Notch1 Deficiency Induces Tumor Cell Accumulation Inside the Bronchiolar Lumen and Increases TAZ Expression in an Autochthonous Kras LSL-G12V Driven Lung Cancer Mouse Model

Purpose: Abrogation of Notch signaling, which is pivotal for lung development and pulmonary epithelial cell fate decisions was shown to be involved in the aggressiveness and the differentiation of lung carcinomas. Additionally, the transcription factors YAP and TAZ which are involved in the Hippo pathway, were recently shown to be tightly linked with Notch signaling and to regulate the cell fate in epidermal stem cells. Thus, we aim to elucidate the effects of conditional Notch1 deficiency on carcinogenesis and TAZ expression in lung cancer. Methods: We investigated the effect of conditional Cre-recombinase mediated Notch1 knock-out on lung cancer cells in vivo using an autochthonous mouse model of lung adenocarcinomas driven by Kras LSL-G12V and comprehensive immunohistochemical analysis. In addition, we analyzed clinical samples and human lung cancer cell lines for TAZ expression and supported our findings by publicly available data from The Cancer Genome Atlas (TCGA). Results: In mice, we found induction of papillary adenocarcinomas and protrusions of tumor cells from the bronchiolar lining upon Notch1 deficiency. Moreover, the mutated Kras driven lung tumors with deleted Notch1 showed increased TAZ expression and focal nuclear translocation which was frequently observed in human pulmonary adenocarcinomas and squamous cell carcinomas of the lung, but not in small cell lung carcinomas. In addition, we used data from TCGA to show that putative inactivating NOTCH1 mutations co-occur with KRAS mutations and genomic amplifications in lung adenocarcinomas. Conclusion: Our in vivo study provides evidence that Notch1 deficiency in mutated Kras driven lung carcinomas contributes to lung carcinogenesis in a subgroup of patients by increasing TAZ expression who might benefit from TAZ signaling blockade.

carcinomas. In addition, we used data from TCGA to show that putative inactivating NOTCH1 mutations co-occur with KRAS mutations and genomic amplifications in lung adenocarcinomas.
Conclusion: Our in vivo study provides evidence that Notch1 deficiency in mutated Kras driven lung carcinomas contributes to lung carcinogenesis in a subgroup of patients by increasing TAZ expression who might benefit from TAZ signaling blockade. Keywords: mouse model, lung cancer, KRAS, NOTCH1, TAZ BACKGROUND Lung cancer is the leading entity in cancer-related mortality in men and women worldwide [1]. Approximately 85% of lung cancer cases belong in the group of non-small cell lung carcinomas (NSCLCs), comprising adenocarcinomas (ADCs), squamous cell carcinomas (SCCs) and large cell carcinomas [2]. Around 15% of lung cancer cases account for small cell lung carcinomas (SCLCs) [3]. About 4-9% of NSCLCs occurs as a mixed adeno-squamous carcinoma [4] and this mixed lung cancer subgroup is associated with a worse prognosis leading to significantly shortened survival after resection compared to carcinoma entities with unilinear differentiation [5].
ADCs are derived from alveolar type II, Clara cells and bronchioalveolar precursors located in the alveolar periphery and the bronchiolar epithelium [6,7]. SCCs arise from basal cells residing below the epithelia of the upper airways expressing specific lineage markers as p63, cytokeratin (CK) 5 and 6 and thrombomodulin [4]. Frequent oncogenic drivers of ADCs are mutated KRAS and EGFR, which are less frequent in SCCs [2].
Inactivating mutations in Notch genes were identified in SCC of both, skin and lung [8]. The Notch pathway consists of four different membrane-spanning receptors (Notch1-4), which transduce canonical signals after binding their ligands (Jagged1 and 2, Delta 1, 3 and 4) on neighboring cells. Upon ligand binding, these receptors are cleaved at two proteolytic sites, which liberates the functionally active Notch intracellular domain (NICD) into the cytoplasm. NICD translocates to the nucleus and builds a DNA-binding complex together with its coactivator Mastermind-like (MAML) to regulate target gene expression. Thereby, Notch signaling fulfills pivotal functions in lung development and in epidermal cell differentiation [9].
Importantly, abrogation of Notch signaling by means of the mechano-activation of transcriptional coactivator with PDZbinding motif (TAZ), encoded by the WWTR1 gene, was recently shown to alter epidermal stem cell fate decisions [10]. TAZ is an effector of the Hippo signaling pathway and has a functional role in organ development. Moreover, TAZ is implicated as an oncogene in NSCLC [11], and TAZ expression is associated with worse prognosis and is additionally an independent prognostic factor for poor survival in patients with resected NSCLC [12].
In our study, we aimed to investigate the effects of conditional Notch1 deficiency on carcinogenesis and TAZ expression using an autochthonous Kras LSL-G12V driven lung cancer mouse model [13].

