YAP1 promotes the stemness of airway epithelial basal cells and spontaneous formation of lung squamous cell carcinoma in a YAP1KITrp53KO mouse model

doi:10.21203/rs.3.rs-3737010/v1

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Research Article

YAP1 promotes the stemness of airway epithelial basal cells and spontaneous formation of lung squamous cell carcinoma in a YAP1^KITrp53^KO mouse model

https://doi.org/10.21203/rs.3.rs-3737010/v1

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Background

Tumorigenic mechanisms and personalized therapeutic strategies for lung squamous cell carcinoma (LSCC) lack clarity. Practical LSCC animal models remain to be developed or improved. We aimed to determine whether Yes-associated protein-1 (YAP1) promotes the stemness of airway epithelial basal cells and LSCC tumor-initiating cells (TICs) and spontaneous tumorigenesis in a self-developed YAP1 knock-in/Trp53 knockout mouse model.

Methods

Airway epithelial basal cells and LSCC TICs were assessed for stemness by immunofluorescence (IF) staining and fluorescence-activated cell sorting. YAP1 expression patterns and levels were evaluated by IF and qRT-PCR. The effect of YAP1 on the tracheosphere-forming ability of airway epithelial basal cells was investigated by YAP1 overexpression and deletion, observed by 3D-matrigel. Homozygous YAP1^KITrp53^KO mice were generated by a special vector design that introduced a ciliated cell-specific promoter FOXJ1. Tumor formation was determined by micro-CT scanning, and histological subtype was confirmed through hematoxylin-eosin (H&E) and immunohistochemical (IHC) staining.

Results

YAP1 promoted the stemness maintenance of airway epithelial basal cells. Overexpression and deletion of YAP1 increased and decreased the tracheosphere-forming ability of airway epithelial basal cells, respectively. YAP1 also contributed to the stemness of LSCC TICs. A homozygous YAP1^KITrp53^KO LSCC mouse model was constructed successfully. After a period of feeding, cancer nests occurred spontaneously in the murine lung. H&E and IHC staining confirmed the LSCC histological subtype, and YAP1 was primarily expressed in the nucleus as evidence of active proliferation.

Conclusions

We established a YAP1^KITrp53^KO mouse model of spontaneous LSCC, providing a convenient tool for investigating novel targets and therapies.

YAP1

lung squamous cell carcinoma

basal cell

mouse model

According to the latest global cancer statistics, lung cancer remains the leading cause of cancer-related deaths and the most frequently occurring cancer secondary to female breast cancer worldwide [1]. Non-small cell lung cancer (NSCLC) accounts for more than 80 to 85% of lung cancers, of which approximately 40% are lung adenocarcinoma (LUAD), 25 to 30% are lung squamous cell carcinoma (LSCC), and 10 to 15% are large cell carcinomas (LCC) [2]. Numerous efforts have been made to improve the treatment strategies for NSCLC, and promising therapies have emerged, such as molecular targeted therapy and immunotherapy, making breakthroughs in treating LUAD. However, overall efficacy is yet to be improved, since 5-year survival is below 22% [3]. LSCC, in particular, lacks personalized therapeutic strategies.

A series of genes and molecular pathways are involved in the genesis, development and drug resistance of lung cancer, and become mutated in LSCC tissues. The mammalian Hippo-YAP signaling pathway has received increasing attention in recent years. First documented in the genetic screening of Drosophila, this pathway has been revealed to play important roles in the regulation of cell proliferation, stem cell renewal and differentiation, organ size, and tumorigenesis [4–7]. Yes-associated protein-1 (YAP1) and transcriptional coactivator with PDZ-binding motif (TAZ) are the major downstream effectors of the Hippo pathway. YAP1, a key effector and transcription co-activator, is highly conserved in mammals [4]. When this pathway is activated, YAP1/TAZ in cytoplasm is phosphorylated for degradation, thus maintaining cell homeostasis and promoting cell differentiation [8, 9]; when this pathway is inhibited, unphosphorylated YAP1/TAZ translocates into the nucleus and binds to other transcriptional factors such as TEAD to regulate the downstream gene expression, thus promoting stemness maintenance, tumor growth and metastasis [10–12]. YAP1 also plays an important role in lung development and alveolus regeneration by regulating the differentiation of epithelial cells. Knocking YAP out of the basal cells in adult mice decreases the number of basal cells and promotes their differentiation into Club cells and ciliated cells. Overexpressing YAP in basal cells triggers their rapid proliferation and inhibits their differentiation [13]. In the context of NSCLC, studies have shown that YAP1 can promote tumor invasion, immigration and metastasis [14–16], and contributes to the stemness maintenance of lung cancer stem cells [17]. The level of nuclear YAP1 is correlated with the poor prognosis of NSCLC [18, 19, 20], and contributes to the hallmarks of NSCLC [21]. However, current research lacks direct evidence that YAP1 could promote the onset of LSCC.

