Down-regulation of YAP prevents smoking- and alcohol-induced carcinogenesis of esophageal paracancerous tissue by promoting cellular pyroptosis

doi:10.21203/rs.3.rs-5277548/v1

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Down-regulation of YAP prevents smoking- and alcohol-induced carcinogenesis of esophageal paracancerous tissue by promoting cellular pyroptosis

https://doi.org/10.21203/rs.3.rs-5277548/v1

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Background

Paracancerous tissues (PCTs) were previously considered benign regions, but recent findings reveal genomic instability in these areas. Smoking and alcohol consumption are closely associated with esophageal cancer (EC) development. This study explored the interplay between the Hippo pathway and pyroptosis in EC, PCTs, and distal normal tissues (DNTs).

Methods

We used molecular epidemiological methods to analyze the effects of smoking and alcohol on these pathways. We found that key genes in both pathways were more altered in smokers and/or drinkers compared to non-smokers and non-drinkers. Additionally, we observed changes in some genes and proteins in PCTs, while the Hippo pathway and pyroptosis had not yet been influenced. We applied 4.0% alcohol combined with various concentrations of cigarette smoke extract (CSE) to PCTs cultured in vitro to observe carcinogenesis and changes in these pathways. Verteporfin, as an inhibitor of YAP, was also used in vitro culture experiments to observe its effects on cellular carcinogenesis.

Results

Among 56 EC patients, 41 had a history of smoking and/or alcohol consumption in this study. Significant alterations in Hippo pathway genes (LATS1, YAP, TAZ) and pyroptosis genes (NLRP3, ASC, GSDMD, Caspase-1) were observed in EC tissues and change of Lats1, Nlrp3, and Asc in PCTs. We also found verteporfin inhibited CSE and alcohol-induced carcinogenesis by downregulating YAP and promoting pyroptosis.

Conclusions

It is speculated that the downregulation of YAP could prevent smoking- and alcohol-induced carcinogenesis in esophageal PCTs by promoting pyroptosis, which may offer new insights for the treatment of esophageal squamous carcinoma.

esophageal cancer

paracancerous tissues

Hippo pathway

pyroptosis

The etiological evidence suggests that alcohol, cigarettes, and hot drinks play a crucial role in esophageal cancer (EC)(Lipenga et al. 2021). Despite improvements in detection, staging, and proactive multimodal interventions for EC, overall survival has not changed significantly over the last few decades(Chen 2022). The main reason is that the mechanism of its occurrence and development remains unclear, resulting in poor treatment outcomes.

The Hippo pathway plays a unique regulatory role in biological processes, such as organ size and tissue regeneration, and is closely related to the occurrence and development of tumours(Piccolo et al. 2023). Inhibition of Hippo pathway activity promotes the proliferation and migration of esophageal squamous cell carcinoma (ESCC) cells cultured in vitro(Sun et al. 2023). Pyroptosis is a type of programmed cell death triggered by inflammasomes, with its biomarker being gasdermin D (GSDMD)(Yu et al. 2021). Emerging studies have shown that certain chemotherapeutic drugs can damage EC cells by inducing pyroptosis(El-Kenawi et al. 2023). The nucleotide-binding and oligomerization domain (NOD) - like receptor family protein 3 (NLRP3) inflammasome is known to be closely associated with EC(Zhou and Fang 2019), which contains apoptosis-associated speck-like (ASC) protein, CASPASE, and NLRP3(Huang et al. 2021). Yes-associated protein (YAP) may be involved in regulating the activation of NLRP3 inflammasomes.