Animal Experiments
This study was carried out in accordance to the recommendations of the Federation of European Laboratory Animal Science Association. The protocol was approved by the local Ethics Committee of Animal experiments and the Landesamt für Natur, Umwelt und Verbraucherschutz of North Rhine-Westphalia in Germany (reference number: 84-02.04.2015.A199). Six to eight weeks old, genetically engineered, male and female mice with C57BL/6J lineage background were anesthetized with Ketamin/Xylazin (100 mg/kg/(body weight) BW i.p./0.5 mg/kg/BW i.p.) and 1 × 10 7 pfu Adeno-Cre was applied intratracheally [14]. Viral vectors were provided by the University of Iowa Viral Vector Core (http://www.medicine.uiowa.edu/vectorcore). Prior to tumor induction, the mice were genotyped. We crossed Kras LSL-G12V mice, commonly called RASLO, which express the Kras G12V mutant after Cre-recombinase exposure [13] (Supplementary Figure S1a) with the conditional Cre-inducible Notch1 knock-out model (Notch1 tm1Agt , commonly called Notch1 fl/fl ) [15] (Supplementary Figure S1b), kindly provided by Freddy Radtke and colleagues. Eight weeks after Cre-application, mice were sacrificed by cervical dislocation and lung tissue was harvested and formalin fixed and paraffin embedded (FFPE). In total, seven mice per group have been analyzed and are indicated as follows: R+ N1w/w (n 7) harboring mutated Kras driven tumors with wild type Notch1 alleles, R+ N1f/w (n 7) harboring mutated Kras driven tumors with heterozygous Notch1 knock-out and R+ N1f/f (n 7) harboring mutated Kras driven tumors with homozygous Notch1 knock-out.
TAZ protein expression was scored by IHC in murine tumor samples and in clinical samples combined in tissue micro arrays (TMAs). Thirty-eight squamous cell carcinomas (SCC), 37 adenocarcinomas (ADC) and 32 small cell carcinomas (SCLC) of patients have been investigated. TAZ expression in the nucleus or the cytoplasm was rated as positive if more than 10% of tumor cells exhibited expression. The TAZ score was determined by two independent investigators and graded on an integer number fourgrade scale (0: no expression, 1: weak expression, 2: moderate expression, 3: strong expression). The TAZ score was obtained by adding the nuclear and the cytoplasmic staining intensity, the nuclear multiplied with 0.75, the cytoplasmic multiplied with 0.25, in order to have a measure of the TAZ activation status [17].