LSCC may originate from airway epithelial basal cells [22]. In our previous work, we isolated and characterized tumor-initiating cells (TICs) in LUAD and obtained the key regulatory miRNAs based on our self-constructed spontaneous LUAD mouse model with the Kras gene conditionally knocked out (LSL-Kras G12D) [23]. Since LSCC has a different genetic origin and regulatory mechanisms from LUAD, we intended to further explore the molecular phenotypes and regulatory mechanisms of airway epithelial basal cells and LSCC TICs. Previously, we demonstrated that the cell surface markers EpCAM Pecy7 and CD49f-APC were effective in identifying mammary epithelial basal cells and breast cancer TICs, and CD49f^highEpCAM^low represented the TIC phenotype [24]. Based on the fact that airway cells and breast cells originate from the same source, basal epithelial cells, we propose that the markers for mammary epithelial basal cells can also be applied to airway epithelial basal cells [25]. Currently, no studies have been reported on the identification of the molecular phenotype of LSCC or transition of LSCC cells from airway epithelial basal cells. We planned to use EpCAM Pecy7 and CD49f-APC to mark and identify airway epithelial basal cells and LSCC TICs in mice.

Furthermore, the overactivation of YAP1 has been demonstrated to cause the loss of contact inhibition and over-proliferation of epithelial basal cells, thus leading to epithelial tumors, such as breast, esophageal and colon cancers [26, 27, 28]. Moreover, YAP1 has been shown to facilitate the stemness of TICs in multiple epithelial tumor types [24, 29]. However, the stemness-promoting effect of YAP1 in airway epithelial basal cells and LSCC TICs, and its oncogenic function in LSCC, have not been investigated.

A recent study of whole exome sequencing in samples from 100 patients with LSCC, and target capture sequencing in another 98 samples, revealed that the Hippo-YAP signaling is one of the frequently mutated pathways in LSCC, in which YAP1 is a frequently mutated gene [30]. Meanwhile, well-documented TP53 is the most significantly altered gene in LSCC, consistent with earlier studies by the TCGA Network on 178 lung squamous cell carcinomas [31] and by Korean researchers in 104 Korean patients with LSCC [32]. As a tumor suppressor gene, TP53 shows genetic alterations in different human cancer types [33] and has the highest alteration rate across all human squamous cell carcinomas (SCCs) [34]. However, mice with the loss of TP53 alone never develop spontaneous SCCs [35]. To address this, a research team from Stanford University constructed an LSCC mouse model by knocking out both Keap1 and Trp53 (Keap1^KO/Trp53^KO) [36]. Another team demonstrated that SOX2 overexpression combined with TP53 knockdown drives bronchial dysplasia in vitro [37]. TP53 mutations in combination with other gene alterations in the progression of LSCC are considered an informative way to probe LSCC tumorigenesis mechanisms [38]. In the present study, we have found that YAP1 can facilitate the stemness of airway epithelial basal cells in mice. Therefore, TP53 knockout combined with knocked in YAP1 can possibly promote the spontaneous development of LSCC and serve as a mouse model design for LSCC.

To date, direct evidence that YAP1 promotes the tumorigenesis of LSCC is still lacking, and no mouse model with YAP1 knocked in and TP53 knocked out has been reported to determine whether YAP1 could facilitate the spontaneous development of LSCC. Thus, we first investigated whether YAP1 could contribute to the stemness maintenance of airway epithelial basal cells in adult mice in vitro and in vivo. Based on this result, we constructed a YAP1 knocked in and TP53 (Trp53 in mice) knockout mouse model to test whether YAP1 could promote LSCC genesis, in which YAP1 was exclusively expressed in airway epithelial cells by the addition of a tissue-specific promoter, FOXJ1. The findings may provide new insights into the role of YAP1 in the formation and progression of LSCC. The YAP1^KITrp53^KO mouse model for spontaneous development of LSCC holds promise of an economic and practical animal model to study the molecular mechanisms underlying YAP1-promoted LSCC onset, progression, metastasis and drug resistance, thus laying the foundations for developing personalized treatment and improved immunotherapy for LSCC.

Mouse tracheal cell isolation and immunofluorescence staining

YAP1 knockout mice were constructed in our laboratory [24]. MMTV-Wnt1 mice (Jackson lab,#002934) were euthanized with carbon dioxide, and the murine skin was sterilized with ethanol. The muscles, esophagus and connective tissues attached to the trachea were removed by scissors and forceps. Both ends were cut off the trachea by sterile scissors, and then rinsed 3 times in PBS, 2 min each time. Next, the trachea open lengthways were cut into two pieces. The tissue was fixed either by perfusion or by immersion in 4% paraformaldehyde (PFA) solutions for a set time period. Fixed tissue was then prepared for cryoprotection by submerging the target tissue in a hydrostabilizing solution. The cryoprotection was completed when the target tissue no longer floated in the stabilizing solution. The tissue was sectioned using cryostat (thickness: 10–15 µm), which provides the best results for clarity and integrity, where sections could be collected directly onto slides, or floated onto slides via a PBS/water bath. Sections on slides were thoroughly air/warm dried on a slide warmer overnight at 40–50°C. Immunofluorescence staining was carried out according to the M.O.M.® (Mouse-On-Mouse) Staining Kit (Fluorometric) instructions (Novus, FMK-2201-NB). Primary antibodies (1:100) against YAP1, Krt5, acetyle-tublin and Hoeschst 33342 (1:1K-10K) were used to show the expression patterns. Secondary antibodies were diluted to the appropriate concentration (1:100–400). Staining results were observed with an LSM 880 laser confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).