To date, no study has reported the mutual regulation of Hippo/YAP and pyroptosis in EC. Recent spatial transcriptomic studies have identified genomic instability in benign para-cancerous tissues (PCTs), suggesting that early cancer evolution may occur in these tissues(Erickson et al. 2022). In this study, a molecular epidemiological approach is proposed to investigate the association of smoking and/or alcohol consumption with the Hippo pathway (large tumor suppressor kinase 1 (LATS1), Plats1, YAP, pYAP and transcription coactivator PDZ binding motif (TAZ)) and pyroptosis (NLRP3, ASC, CASPASE-1, GSDMD) in EC tissues, PCTs and distal normal tissues (DNTs). Furthermore, alcohol combined with cigarette smoke extract (CSE) will act on PCTs cultured in vitro in order to observe the cancerous transformation of PCTs and alterations in Hippo pathway and pyroptosis. Finally, Verteporfin (VPF), a YAP inhibitor, will be applied to PCTs cultured in vitro to assess its impact on cellular carcinogenesis, thereby further validating the role of YAP in the regulation of tumor development in ESCC, particularly in patients with a history of alcohol and/or cigarette consumption.

Study design and participants

Fifty-six ESCC patients were admitted to the Department of Thoracic Surgery at Shanghai Changzheng Hospital, China, between January 2022 and August 2023. All individuals and/or their closest relatives provided informed consent to participate in this study, and approval was obtained from the Medical Research Ethics Committee of Shanghai Changzheng Hospital. Informed consent forms signed by patients or their families were collected, and confidentiality agreements were signed with all participants. The patient inclusion criteria and the determination of smoking and/or alcohol consumption are detailed in Crit.S1 and S2 of supplementary materials, respectively. All patients with a history of smoking reported that they unconsciously swallow saliva containing smoke.

Collection blocks of EC, PCT, and DNT

PCT defined as tissue ≤ 2.5 cm from the tumour margins; DNT defined as tissue > 5cm from the tumour margins. All patients underwent radical EC surgery, and their tumour tissue, PCT and DNT blocks were obtained. These three tissue types were promptly cleaned with sterile physiological saline, and each tissue was then divided into three small blocks. The middle block was immersed in 4% paraformaldehyde, while the two blocks on either side were placed in TRIzol reagent (Invitrogen, USA) and radio immunoprecipitation assay (RIPA) lysate (Beyotime Biotechnology, China), respectively. Information on patient smoking and alcohol consumption strictly corresponded to the EC blocks, PCT blocks, and DNT blocks. Blocks used for western blot assays should adhere to the conditions outlined in Crit.S3.

CSE preparation

According to the reference(Sun et al. 2019), CSE was made with two cigarettes manufactured in China with a tar content of 10 mg/cigarette.

In vitro carcinogenesis experiments of esophageal PCTs

Tissue block cultured in vitro was conducted according to the reference(Lambrot et al. 2006). The ranges for daily smoking and alcohol consumption in all EC patients surveyed in this study were 16–29 cigarettes and 100–169 ml (100% alcohol, v/v) (Tab.S3), respectively. After calculating, the in vitro carcinogenesis of PCTs was divided into six groups: control (C) group (with the same volume of DMEM medium which dissolved CSE as the other groups), 5.8% CSE, 4.0% alcohol, 4.0% alcohol + 3.2% CSE, 4.0% alcohol + 4.0% CSE, and 4.0% alcohol + 5.8% CSE groups. After 7 d of incubation, the tissue blocks were harvested and immediately washed with PBS (pH 7.4). Some of them were placed in TRIzol reagent, another in RIPA lysate, and the other in 4% paraformaldehyde.

VPF inhibited YAP in PCTs cultured in vitro

The 4.0% alcohol + 5.8% CSE-induced cancer tissues were selected for YAP inhibition. According to the reference(WeiX. Li 2020), this study established a positive control (PC) group (4.0% alcohol + 5.8% CSE), a low-dose group (3 µ M VPF + 4.0% alcohol + 5.8% CSE, a medium dose group (6 µ M VPF + 4.0% alcohol + 5.8% CSE), and a high-dose group (9 µ M VPF + 4.0% alcohol + 5.8% CSE). VPF was dissolved in dimethyl sulfoxide (DMSO) and added to the culture medium, with 0.1% (v/v) DMSO in each group. After 7 d of incubation, the tissue blocks were harvested.

Total RNA extraction and quantitative PCR (qPCR)

The RNA was extracted from all samples frozen in TRIzol reagent and then reverse transcribed into cDNA. Quantitative PCR (qPCR) was performed using our previous SYBR Green method(Li et al. 2022), with the primer sequences detailed in Table S1. The amount of the target gene relative to the internal reference gene was calculated using the formula Y = 2 ^{− ΔΔCt}.