Cells and Reagents
Lung cancer cell lines were kindly provided by Roman K. Thomas (Department of Translational Genomics, University of Cologne, Germany) and Reinhard Büttner (Institute for Pathology, University Hospital Cologne, Germany). Cells were cultivated in RPMI medium (Life Technologies, Carlsbad, United States) supplemented with 10% FCS (PAA laboratories, Cölbe, Germany). WWTR1/TAZ and NOTCH1 siRNAs (100 nM, Santa Cruz Biotechnology) were transfected using Lipofectamine 3000 (Life Technologies). Plasmid transfection and vector control were performed as previously described [18]. pTight-hASCL1-N174 [19] was a gift from Jerry Crabtree (Addgene plasmid #31876; http://n2t.net/addgene:31876; RRID: Addgene_31876). Cell proliferation was determined by Cell Proliferation kit I [(MTT) assay (Hoffmann-La Roche, Basel, Switzerland)]. MTT assays were performed in 96-well plates with 3,000 cells per well. Cells have been seeded after validated TAZ knock-down in medium containing tetrazolium salt, the reagent which enables the colorimetric detection of proliferating cells. Tetrazolium salt is cleaved to the water insoluble dye formazan by succinate-tetrazolium reductase system. This enzyme is only active in viable cells. The cell proliferation was determined after 24, 48 and 72 h, by measuring the absorbances at A550/ 690 nm using a plate reader. The protocol was performed according to the manufacturer's instructions.
For the generation of cell pellet blocks, 1 × 10 7 cells were centrifuged and fixed in 4% paraformaldehyde (PFA) over night. PFA was removed by centrifugation for 15 min at 2400 rpm and cells were washed with 2 ml 96% ethanol with three drops of glycerin. Cells were again centrifuged for 15 min at 2400 rpm and transferred to tissue capsules and were further processed for paraffin embedding by standard FFPE biopsy protocol.

Ethics
Clinical samples were acquired and analyzed as part of routine diagnostic procedures (Institute of Pathology, University Hospital Cologne, Germany) according to the guidelines and with approval of the local ethics committee (reference number: 13-091).

Statistics
Statistical analysis was performed with Graph Pad Prism (STATCON, Witzenhausen, Germany) and SPSS (IBM Corp., Armonk, United States). The two-sided Student's t-test and the Pearson Correlation Coefficient were used to analyze data for significant differences and correlations. Error bars indicate standard error of the mean (SEM). p-values <0.05 were regarded as significant and indicated in the figures as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Conditional Notch1 Deficiency Significantly Increases Tumor Cell Accumulation Inside the Bronchiolar Lumen in a Mutated Kras Driven Lung Cancer Mouse Model
Mice, carrying the conditional Kras LSL-G12V expression construct [13] were crossed with conditional Notch1 knockout mice [15]. Hence, both, the expression of the Kras mutant and the Notch1 knock-out depend on adenoviral Cre-recombinase application. Eight weeks after intratracheal inhalation of adenoviral Cre-recombinase, lung tissues were harvested ( Figure 1A). The size of single tumor lesions was determined and tumors were analyzed histologically ( Figure 1B). Immunohistochemical analysis of the intracellular domain of Notch1 (NICD1), shown to be the active Notch1 signaling component, confirmed that Notch1 was efficiently knocked out in the Kras mutant derived tumors ( Figure 1C). There was no significant difference in tumor size observed between the mutated Kras driven tumors with wild type Notch1 alleles (R+ N1w/w), the mutated Kras driven tumors with heterozygous Notch1 knock-out (R+ N1f/w) and the mutated Kras driven tumors with homozygous Notch1 knock-out (R+ N1f/f).
Morphologically, the tumors presented as papillary ADCs and showed cuboidal cell shape in R+ N1w/w mice harboring active Notch1 signaling. Notch1 deficiency resulted in a mosaic-like pattern and complete loss of NICD1 in R+ N1f/w and R+ N1f/f mice, resulted in papillary ADCs comprised of cuboidal and columnar tumor cells, respectively ( Figure 1C).
The determined tumor area upon Notch1 knock-out was slightly decreased from 24.68 to 17.90% (p 0.1672) as well as tumor cell proliferation, indicated by the KI67 index, from 21.00 to 17.86% (p 0.3034) in a dose-dependent manner, though not significantly, comparing R+ N1w/w and R+ N1f/f mice (Figures 2A-C). Intriguingly, tumor cell accumulation inside the bronchiolar lumen with proliferating cells which protrude from the bronchiolar lining, was significantly increased both upon the loss of one Notch1 allele [from 9.82 to 28.40% (p 0.0390)] and the loss of two Notch1 alleles [from 9.82 to 40.34% (p 0.0020)] ( Figure 2D). The cell lineage markers, Surfactant Protein C (SPC) and Clara Cell 10 KDa Secretory Protein (CC10) were used to identify the differentiation of dysplastic cells accumulations inside the bronchiolar space. SPC+ cells representing alveolar type II cells, were located in the alveolar periphery (arrow 1) and CC10 + Clara cells lined the bronchiolar walls (arrow 2). ADCs which grew predominantly peri-bronchiolarly were composed of SPC+ tumor cells (arrow 3). In R+ N1w/w mice, bronchiolar airways were rarely invaded by SPC+ tumor cells whereas in R+ N1f/f mice, SPC+ cells which were located within the bronchiolar lining formed epithelial carcinoma in situ lesions protruding from the bronchiolar wall (arrow 4) and budding into the bronchiolar lumen (arrow 5) ( Figure 2E).
Taken together, these data suggest that conditional Notch1 deficiency in this autochthonous Kras LSL-G12V driven lung cancer mouse model induces papillary ADCs accompanied by SPC+ tumor cell accumulation inside the bronchiolar lumen, which is not found under Notch1 wild-type conditions.