The trachea tissue isolated by the above method was incubated in 1.5 mL Dispase (BD Biosciences, 16 U/mL, pre-heating at 37°C) in PBS (30–45 min) at room temperature. Next, the trachea tissues were moved into 2 mL DMEM/F12 (Invitrogen) with 5% FBS in a 6-well plate. Epithelium was peeled off with forceps and pipetted into EP tube with DMEM. The epithelial cells were then centrifuged at 3000 rpm for 5 min at room temperature. After discarding the suspension, the cells were incubated in 900 µL of digestive enzymes (0.1% trypsin, 1.6 mM EDTA) for 15–20 min at 37°C. Digestion was stopped by adding 100 µL of FBS. Then the cells were passed through a 40-µm cell strainer into flow tube. The cells were centrifuged at 1250 rpm (300g) for 5 min at 4°C, and the suspension was discarded. Later, 100 µL of flow buffer (HBSS + 2%FBS), and 3 µL of blocking solution (Rat IgG for stable fragment) were added for 10 min on ice. Then the fluorescence antibodies (2 µL) were added for 10–15 min on ice. After adding flow buffer into the flow tube to almost full, the cells were centrifuged at 1250 rpm (300g) for 5 min at 4°C, and the suspension was discarded. DAPI solution (200 µL) was added for FACS. The basal cells (EP-CAM^lowCD49f^high) were sorted. Quantitative RT-PCR was performed as described previously. The primer sequences are listed in Supplementary Table S1.

Three-Dimensional (3D) tumor spheroid assay in matrigel

As previously described, lentiviruses were used to infect 293T cells to achieve YAP1 overexpression [24]. FACS-sorted basal cells were plated into solidified matrigel (100 µL/well) (BD Bioscience, #356237). The plate was then incubated at 37°C for 10–14 days, and the colonies in the 1st and 2nd passages were counted via an IX83 inverted light microscope (Olympus, Tokyo, Japan).

YAP1^KITrp53^KO mice construction and genotyping identification

YAP1^KI mice were commissioned to be constructed by Cyagen Biosciences Inc. (Guangzhou, China), and the F1 generation (heterozygote) with Neo gene removed from the target vector was obtained by hybridization between chimeric mice and Flp deleted mice. A pair of F1 generation mice was selected for self-crossing, and the F2 generation YAP1^KI mice with Neo removal homozygote were obtained by PCR identification and screening (weight 18-22g, aged 150–200 days, SPF grade). In the whole process of this experiment, the animal disposal methods conformed to the guidelines approved by the Ethics Committee of Army Medical University. The experimental animal license number was STXK (Chongqing, China) 2017-0010. Trp53-C57BL/6N mice were purchased from Cyagen Biosciences Inc.

YAP1^KI mice were mated with C57BL/6-Trp53^KO mice (Cyagen, KOCMP-20953-Trp53) at a male/female ratio of 2:1. The homozygous YAP1^KI female mice and heterozygous Trp53^KO male mice were mated with each other after meeting the criteria of good health, normal body shape, active sports, bright eyes, thick and glossy fur, normal respiration and feces, no skin diseases, no tumors, and normal genitals (7–8 weeks in mice) using the 2:1 long-term cohabit method.

At about 7 days old, mouse tissue was obtained by the method of severed toe for identification using the Mouse Direct PCR Kit (bimake, B40013), and the mice were numbered independently at the same time. The molecular weight of DNA bands was determined by agarose gel electrophoresis, at a 2% agarose (Beyotime, ST04L) concentration. DNA ladder (TIANGEN, MD114) was used to determine whether the DNA position was correct. TAE (Beyotime, ST716) was used as buffer solution for 30 min at 110V. The primer sequences are listed in Supplementary Table S2. Electrophoretic bands showed YAP1^KIWT 155bp, homozygous YAP1^KI 251bp, heterozygous YAP1^KI 251bp/155bp, Trp53^KOWT 759bp, homozygous Trp53^KO 998bp, and heterozygous Trp53^KO 998bp/759bp.

Micro-CT scanning of murine lung volume and pathological identification of YAP1^KITrp53^KO mice

A total of 15 sexually mature mice were genetically identified and assigned into three groups, wild type (WT), heterozygotes, and homozygotes (n = 5). Lung volume was scanned using a Bruker-micro-CT machine and data were recorded. The lungs of the double homozygous YAP1^KITrp53^KO mice were dissected and sliced. Hematoxylin-eosin (H & E) and immunohistochemical (IHC) staining was completed by Chengdu Lilai Biotechnology Co. Ltd. The staining results were explained and confirmed by a professor in Pathology Department of the First Affiliated Hospital of AMU.

Statistical analysis

Statistical analyses were performed using the GraphPadPrism8 software program. Experimental data are expressed as x ± s. Unpaired T-test was used to compare two groups, and one-way analysis of variance (ANOVA) was used to compare multiple groups. p < 0.05 was considered statistically significant.

1. YAP1 was expressed in airway epithelial basal cells, but not localized in nuclei during homeostasis

In normal mice, immunofluorescent staining of the airway epithelial cells showed that YAP1 was expressed in the cytoplasm of airway epithelial basal cells, not in the nucleus during homeostasis (Fig. 1A and B). After being isolated, the cells underwent double staining with CD49f-APC and EpCAM-FITC markers for flow cytometry. FACS allowed the airway epithelial cells to be sorted into two subpopulations: CD49f^highEpCAM^low basal cells and CD49f^lowEpCAMl^high luminal cells (Fig. 1C). Further staining with stemness markers showed that the basal cells expressed stemness phenotypes, including Krt5, p63 and Slug, while the luminal cells exhibited differentiation phenotypes, such as Krt18 and Ecad (Fig. 1D). Thus, YAP1 was expressed in the normal airway epithelial basal cells, primarily in cytoplasm during homeostasis, indicating the involvement of YAP1 in the homeostasis maintenance of airway epithelial basal cells.