Western Blotting

The samples placed in RIPA were homogenised and centrifuged at 4°C, and the supernatant was collected as the total protein solution for western blot analysis. All antibodies were purchased from CST Corporation (USA). Monoclonal antibodies including anti-pLATS1, anti-LATS1, anti-YAP, anti-pYAP, anti-TAZ, anti-NLRP3, anti-GSDMD, anti-CASPASE-1, and anti-ASC were diluted to a concentration of 1:1000, while β-ACTIN monoclonal antibodies were diluted to a concentration of 1:3000.

Immunofluorescence analysis

Tissue sections were deparaffinized, hydrated, and treated to eliminate endogenous peroxidase. Antigen retrieval was performed using sodium citrate buffer (pH 6.0), followed by blocking with 3% BSA. The sections were then incubated separately with YAP and GSDMD polyclonal antibodies (diluted 1:200) at 4°C overnight. Afterwards, they were incubated with a fluorescent secondary antibody and stained with 4’, 6-diamido-2-phenylindole (DAPI). Finally, the sections were observed and photographed using a fluorescence microscope (Leica, Germany).

Statistical analysis

The data were statistically analyzed using SPSS software (version 26.0). All measurement results are presented as mean ± standard deviation. Confidence intervals for smoking and alcohol consumption were calculated using the frequency method, and the multi-sample Kruskal-Wallis rank sum test was used for comparison of count data. Comparisons between two groups were conducted using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Counting data were analyzed using the chi-square test. The significance level in this study was set at α = 0.05.

Hippo pathway and pyroptosis changes in tumour tissue of EC

Investigation of smoking and alcohol consumption in EC patients and Pathological observation of EC tissue, PCTs, and DNTs.

Among the patients, three (5.4%) were only smokers, and nine patients (16.1%) were only drinkers, twenty-nine patients (51.8%) were both smokers and drinkers (Tab.S2). The median number of cigarettes smoked per day by smokers was 20, with a 95% confidence interval of 16–29 cigarettes (Tab.S3). The median daily alcohol consumption of the patients was 125 ml (equivalent to 100% alcohol), with a 95% confidence interval of 100–169 ml (Tab.S3). Compared to DNT cells, PCT cells did not exhibit significant heterogeneity, whereas cells in cancerous tissues showed marked differences. (Fig. 1A).

Changes in genes related to the Hippo pathway and pyroptosis in EC and PCTs

Compared with DNTs, the expression of Lats1 decreased (P < 0.05), while the levels of Yap and Taz increased (P < 0.05) in EC. The abundance of the pyroptosis genes Nlrp3 and Asc increased (P < 0.05), while the expression of Gsdmd and Caspase-1 decreased (P < 0.05) (Fig. 1B and C, Tab.S4). All seven genes were altered in 3 cases (100%) of smokers only, 8 cases (88.9%) of drinkers only, 28 cases (96.6%) of both smokers and drinkers, and 10 cases (66.7%) of non-smokers and non-drinkers (Tab. S5).

Meanwhile, the expression of Lats1 in the Hippo pathway was reduced (P < 0.05), whereas the abundance of the pyroptosis genes Nlrp3 and Asc was increased (P < 0.05) in PCTs (Fig. 1B and C, Tab.S4). These three genes were altered in 3 patients (100%) who were smokers only, 7 patients (77.8%) who were alcohol drinkers only, 28 patients (96.6%) who were smokers and drinkers, and 9 patients (60.0%) who were non-smokers and non-drinkers (Tab.S5).

Compared with DNTs, not only was the Hippo pathway inhibited (LATS1, pLATS1 and pYAP decreased, while YAP and TAZ increased) in ESCC tissues, but pyroptosis was also suppressed (NLRP3 and ASC increased, while CASPASE-1 and GSDMD declined). However, only a few genes/proteins were altered in PCTs, and neither the Hippo pathway nor cellular pyroptosis was inhibited (Fig. 1B-G, Tab.S4-6).

