present more evidence, possibly using different PKC inhibitors, to reproduce the observed results with Gö 6983. This could help address potential concerns or discrepancies and contribute to a more comprehensive understanding of the role of PKC in nicotine-induced ISC expansion.

Gö 6983 is a pan-PKC inhibitor against for PKCα, PKCβ, PKCγ, PKCδ and PKCζ with IC50 of 7 nM, 7 nM, 6 nM, 10 nM and 60 nM, respectively. Since we used Gö 6983 at the concentration of 10nM in our experiment, we consider PKCζ may not be possible target of nicotine. Additionally, we treated using 5nM Sotrastaurin, another pan-PKC inhibitor, which is supposed not to affect PKCζ. The observed result with Gö 6983 was reproduced by Sotrastaurin (Supplemental Figure 3E).

An additional avenue that could enhance the clinical relevance of the study is the exploration of human datasets. Specifically, leveraging scRNA-seq datasets of the human intestinal epithelium (DOI: 10.1038/s41586-021-03852-1) could provide valuable insights. Analyzing the expression patterns of nAChRs across diverse regions and cell types in the human intestine may offer a potential clinical implication.

We analyzed distribution pattern nAChRs of by scRNA-seq datasets of the human intestinal epithelium (DOI: 10.1038/s41586-021-03852-1). In consistent with mouse data (Figure3C), the expression of human α7-nAChR is higher than that of other nAChRs. The difference of the expression between ISCs and Paneth cells is not clear as in that of mouse (Supplemental Figure4A and B). From mouse and human data, we speculate the induction of specific nAChR by nicotine is essence of ISC response to nicotine, rather than the distribution of nAChRs.

Reviewer #2 (Recommendations For The Authors):

The manuscript could benefit from addressing a few minor points to enhance its quality before publication:

(1) Ensure all images are presented in higher resolution to improve visual clarity.

We replaced all images by those with higher resolution.

(2) Quantify Western blot results accurately for rigor and precision in data representation.

We quantified all blots.

(3) Include error bars in control groups where missing, particularly in Figures 3C and 4D, to enhance data interpretation.

We included error bars in control groups in new Figure 3C and 4D.

(4) The layout of Figure S3B, S4A and S4B should be corrected.

We corrected the layout of those Figures.