2. Overexpression of constitutively active YAP1 increased tracheosphere formation by airway epithelial basal cells

After overexpressing constitutively active YAP1 in airway epithelial basal cells, we used 3D-matrigel to demonstrate the tracheosphere formation of different groups of cells. In the first passage, the YAP1-overexpression group formed a larger number of, and more densified colonies than, the control and vector control groups (Fig. 2A). In successive passages, YAP1 overexpression continued to increase the number and density of colonies (Fig. 2B and C). Moreover, immunofluorescent staining of the 3D-matrigel sections showed that the YAP1-overexpressed airway epithelial basal cells formed colonies in a more disordered and densified manner and had increased proliferative activity compared with the vector controls, as demonstrated by the increased expression of p63 and Krt14 (Fig. 2D). These findings suggest that overexpression of constitutively active YAP1 could enhance the tracheosphere formation by airway epithelial basal cells.

3. Deletion of endogenous YAP1 inhibited tracheosphere formation of airway epithelial basal cells

Using the Cre-Lox system, we established a YAP1 knockout mouse model. Induced with tamoxifen, Cre-recombinase was activated by fusing Cre to a mutant form of the estrogen receptor (ER) and translocated into the nucleus wherein it catalyzes loxP-specific recombination events upon binding of the active tamoxifen metabolite 4-hydroxytamoxifen (4-OHT; tamoxifen-OH). To track the Cre-recombination or the YAP1-knockout efficacy, we crossed CreERT2 mice with ROSAmT/mG reporter mice. Prior to Cre recombination, cell membrane-localized tdTomato (mT) fluorescence expression was widespread in cells, showing a bright red color. After Cre recombination, cell membrane-localized EGFP (mG) fluorescence expression replaced the red fluorescence with green fluorescence. The airway tissues were obtained from the YAP1-knocked out mice after sacrificing, then the airway epithelial basal cells were isolated and purified and seeded into 24-well plates. 3D-matrigel was also added into the plate to allow tracheosphere formation. Tamoxifen-OH was added into the culture plate to activate Cre-recombination in vitro. Tracheospheres with GFP fluorescence were observed under a microscope, suggesting that YAP1 was successfully knocked out of the mouse airway epithelial basal cells (Fig. 3A). The colonies were then processed to dissociate the cells for cell passage. Meanwhile, WT mice with mTmG reporter genes were also sacrificed to obtain the airway epithelial basal cells and the colonies formed by them. Adding 2 µM of tamoxifen-OH to the ROSA-CreERT2 cells to allow for YAP1 knockout, the number of tracheospheres was decreased remarkably compared with no tamoxifen-OH addition (Fig. 3B). Adding 4 µM of tamoxifen-OH to the WT cells, the number of tracheospheres showed no obvious change compared with no tamoxifen-OH addition (Fig. 3B). These results indicate that deletion of endogenous YAP1 inhibited tracheosphere formation of airway epithelial basal cells.

4. YAP1 was related to the stemness of CD49f^highEpCAM^low cells in human LSCC tissues

Using cancer tissue samples from 18 cases of LSCC and LUAD, we prepared cell suspension, and performed double or multiple staining with TIC markers, including CD49f-APC, EpCAM-FITC, Muc1, CD44, CD24, THy and CD271. Flow cytometry analysis of the cells revealed that the proportion of CD49f^highEpCAM^low cells was significantly higher in LSCC than in LUAD (3.43% vs 0.23%) (Fig. 4A). Furthermore, stemness identification confirmed that CD49f^highEpCAM^low was the molecular phenotype of LSCC TICs. In addition, the CD49f^highEpCAM^low subpopulation of LSCC highly expressed YAP1 and TIC transcriptional factors, such as Oct-4, Sox-2, Nanog and Klf4 (Fig. 4B). 3D-matrigel culture showed that the CD49f^highEpCAM^low subpopulation formed obviously more colonies than the CD49f^lowEpCAM^high subpopulation (Fig. 4C), demonstrating a strong colony-forming ability. To assay the TIC frequency, TICs (CD49f^highEpCAM^low) and non-tumor cells (NTCs; CD49f^lowEpCAM^high) were inoculated into nude mice. For each cell type, the mice were divided into three groups, each group inoculated with 50,000, 5,000, and 500 cells, respectively (n = 8). In each group, the number of tumors formed in the mice was significantly larger for the TICs than for the NTCs. Thus, the TIC frequency was 100 times higher in the CD49f^highEpCAM^low subpopulation than in the CD49f^lowEpCAM^high subpopulation, validating the stemness features of CD49f^highEpCAM^low cells (Fig. 4D). These results suggest that YAP1 was closely related to the stemness of TICs in human LSCC.

Combining all these above results, we speculated that YAP1 maintains and promotes colony formation by airway epithelial basal cells in the normal airways of mice. YAP1 overexpression led to disordered proliferation of airway epithelial cells, suggesting that YAP1 may promote lung cancer tumorigenesis, especially LSCC. Therefore, we planned to establish a YAP1^KITrp53^KO mouse model to further explore this role of YAP1.