Alcohol and/or CSE may induce cellular carcinogenesis in PCTs cultured in vitro by inhibiting the Hippo pathway and pyroptosis

In vitro experiments showed that 5.8% CSE, 4.0% alcohol + 3.2% CSE, 4.0% alcohol + 4.0% CSE, and 4.0% alcohol + 5.8% CSE-treated groups all showed a certain degree of anisotropy, and the degree of anisotropy gradually increased or even became cancerous, whereas the cells of the control group, 4.0% Alcohol-treated group had no obvious anisotropy and resembled normal squamous epithelium (Fig. 2A).

Compared to the control group, one member of Lats1 in the Hippo pathway decreased, and the other two members, Yap and Taz increased in the 5.8% CSE, 4.0% alcohol + 3.2% CSE, 4.0% alcohol + 4.0% CSE, and 4.0% alcohol + 5.8% CSE groups (P < 0.05) (Fig. 2B, Tab.S7). In contrast, the levels of the pyroptosis-related genes Gsdmd and Caspase-1 were reduced compared to the controls, while Nlrp3 and Asc abundances were increased (P < 0.05) (Fig. 2C, Tab.S7).

Compared with the control group, the expression of three key proteins of the Hippo pathway, pLATS1, LATS1, and pYAP, was decreased in the 5.8% CSE, 4.0% alcohol + 3.2% CSE, 4.0% alcohol + 4.0% CSE, and 4.0% alcohol + 5.8% CSE groups compared to the control group (P < 0.05), whereas YAP and TAZ levels were elevated (P < 0.05) (Fig. 2D and E, Tab.S8). The expression of two key pyroptosis proteins, GSDMD and CASPASE-1, decreased compared to that in the control group (P < 0.05), whereas the levels of NLRP3 and ASC increased (P < 0.05) (Fig. 2F and G, Tab.S8).

These results illustrated that 5.8% CSE, 4.0% alcohol + 3.2% CSE, 4.0% alcohol + 4.0% CSE, and 4.0% alcohol + 5.8% CSE caused inhibition of the Hippo pathway and pyroptosis in the PCTs, which in turn stimulated cellular proliferation and heterogeneity/carcinogenesis (Fig. 2, Tab.S7-8). Smoking and/or alcohol consumption may be risk factors for these changes.

There are a few alterations in the Hippo pathway and pyroptosis gene/protein in PCTs, which is supported by the emerging study(Erickson et al. 2022). CSE and/or alcohol induced activation of the Hippo pathway in PCTs, and up-regulated YAP mediated a decrease in NLRP3, ASC, CASPASE-1, and GSDMD(Hong et al. 2021), resulting not only in cellular oncosis/heterogeneity but also in reduced pyroptosis and promoted survival.

VPF may prevent CSE and/or alcohol induced cell anisotropy/carcinogenesis in PCTs by inhibiting YAP

When 4.0% alcohol + 5.8% CSE induced carcinogenesis in PCTs, different doses of VPF are added. The cells in the positive control (PC) group were disordered and showed heavy heterogeneity or cancerous changes. The addition with low-dose (3 µM) VPF reduced cellular atypicality compared with the PC group, but the heteromorph was still obvious and the ratio of nucleus to cytoplasm was still enlarged. After adding a medium dose (6 µM) of VPF, compared to the PC group, the cell arrangement was more ordered, the cellular heterogeneity decreased, and the nuclear cytoplasmic ratio of some cells increased. After adding high-dose (9 µM) VPF, the arrangement and morphology of the cells were close to normal squamous epithelium, and no obvious heterogeneity were observed (Fig. 2A).