Co-Occurrence of Putative Loss-of-Function Mutations in NOTCH1 with Genomic KRAS Aberrations in Lung ADCs
To determine whether Notch1 deficiency due to genomic aberrations can also be observed in human mutated KRAS driven ADCs, we used TCGA database and investigated the publicly available lung ADC dataset containing 230 cases [22] for genomic aberrations in KRAS and NOTCH1. We found ten genomic alterations in NOTCH1 (4.3%) ( Table 1), of which five were putative loss-of-function aberrations (2.15%). One of the NOTCH1 alteration was annotated as an oncogenic likely loss-offunction frame shift insertion. The other four of the NOTCH1 alterations were missense mutations in domains coding for the extracellular EGF-like repeats, which may result in interference with Notch ligand binding and reduce NOTCH1 signaling [18]. In four out of five cases harboring putative NOTCH1 loss-of-function alteration, KRAS was also altered by genomic amplification and mutation ( Table 1). In 178 cases of SCCs obtained from TCGA database [23], genomic aberrations in NOTCH1 and KRAS are mutually exclusive and do not co-occur (Supplementary Table S1).
Taken together, this data suggests that Notch1 deficiency by putative inactivating NOTCH1 alterations occurs together with KRAS aberrations particularly in human ADCs of the lung, thus giving clinical importance to our findings.