5. Generation of homozygous YAP1^KI mice and establishment of YAP1^KITrp53^KO mouse model

The conditional YAP1 knock-in was designed by Cyagen Biosciences (Guangzhou, China) (Supplementary Fig. S1A). The targeting vector was first constructed (Supplementary Fig. S1B). The YAP1 targeting construct was linearized by restriction digestion with NotI, followed by phenol/chloroform extraction and ethanol precipitation. The linearized vector was transfected into C57BL/6 ES cells according to Cyagen’s standard electroporation procedures. The transfected ES cells were subject to G418 selection (200 µg/mL) 24 hours post electroporation. A total of 94 G418-resistant clones were picked and amplified in 96-well plates. Two copies of 96-well plates were made, one copy frozen down and stored at − 80°C and the other copy used for DNA isolation and subsequent PCR screening for homologous recombination. PCR screening identified 25 potential targeted clones (Supplementary Fig. S1C), from among which six were expanded and further characterized by Southern blot. Five of the six expanded clones (1B6, 1C104, 1D4, 1D8 and 1H3) were confirmed to be correctly targeted (Supplementary Fig. S1D).

Targeted ES cell clone 1D8 was injected into C57BL/6 albino embryos, which were then re-implanted into CD-1 pseudo-pregnant females. Founder animals were identified by their coat color, and their germline transmission was confirmed by breeding with C57BL/6 females and subsequent genotyping of the offspring. The Neo cassette was self-deleted in germ cells so the offspring were Neo cassette-free. Four male and three female heterozygous targeted mice were generated from clone 1D8 as final deliverables. Thus, the Yap knocked-in (TurboKnockout®) mice were obtained. Next, the F1 mice of heterozygous YAP1^KI genotype were crossed. The F2 mice were genotyped and crossed, and later the F3 were bred and crossed to generate sufficient homozygous mice. Genotyping of mouse tail DNA via PCR showed that 2 and 7 homozygous YAP1^KI mice were obtained.

Mice with Trp53 conditionally knocked out (Trp53^flox/flox) were purchased from Cyagen Biosciences. The homozygous YAP1^KI mice were mated with Trp53^KO mice. Their offspring underwent genotyping, and YAP1^KITrp53^KO mice were selected (Supplementary Fig. S1E). The whole process of YAP1^KI Trp53^KO mouse model establishment is shown in Supplementary Figure S1F.

6. Tumor formation was detected in YAP1^KITrp53^KO mice by lung volume measurement

After genotyping confirmation, the homozygous and heterozygous YAP1^KITrp53^KO mice were selected and bred for 1 year. Micro-CT for animals was used to perform scanning of the murine lungs in vivo. Mice that received micro-CT scanning were divided into three groups (5 mice in each group): double homozygous YAP1^KITrp53^KO mice, heterozygous YAP1^KITrp53^KO mice (heterozygous for both YAP1 and Trp53), and WT mice, with the latter two as controls. By micro-CT imaging, the lung volume for each mouse was observed and delineated (Table 1). Coronal view of the murine lungs showed that the lung volume was largest in the homozygous group and smallest in the heterozygous group (Fig. 5A). Further statistical analysis showed that the lung volume was significantly larger in the homozygous group than in the WT group and heterozygous group (p < 0.05), and was significantly larger in WT than in heterozygous mice (p < 0.05) (Fig. 5B). These results indicated that lung tumors formed in double homozygous YAP1^KITrp53^KO mice, and tumor formation was probably facilitated by YAP1 knock-in and Trp53 knock-out.

Table 1

The lung volume of the wild-type (WT), heterozygous and homozygous C57 mouse (V/cm³)
WT	Heterozygote	Homozygote
373.21	266.06	448.82
372.11	213.03	528.91
331.29	134.18	354.86
310.77	177.69	470.44
322.63	116.68	392.59

7. Pathohistological detection revealed that LSCC was formed in YAP1^KITrp53^KO mice

After the discovery of tumor formation in YAP1^KITrp53^KO mice by micro-CT imaging, we further validated tumor formation and identified the histological tumor subtype by pathohistological staining. The mice were sacrificed, and the lungs from YAP1^KITrp53^KO and WT mice were dissected. Compared with the lungs from WT mice, the volume of the lung was larger in YAP1^KITrp53^KO mice, and the tumor xenograft could be identified clearly (Fig. 6A). The lung tissues were then processed and sliced into sections. H&E staining demonstrated that a tumor nest was formed (Fig. 6B). IHC staining was performed for the detection of pan-CK, CK5, p63, Ki67, p53, CK7, YAP1, TTF-1, NCAM1, NSE, and CgA proteins. The p53 protein was not expressed in either the nucleus or the cytoplasm (Fig. 6G), validating the successful knockout of the Trp53 gene. In contrast, the YAP1 protein was expressed primarily in the nucleus (Fig. 6I), indicating the successful knock-in of the YAP1 gene and the proliferative activity of the cells. The proteins p63 and Ki67, which are generally expressed in LSCC, were positive in the xenograft tumor tissue (Fig. 6E and F). Moreover, the LSCC-related proteins pan-CK and CK5 were also expressed in the tumor tissue (Fig. 6C and D). Meanwhile, the proteins CK7, TTF-1, NCAM1, NSE and CgA, which are mainly expressed in LUAD, were negative in the tumor tissue (Fig. 6H, J, K, L, and M). All these results confirmed the formation of lung cancer in YAP1^KITrp53^KO mice and indicated that the pathohistological subtype of the cancer was LSCC.