Compared with the control group, the abundance of the key Hippo gene Lats1 increased in the high-dose VPF group, while the expression of Yap and Taz decreased (P < 0.05) (Fig. 2B, Tab.S9). The abundance of pyroptosis-related genes Gsdmd and Caspase-1 was elevated, whereas the expression of Nlrp3 and Asc was lower than that in the control group (P < 0.05) (Fig. 2C, Tab.S9). The levels of the Hippo pathway proteins pLATS1, LATS1, and pYAP were elevated in the high-dose VPF group compared with those in the control group, and the expression of YAP and TAZ was lower than that in the control group (P < 0.05) (Fig. 3D and E, Tab.S10). The expression of the pyroptosis-related proteins GSDMD and CASPASE-1 was elevated compared to that in the control group, whereas the levels of NLRP3 and ASC were lower than those in the control group (P < 0.05) (Fig. 3F and G, Tab.S10). Immunofluorescence staining showed that YAP and GSDMD were mainly localised in the cytoplasm of normal, heterotypic, and cancer cells, and their expression was consistent with the western blot results. The YAP fluorescence intensity was significantly weakened in the high-dose (9 µM) VPF-treated group compared to the PC group, while the GSDMD fluorescence intensity was significantly enhanced (Fig. 2H and I).

In summary, VPF prevented cellular heterogeneity/carcinogenesis in PCTs induced by the combination of CSE and alcohol (Fig. 3A), possibly because it inhibited YAP and consequently activated the Hippo pathway and pyroptosis (Fig. 3B-I, Tab. S7-8, Fig. 2E and G)(Zhang et al. 2022). It is suggested that inhibition of YAP promotes cellular death of PCTs and consequently inhibits cellular heterogeneity and carcinogenesis, reduces cell proliferation, and thus prevents the transformation of PCTs to tumors.

PCTs have long been considered benign regions, with previous molecular biology studies suggesting only slight differences at the molecular level compared to normal tissue. However, recent research has uncovered genomic instability in benign prostate PCTs, and we have observed alterations in certain genes and proteins involved in the Hippo pathway and pyroptosis in esophageal PCTs. Smoking and alcohol consumption are well-known major risk factors for esophageal cancer, with prolonged exposure significantly increasing the likelihood of developing the disease(Abnet et al. 2018; Thrift 2017). In our in vitro experiments, CSE and/or alcohol induced cellular heterogeneity and cancerous changes in PCTs. Consequently, we shifted our focus to the PCTs in ESCC, aiming to explore the mechanisms through which atypical esophageal epithelial cells may further progress into squamous carcinoma cells.

Most of the ESCC patients in this study had a history of smoking and/or alcohol consumption, with 6.8% smoking only, 18.2% drinking only, and 75.0% smoking and drinking both. In the in vitro experiments, the cells in the PCTs of the esophagus in all groups except the control and 4.0% alcohol groups showed heterogeneity or even cancerous changes, which may be attributed to the fact that alcohol has a single component and a shorter duration of action, whereas CSE contains a variety of oncogenic components that make its oncogenic ability significantly higher than that of alcohol(Viner et al. 2019).

YAP binds to TEADs to form "promiscuous" transcription factors that regulate several growth-related target genes. YAP expression is significantly upregulated in various malignancies, and alcohol stimulates severe dysregulation of the Hippo/YAP pathway in the liver(Bou et al. 2021). In this study, patients who consumed only alcohol showed a decrease in YAP expression in ESCC, activation of the Hippo pathway, and inhibition of pyroptosis. However, alcohol alone could not alter the Hippo pathway and pyroptosis in PCTs cultured in vitro for over 7 days. This may be because short-term exposure to alcohol does not alter the Hippo pathway, and few available studies have verified such changes in hepatic Hippo/YAP after long-term exposure of mice to alcohol(Bou et al. 2021; Kusumanchi et al. 2021). Although short-term alcohol exposure in cultured human and mouse normal hepatocytes triggers pyroptosis, alcohol-induced liver pyroptosis in mice, in vivo, still requires long-term exposure(Zhao et al. 2022; Chu et al. 2021). In this study, short-term exposure to alcohol, in vitro, did not induce changes in pyroptosis-related genes/proteins in cultured esophageal PCTs. This may be because the cells in the esophagus are not as sensitive to alcohol as those in the liver, and the metabolic capacity of the tissue is stronger than that of the cells.