Deletion of Notch1 Triggered TAZ Expression in Mutated Kras LSL-G12V Driven Lung Cancer
Since TAZ and Notch signaling cooperate in epidermal cell fate decisions [10], we aimed to decipher whether upon Notch1 deletion TAZ expression is altered in mutated Kras driven ADCs. ASCL1 is a master regulator in Notch signaling [18] and is used as a positive control for Notch1 deficiency in the following analyses.
We investigated TAZ expression in R+ N1w/w, R+ N1f/w and R+ N1f/f mice by IHC and determined a total protein expression score per single lesion. We found that partial and full Notch1 knock-out in mutated Kras driven ADCs increased TAZ expression significantly (t-test, two-sided, p 0.0012). In addition, Pearson Correlation analysis revealed a weak correlation of moderate and high TAZ expression to partial and full Notch1 knock-out R 0.27231; p 0.00029) (Figure 3). Similar results were obtained in vitro using the ADC cell line PC9 which has NOTCH1 expression and low levels of ASCL1 (Supplementary Figure S2a). ASCL1 is negatively regulated by NOTCH1 [18]. Thus, PC9 cells showed increased ASCL1 and WWTR1/TAZ expression upon NOTCH1 knock-down (Supplementary Figure  S2b). Upon plasmid derived ASCL1 expression in PC9 cell clones we detected WWTR1/TAZ up-regulation, as well (Supplementary Figure S2c). These data show that TAZ expression is regulated by Notch1.
Furthermore, we investigated TAZ expression in cell blocks of human lung carcinoma cell lines, representing the most frequent lung carcinoma entities, including four SCC cell lines (H226, HCC15, HCC95, H1703), four ADC cell lines (H1975, PC9, H441, A549) and four SCLC cell lines (GLC8, GLC1, N417, DMS114). All SCC cell lines exhibited high TAZ expression,  among the ADC cell lines, A549 and H441 cells, both harboring KRAS mutations, showed the highest TAZ expression. Among the SCLC cell lines only DMS114 cells showed TAZ expression ( Figures 4A-C). Knock-down of TAZ in H1703, A549 and DMS114 cells using three pooled target specific siRNAs significantly reduced tumor cell proliferation after 72 h determined by MTT assay (Figures 4D-F). In the cell lines GLC8 and GLC1, that showed only marginal TAZ expression, TAZ knock-down did not affect tumor cell proliferation (Supplementary Figure S3). Thus, we hypothesize that TAZ protein expression correlates to the inhibitory effect of TAZ knock-down on cell proliferation and that TAZ protein expression might serve as a predictive marker for treatment with TAZ signaling inhibitors.
To show whether this TAZ distribution also applies to clinical patient samples, we investigated TAZ expression in lung carcinoma samples acquired during routine diagnostics. Patients' lung SCCs, ADCs and SCLCs were analyzed by IHC using TMAs with regard to the increased TAZ expression and nuclear TAZ localization ( Figure 5; Table 2; Supplementary Figure S4) because the nuclear translocation of TAZ indicates activation of the Hippo signaling pathway [24]. Within the clinicopathologic parameters we found no association of TAZ expression to age and gender ( Table 2).
Importantly, high total and nuclear TAZ protein expression was detected in ADCs, associated to the SCC entity using Pearson Correlation (R total 0.66464; R nucleus 0.5999; p < 0.0001), but less present in the SCLC cohort ( Figures 5C, D; Table 2).
Addressing TAZ expression and survival in publicly available TCGA data [22,23], WWTR1/TAZ amplification served as a negative prognostic factor in ADCs (Logrank test; p 0.0533), but had no impact on survival in SCCs (Logrank test; p 0.988) (Supplementary Figure S5).
This data demonstrates that TAZ expression is found in SCCs, ADCs and SCLCs of the lung, serves as a negative prognostic factor in ADCs and is mainly associated to the SCC entity. Interestingly, TAZ expression which is related to a poor prognosis in ADCs, might be regulated by NOTCH1 deficiency.