8. Genome sequencing and bioinformatic analysis of the YAP1^KITrp53^KO mouse

To further investigate the cancer-related genes, we performed genome sequencing and comprehensive bioinformatic analysis on the YAP1^KITrp53^KO mice and compared the data with those from C57 WT mice. Protein-protein interaction (PPI) network analysis generated a network of 300 differentially expressed genes (DEGs) between LSCC and normal tissues (Supplementary Fig. S2A). Further analysis revealed the top 10 primary modules of PPI sub networks, including Snap25, Syt2, Sv2b, Sv2a, Vamp2, Dlg4, Syn1, Slc17a7, Dnm1, and Stxbp1 (Supplementary Fig. S2B). The corresponding top 10 genes with a higher degree of connectivity are shown in Supplementary Table S3. The GO enrichment analysis of biological processes (BP), cellular components (CC), and molecular functions (MF) of the DEGs showed that the DEGs were mainly related to neurological functions (Supplementary Fig. S2C). GO analysis results of DEGs associated with tumor-related pathways are shown in Supplementary Table S4. YAP1 is constitutively expressed, which may partially explain the absence of YAP1 in the top 10 DEGs. The comparison between transgenic and WT mice may also produce inaccurate results. KEGG pathway enrichment analysis showed that DEGs were most enriched in pathways of neurodegeneration-multiple diseases, with significant differences (Supplementary Fig. S2D). KEGG pathway analysis revealed 24 genes related to tumorigenesis, and PPI network analysis screened out 10 genes most closely related to tumorigenesis (Supplementary Fig. S2E), among which three genes overlapped, Sv2b, Sv2a, and Dlg4 (Supplementary Fig. S2E). DEGs associated with tumor-related pathways revealed by KEGG analysis are shown in Supplementary Table S5.

In the present study, we assessed whether YAP1 could promote the stemness of airway epithelial basal cells in adult mice in vitro and in vivo. We found that YAP1 was involved in homeostasis maintenance and promoted the stemness of airway epithelial basal cells. Based on this result and previous evidence that TP53 has the highest alteration rate across all human SCCs, we constructed a mouse model with simultaneous YAP1 knock-in and Trp53 knockout to determine whether YAP1 could promote LSCC tumorigenesis. As a result, YAP1 promoted tumorigenesis and cell proliferation in LSCC. Conditionally knocking in YAP1 and knocking out Trp53 could facilitate the spontaneous formation of LSCC in mice. We have successfully constructed such a double homozygous YAP1^KITrp53^KO mouse model, in which LSCC developed spontaneously.

Consistent with our findings, previous studies have demonstrated the essential role of YAP1 in maintaining the stemness of an adult mammalian stem cell. Cytoplasmic YAP1 promotes the maturation of the developing lung epithelium by curbing β-catenin and Fgf10 signaling [39]. Loss of YAP1 leads to unrestrained differentiation of adult airway basal stem cells, and YAP1 overexpression increases stem cell self-renewal and blocks terminal differentiation [40]. Abnormal accumulation of nuclear YAP1 prevented airway epithelial cell differentiation in mammalian lung [41].

YAP1 is also associated with the properties of tumor TICs and tumorigenesis in epithelial tumors. Overactivation of YAP1 could lead to excessive cell proliferation, contact inhibition loss and hyperplasia, thus inducing the formation and development of epithelial tumors. For example, nuclear YAP localization and overall YAP expression levels are involved in the development and progression of invasive lobular breast cancer [26]. YAP1 promotes the survival and self-renewal of breast TICs [24]. Also, YAP1 is a major inducer of TIC properties in non-transformed cells and esophageal cancer cells by upregulating SOX9 expression [29].

Distinct epithelial tumors possibly share similar mechanisms of tumor formation due to the identical cellular constitution and origin of tumor cells. As a major component of the epithelium, epithelial basal cells are multipotent stem cells that can give rise to other major subpopulations and contribute to the self-renewal of epithelial cells. In LSCC, airway epithelial basal cells are responsible for airway injury repair and can transform into TICs after accumulating malignant mutations, thus generating LSCC tissues [42–43]. Likewise, in other tumor types, such as breast cancer, esophageal cancer, and small bowel cancer, epithelial basal cells are also considered the origin of TICs and cancer cells [44]. Therefore, combined with our current findings, YAP1 most probably promotes the transformation of airway epithelial basal cells into TICs and the subsequent tumorigenesis of LSCC.

Based on these findings and theories, our YAP1^KITrp53^KO mouse model has several advantages. First, it successfully mimics the progressive nature of LSCC formation in humans, allowing for research on the different stages and the developmental process of LSCC. To ensure the formation of LSCC in the trachea exclusively, we introduced an airway-specific promoter FOXJ1 into the targeting vector. This promoter restricts the spatial location of LSCC formation without affecting the timing, since no switch for time control was used, which is different from existing LSCC mouse models [45] and highlights and advance in this field [38]. The natural formation of LSCC in our mouse model enables more accurate research of the molecular mechanisms underlying tumor development. In addition, the expression of YAP1 specifically in the airway mostly ensured the homogeneous histology of newly formed lung cancer.

