Fig 2: Epithelial self-renewal is regulated by Bmal1. (A) Heat map showing 24-hour fold change in relative mean expression of Bmal1-/- organoids compared with control organoids. Circadian clock genes were down-regulated, and inflammation markers were up-regulated in Bmal1 mutant organoids compared with controls. (B) Heat map showing 24-hour fold change in relative mean expression of Apcmin + Bmal1-/- organoids compared with Apcmin control organoids. Wnt pathway targets were down-regulated, whereas Hippo pathway targets were up-regulated. Cell proliferation genes also showed an increase in Apcmin + Bmal1-/-. (C) GO (Gene Ontology) analysis of pathways down-regulated by Apcmin + Bmal1 mutant tumor organoids, relative to Apcmin controls. Wnt signaling was decreased, whereas cell proliferation was increased in Bmal1 mutants, together with changes in transcriptional regulation. A Benjamini cut-off value of 0.05 was used for GO analysis. (D) Apcmin + Bmal1-/- have up-regulated Yap signaling compared with Apcmin controls (enrichment score, 0.44; normalized enrichment score, 2.083), nominal P value = .000, False Discovery Rate (FDR) q value = 0.000, Family Wise Error Rate (FWER) P value = .000). (E) After derivation, individual organoids from each genotype were dissociated into single cells and plated separately to assess the number of subclones, and the number of crypt buds per subclone. (F) Representative images of organoid subclones. Scale bar: 100 μm. (G) Individual Bmal1-/- organoids developed more subclones (t test, P = .0004), indicating increased self-renewal ability. (H) Bmal1-/- subclones had more crypt buds, indicating further increased self-renewal of ISCs (t test, P < . 0075). (I) Schematic shows clonal analysis of Apcmin organoids. (J) Representative images of Apcmin and Apcmin + Bmal1-/- organoid subclones. Scale bar: 100 μm. (K) Apcmin + Bmal1-/- tumor organoids produce more subclones indicating increased self-renewal (t test, P = .0444). (L) Apcmin control and Apcmin + Bmal1-/- mutant organoids were treated with Yap inhibitor; dimethyl sulfoxide (DMSO) is the vehicle control. Apcmin + Bmal1-/- organoids showed increased expression of the Yap target gene Ereg, which was reduced by Yap inhibitor (ANOVA, f(3,10) = 19.55; P = .002). (M) Organoids were pretreated with Yap inhibitor before subcloning. Apcmin + Bmal1-/- organoids showed increased self-renewal, which was disrupted by pretreatment of Yap inhibitor (ANOVA, f(3, 24) = 7.087; P = .00144). Data show n = 10 clones from 2 to 3 lines for quantifications. All error bars represent SEM. Inflam, Inflammation; qPCR, quantitative polymerase chain reaction; Red-Ox, Reduction-Oxidation; Rel Mean Expr, Relative Mean Expression; Sig, Signalling; Yap-i, Yap-inhibitor.

Fig 3: Yap activity is increased in Bmal1 mutant tumors. (A and B) Representative images showing increased Yap+ nuclear signal in the cells of Apcmin + Bmal1-/- tumors (arrowheads show the cell nucleus in inset image ). Yap-activated nuclei are present in higher numbers (t test, P = .0186). Scale bar: 50 μm. (C) Western blots showing increased levels of total and activated Yap in Apcmin; Bmal1-/- mutant tumors. (D and E) Representative images showing increased Yap+ nuclear signal in the cells of Apcmin + Bmal1 cKO that are present in higher numbers (t test, P = .0009). Scale bar: 50 μm. (F and G) Control tumors show higher expression of Lgr5 RNA (blue) while Apcmin + Bmal1 cKO tumors show higher Tead4 RNA expression (red). The area of Tead4+ cells is significantly higher in Apcmin + Bmal1 cKO (ANOVA f(3, 36) = 9.241; P = .0001). Scale bar: 50 μm. (H and I) Apcmin + Bmal1 cKO tumors show higher levels of the Yap target gene Ereg (blue), but lower levels of the Wnt target gene, Axin2 (red), relative to controls. The area of Ereg+ cells is significantly higher (ANOVA f(3, 36) = 9.422; P < .0001). Histologic quantification shown for n = 5–10 regions per animal, and n ≥ 3 animals per condition. Scale bar: 100 μm. All error bars represent SEM.

Fig 4: Expression of SuperHippo activates Hippo signaling in mesothelioma cell lines(A) Schematic diagram illustrating full-length WWC1 and SuperHippo (1–200 aa of WWC1).(B) Schematic diagram illustrating the activation of the Hippo signaling by SuperHippo via its functional engagement in the HPO1 signaling module.(C) SuperHippo expression in mesothelioma cell lines inactivates YAP.(D) Decreased mRNA levels of YAP/TAZ target genes ANKRD1, CTGF, and CYR61 upon SuperHippo expression in mesothelioma cell lines.(E) SuperHippo expression induces cell death in mesothelioma cell lines. Cell counting kit-9 (CCK8) assays were performed.(F) SuperHippo expression represses colony formation of mesothelioma cell lines. Data are presented as mean ± SEM from three independent experiments. Statistical significance: ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001. The Student’s t test was used.See also Figure S4.

Fig 5: Mitigation of tumor progression by AAV6-SuperHippo-P2A-NF2 in an Nf2;Trp53 dKO mouse DPM model(A) Schematic diagram indicating the experimental procedure. Adeno-Cre was injected intrathoracically into 4-week-old Nf2fl/fl;Trp53fl/fl mice to initiate DPM, and, one week later, AAV6-control or AAV6-SuperHippo-P2A-NF2 was injected intrathoracically. Lungs and hearts were collected for histological analysis after another 8 weeks. n = 5–7 for each group.(B) Body weight measurement of mice used.(C) H&E and IHC staining of lung and heart tissue sections from control and treated mice. IHC for WT1, Mesothelin, YAP, and Ki67 were shown. Scale bar, 5 mm for gross lung and heart images, and 50 μm for H&E or IHC images.(D and E) Measurement of the average thickness of lung and heart mesothelium in mice from control and treated groups. Mesothelium thickness is primarily defined by Mesothelin staining signals.(F) Overall survival curve of mice treated with AAV6-control or AAV6-SuperHippo-P2A-NF2.(G) Immunoblotting analysis of SuperHippo-P2A-NF2 expression and cleavage in lungs and hearts from mice treated with AAV6-SuperHippo-P2A-NF2. Expression of YAP/TAZ targets, CTY61 and CTGF, was also reduced in treated samples.Data are presented as mean ± SEM. Statistical significance: ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001. The log rank (Mantel-Cox) test was used for the overall survival curve (F), and the Student’s t test for other data.See also Figure S7.