DISCUSSION
In this study, we investigated the effects of functional Notch1 deficiency in an autochthonous Kras LSL-G12V driven lung adenocarcinoma mouse model.
Mutated Kras driven murine lung tumors are considered to be weakly, if at all, invasive and it has been discussed that additional deletion of Tp53 is required for a full stromal invasive growth [14,25]. An alternative highly aggressive pathway for invasion in humans involves the spread of tumor cells via air spaces (STAS), for example presenting with micropapillary growth pattern associated with poor survival probability [26,27].
Spread through air spaces is thought to involve tumor budding at the invasive front of tumors [28] and we also observed tumor cells accumulating inside the bronchiolar lumen. Mutated Kras driven tumors generally arise in the periphery of the lung and are less frequently observed in the central major airways which is thought to be due to the cellular origin of lung tumors [27]. SPC+ alveolar type II cells and CC10+ SPC+ bronchioalveolar stem cells are suggested to be the major cell type of lung cancer origin in humans and mice [6,7,27]. In line with this notion, the tumors observed in the Kras driven lung cancer mouse model used here, positively stain for SPC, indicating canonical pathophysiological tumorigenesis with respect to the cell of origin.
Carcinogenesis in lung cancer, like in many other solid tumors, has been shown to occur through a multistep process [29]. In the used lung cancer mouse model, the activating Kras G12V mutation induce an adenomatous change in the lung parenchyma because the proliferating cells are not eliminated. Interestingly, a similar mouse model with Kras activation in the gut does not lead to tumor formation since the proliferating cells are just shed, unless an inactivating genomic aberration in adenomatous polyposis coli (Apc) is present in addition [30,31].
Similarly, in the central airways of the lung, proliferative cells can be easily shed into the lumen and therefore no dysplastic cell mass is able to accumulate, which is the required condition for further mutation hits resulting in a multistep carcinogenesis. Our findings on lung cancer in mice upon Kras activation combined with Notch1 inactivation clearly show an accumulation of tumor cells in the lumen of bronchioles protruding from the bronchiolar epithelium. The enrichment of tumor cells in the bronchiolar lumen is reasoned by Notch1 deficiency on lateral inhibition in the tumor cells [32]. Moreover, the reduced cell contact inhibition, caused by the induced TAZ expression upon Notch1 deficiency followed by the Hippo pathway alteration, facilitates protruding of proliferating tumor cells from the bronchiolar lining [33,34].
Interestingly, TAZ regulates lineage decisions in mammary epithelium, being active in basal cells, the likely cell of origin of SCCs, and mediating luminal differentiation upon downregulation [24]. Recently, Totaro and colleagues linked YAP/ TAZ and Notch signaling to epidermal cell fate decisions showing that active YAP/TAZ induced Notch inhibition [10], which is in line with our findings.

Pathology & Oncology Research
April 2021 | Volume 27 | Article 596522 mutant mouse model. Under healthy conditions Maml1 forms a signaling complex together with Notch, but linked to the dominant negative mutant of Maml1, Notch signaling was abrogated completely in the mutated Kras driven lung tumors. They identified a squamous cell differentiation in 5-17% of tumor cells accompanied by reduced tumor burden upon deletion of Notch signaling [35]. Moreover, the inactivating NOTCH mutations described in human lung tumors have been found mainly in centrally located tumor represented by neuroendocrine differentiated or squamous cell carcinomas [8,18], which is consistent with the findings in the mouse model described here. Therefore, we show here for the first time in an in vivo model that Notch1 deficiency is able to induce tumor cell accumulation even in central airways in a mutated Kras driven lung cancer mouse model, which may represent the early steps of a typical multistep carcinogenesis. Since putative loss-of-function mutations co-occur in a small fraction of lung ADCs with KRAS mutations and genomic amplifications, there is a rationale to use this model to test therapy regiments targeting TAZ. Since therapeutic interventions directly blocking TAZ signaling are missing [36], treatments acting upstream and downstream of TAZ signaling might be investigated in this model.

CONCLUSION
Our study provides a genetically engineered mouse model to investigate tumorigenesis of autochthonous Kras LSL-G12V driven lung cancer upon conditional Notch1 knock-out. Particularly, we found that Notch1 deficiency triggered tumor cell accumulation inside the bronchiolar lumen and expression of the Hippo pathway transcription factor TAZ, which may represent a target for cancer therapy in this specific subgroup of lung ADCs.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material. (B) TAZ stained sections were scanned, analyzed using the Pannoramic Viewer and scored in terms of TAZ nuclear localization with 0 (no expression), 1 (weak expression), 2 (moderate expression), 3 (strong expression). Statistical analysis was performed using Pearson Correlation (ns-not significant; *p < 0.05; **p < 0.01, ***p < 0.001).