Second, we used a targeting vector with a distinctive structure. The YAP1 targeting construct was linearized by restriction digestion with NotI, followed by phenol/chloroform extraction and ethanol precipitation. The linearized vector was transfected into C57BL/6 ES cells according to Cyagen’s standard electroporation procedures. Despite lower transfection efficiency, the above technique, which is less advanced than CRISPR/Cas9, was used due to its better accuracy for targeting.

Third, our model is the first mouse model of spontaneous LSCC available to different institutions in China. Most existing LSCC mouse models can only be obtained from overseas laboratories, limiting the availability and increasing the cost [29]. Thus, our current model can facilitate domestic LSCC studies by providing easier access and reduced costs, enriching the types of available LSCC mouse model.

Fourth, our model enables convenient application. LUAD mouse models require delicate techniques, such as adenovirus delivery, which produce high failure rates, virus contamination risks, and demand for environmental conditions. However, our current model does not require extra interventions and can be applied after a certain period of growth.

It is important to acknowledge several limitations of our study. Since YAP1 is a constitutively expressed molecule, it was difficult to identify significant differences in YAP1 expression levels between airway epithelial basal cells and luminal cells, and between LSCC TICs and tumor cells. The tissue used to compare YAP1 expressions was from the murine lung not specifically the trachea, which might also affect the results. We could only detect the different YAP1 expression locations in different cell types. In addition, we used tamoxifen to induce YAP1 deletion and did not use a fluorescent system to track YAP1 expression, which limited our observational results. Furthermore, the approach we used to determine tumor formation and growth can be improved. Micro-CT scanning to compare the size of the lung was hard to manipulate and provided only indirect evidence of tumor formation. However, other existing mouse models have also not reported an efficient detection tool. Finally, the tumor formation rate of our model was not as high as expected. Chemical induction can be added to accelerate the tumorigenesis process.

Together, the successfully constructed YAP1^KITrp53^KO mouse model of spontaneous LSCC can provide an economic and convenient preclinical tool for research on potential molecular targets and novel therapeutic options for LSCC, holding the promise of wide application. YAP1 promotes the stemness maintenance of airway epithelial stem cells and LSCC TICs, warranting further studies to clarify the role of high YAP1 expression as a predictor of LSCC.

NSCLC

non-small cell lung cancer

LUAD

lung adenocarcinoma

LSCC

lung squamous cell carcinoma

SCC

squamous cell carcinoma

YAP1

Yes-associated protein-1

TIC

tumor-initiating cell

FACS

fluorescence-activated cell sorting

qRT-PCR

quantitative RT-PCR

wild type

H & E

hematoxylin-eosin

IHC

immunohistochemical

immunofluorescence

NTC

non-tumor cell

PPI

protein-protein interaction

DEG

differentially expressed gene

biological process

cellular component

molecular function

Ethics approval and consent to participate

In the whole process of the experiment, the animal disposal methods conformed to the guidelines approved by the Ethics Committee of Army Medical University. The current study did not involve human subjects.

Consent for publication

Not applicable.

Availability of data and materials

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

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by the National Natural Science Foundation of China (82172670, 81972858, 82202951 and 81773245), the Technology Innovation and Application Development Project of Chongqing (2023DBXM002 and CSTB2022TIAD-KPX0176), the Cultivation Program for Clinical Research Talents of Army Medical University (2018XLC1010) and Red Medical Talents Development Program of Army Medical University.

Authors' contributions

XWC and CLJ analyzed the data and drafted the manuscript. LYZ performed the confirmation experiments. LYZ, FL and LCL prepared parts of the figures. LCL performed the bioinformatic analysis. FL and XYL contributed to performing experiments. XC, LPZ, JXC and CRY helped perform experiments and/or interpreted imaging data. FLL, DQC, LYS, YXQ and KN provided critical advice and aided in manuscript preparation. JGS designed and supervised the experiments, provided financial and administrative support. All authors approved the submitted version.

Acknowledgments

We would like to sincerely thank Prof. Cheng-Ping Xu (Pathology Department, Southwest Hospital, Army Medical University) for his guidance on data analysis. The authors also thank Dr. David P. Figgitt, Content Ed Net, for English language editing.