Previous studies have found elevated YAP not only in smoking-induced non-small cell lung cancer(Bai et al. 2023), but also in EC(Zhao et al. 2014). In this study, both the EC tissues of smoking patients and the PCTs induced by CSE in vitro showed an increase in the Hippo/YAP pathway members, suggesting that smoking may promote the development of EC by inhibiting the Hippo pathway (increasing YAP). In general, smoking induces some diseases/injuries by increasing NLRP3, ASC, CASPASE-1, and GSDMD, leading to pyroptosis(Wang et al. 2023; Liu et al. 2023), while activating YAP can inhibit pyroptosis(Zhang et al. 2022). In the present study, pyroptosis-related proteins/genes were reduced in cancerous tissues, which may be attributed to the inhibition of the Hippo pathway (YAP activation), which regulates the reduction in NLRP3, ASC, CASPASE-1, and GSDMD(Hong et al. 2021). Smoking is a stronger risk factor for ESCC than other risk factors, such as alcohol consumption; and concurrent smoking and alcohol consumption not only increases the risk of all cancers(Viner et al. 2019), but also changes in molecular level/mechanisms(Abreu-Villaca et al. 2017). In this study, the short-term simultaneous exposure of PCTs to CSE and alcohol in vitro further inhibited the Hippo pathway and pyroptosis.

Recently, VPF has been identified as a YAP inhibitor that disrupts YAP-TEAD interactions.(Wang et al. 2023). In this study, VPF inhibited YAP and regulated the increase in pyroptosis, thereby counteracting the cancer-promoting effects of CSE and alcohol on esophageal PCTs in vitro. These findings also confirm that YAP activation inhibits the Hippo pathway and pyroptosis in vitro, leading to the malignant transformation of PCTs. Based on these results, we hypothesized that inhibiting YAP could prevent tumour progression in the PCTs of ESCC by activating Hippo pathway and further promoting pyroptosis. This could provide new insights into the treatment of early-stage esophageal squamous carcinoma. However, we have not yet observed any significant effects of VPF or other YAP inhibitors on EC or esophageal precancerous lesions in experimental animals, nor have we found relevant reports in the literature.

In conclusion, we found that both the Hippo pathway and pyroptosis were inhibited in ESCC, with some alterations in Hippo pathway and pyroptosis genes/proteins also observed in PCTs. The inhibitions of Hippo pathway (activation of YAP) and pyroptosis were also observed in the cellular heterogeneity/carcinogenesis of esophageal PCTs induced by CSE alone or in combination with alcohol in vitro. VPF, a YAP inhibitor, was effective in counteracting CSE and alcohol-induced cellular heterogeneity and carcinogenesis by inhibiting YAP (activating the Hippo pathway) and further promoting pyroptosis.

Ethics approval and consent to participate

All individuals and/or their parents provided informed consent to participate in this study and approval was provided by the Medical Research Ethics Committee of Shanghai Changzheng Hospital (Second Affiliated Hospital of Naval Medical University), China. Informed consent form signed by the patients or their families has been obtained.

Competing interests

The authors declare no competing interests.

Funding

None

Author Contribution

Zihao Chen and Yongfei Zhu, designed the research project, supervised experiments, and revised the manuscript. Xinyu Ding and Ying Li, questionnaire investigation, obtained samples, conducted part experiments, acquired data, analyzed data, and wrote the manuscript. Xuning Wang and Fan Li, conducted part experiments, acquired data, analyzed data.

Availability of data and materials

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

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Down-regulation of YAP prevents smoking- and alcohol-induced carcinogenesis of esophageal paracancerous tissue by promoting cellular pyroptosis

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Study design and participants

Collection blocks of EC, PCT, and DNT

CSE preparation

VPF inhibited YAP in PCTs cultured in vitro

Total RNA extraction and quantitative PCR (qPCR)

Western Blotting

Immunofluorescence analysis

Statistical analysis

Results

Hippo pathway and pyroptosis changes in tumour tissue of EC

Changes in genes related to the Hippo pathway and pyroptosis in EC and PCTs

VPF may prevent CSE and/or alcohol induced cell anisotropy/carcinogenesis in PCTs by inhibiting YAP

Discussion

Conclusions

Declarations

Ethics approval and consent to participate

Competing interests

Funding

Author Contribution

Availability of data and materials

References

Additional Declarations

Supplementary Files

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Version 1