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SFig.1.tif
Supplementary Figure 1 Generation of homozygous YAP1^KImice and construction of the YAP1^KITrp53^KOmouse model. A) Overview of the targeting strategy for conditional YAP1 knock-in. The targeting vector was first constructed. Then the linearized vector was introduced into the C57BL/6 embryonic stem (ES) cells via electroporation. Next, the correctly targeted cells were selected in the presence of drug and by PCR, and confirmed by Southern blot. Finally, the targeted clones were microinjected with C57BL/6 blastocytes to yield chimeric mice. B) The sequence of the linear targeting vector for YAP1 knock-in. Human SP-C promoter-mutant mouse YAP1 CDS-p loyA box was cloned into the intron of ROSA26 gene. The ciliated cell-specific promoter FOXJ1 was inserted upstream of the box to ensure exclusive YAP1 expression in airway surface epithelium. The point mutation of S112A (TCC to GCC) was introduced into YAP1 CDS of the mutant mouse. Then, the bacterial artificial chromosome (BAC) clone from the C57BL/6 library was used as a template to generate homologous arms (HAs) by PCR. The mouse genome fragment containing HAs was amplified from the BAC clone using a high-fidelity DNA polymerase. The amplified fragment was then assembled into the targeting vector with its downstream recombinant site SDA and selection marker Neo. C) Nucleic acid electrophoresis to validate the YAP1 knock-in ES vector. Lane 1: ApaLI: 9.7/5.3/4.5/1.2 (kb); Lane 2: DrdI: 8.9/3.7/3.3/2.8/2.0 (kb); Lane 3: NcoI: 8.6/3.6/2.3/2.1/1.5/1.0/0.8/0.6/0.3 (kb); Lane 4: AgeI/ScaI: 8.4/3.4/2.7/2.1/1.4/1.2/0.9/0.6 (kb); Lane 5: AhdI: 9.8/4.6/2.9/1.7/1.6 (kb); Lane 6: NotI: 20.7 (kb). D) Five of the six ES clones (1B6, 1C104, 1D4, 1D8 and 1H3) were confirmed correct by Southern blot. E) Genotyping and selection of the YAP1^KITrp53^KOmouse. Left: YAP1^KIWT: 155bp; YAP1^KIHomo: 251bp; YAP1^KIHeter: 251bp/155bp. Right: Trp53^KOWT: 759 bp; Trp53^KOHomo: 998bp; Trp53^KOHeter: 998bp/759bp. F) Flow chart of the construction of the YAP1^KITrp53^KOmouse model.
SFig.2.tif
Supplementary Figure 2 Genome sequencing and bioinformatic analysis of the YAP1^KITrp53^KO mouse. A) Protein-protein interaction (PPI) network of 300 differentially expressed genes (DEGs). B) Top 10 primary modules of PPI sub-networks. C) The DEGs GO enrichment analysis of biological processes (BP), cellular components (CC) and molecular functions (MF). D) KEGG pathway enrichment of DEGs. The size of the circle indicates the numbers of DEGs, and the color represents the P-value. p≤0.05 was considered significantly enriched. E) A Venn diagram representing the overlapped genes between KEGG pathway genes and PPI network top 10 genes. KEGG pathway analysis found 24 tumorigenesis-related genes (blue), while PPI network analysis screened out top 10 tumor-related genes (red), with 3 overlapping genes in total (blue-red), Sv2b, Sv2a and Dlg4.
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YAP1 promotes the stemness of airway epithelial basal cells and spontaneous formation of lung squamous cell carcinoma in a YAP1^KITrp53^KO mouse model

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Mouse tracheal cell isolation and immunofluorescence staining

Three-Dimensional (3D) tumor spheroid assay in matrigel

YAP1^KITrp53^KO mice construction and genotyping identification

Micro-CT scanning of murine lung volume and pathological identification of YAP1^KITrp53^KO mice

Statistical analysis

Results

2. Overexpression of constitutively active YAP1 increased tracheosphere formation by airway epithelial basal cells

3. Deletion of endogenous YAP1 inhibited tracheosphere formation of airway epithelial basal cells

4. YAP1 was related to the stemness of CD49f^highEpCAM^low cells in human LSCC tissues

5. Generation of homozygous YAP1^KI mice and establishment of YAP1^KITrp53^KO mouse model

6. Tumor formation was detected in YAP1^KITrp53^KO mice by lung volume measurement

7. Pathohistological detection revealed that LSCC was formed in YAP1^KITrp53^KO mice

8. Genome sequencing and bioinformatic analysis of the YAP1^KITrp53^KO mouse

Discussion

Conclusions

List Of Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

YAP1 promotes the stemness of airway epithelial basal cells and spontaneous formation of lung squamous cell carcinoma in a YAP1KITrp53KO mouse model

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Mouse tracheal cell isolation and immunofluorescence staining

Three-Dimensional (3D) tumor spheroid assay in matrigel

YAP1KITrp53KO mice construction and genotyping identification

Micro-CT scanning of murine lung volume and pathological identification of YAP1KITrp53KO mice

Statistical analysis

Results

2. Overexpression of constitutively active YAP1 increased tracheosphere formation by airway epithelial basal cells

3. Deletion of endogenous YAP1 inhibited tracheosphere formation of airway epithelial basal cells

4. YAP1 was related to the stemness of CD49fhighEpCAMlow cells in human LSCC tissues

5. Generation of homozygous YAP1KI mice and establishment of YAP1KITrp53KO mouse model

6. Tumor formation was detected in YAP1KITrp53KO mice by lung volume measurement

7. Pathohistological detection revealed that LSCC was formed in YAP1KITrp53KO mice

8. Genome sequencing and bioinformatic analysis of the YAP1KITrp53KO mouse

Discussion

Conclusions

List Of Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

YAP1 promotes the stemness of airway epithelial basal cells and spontaneous formation of lung squamous cell carcinoma in a YAP1^KITrp53^KO mouse model

YAP1^KITrp53^KO mice construction and genotyping identification

Micro-CT scanning of murine lung volume and pathological identification of YAP1^KITrp53^KO mice

4. YAP1 was related to the stemness of CD49f^highEpCAM^low cells in human LSCC tissues

5. Generation of homozygous YAP1^KI mice and establishment of YAP1^KITrp53^KO mouse model

6. Tumor formation was detected in YAP1^KITrp53^KO mice by lung volume measurement

7. Pathohistological detection revealed that LSCC was formed in YAP1^KITrp53^KO mice

8. Genome sequencing and bioinformatic analysis of the YAP1^KITrp53^KO mouse