WWTR1(TAZ)-CAMTA1 reprograms endothelial cells to drive epithelioid hemangioendothelioma

Abstract

Epithelioid hemangioendothelioma (EHE) is a poorly understood and devastating vascular cancer. Sequencing of EHE has revealed a unique gene fusion between the Hippo pathway nuclear effector TAZ (WWTR1) and the brain-enriched transcription factor CAMTA1 in ∼90% of cases. However, it remains unclear whether the TAZ-CAMTA1 gene fusion is a driver of EHE, and potential targeted therapies are unknown. Here, we show that TAZ-CAMTA1 expression in endothelial cells is sufficient to drive the formation of vascular tumors with the distinctive features of EHE, and inhibition of TAZ-CAMTA1 results in the regression of these vascular tumors. We further show that activated TAZ resembles TAZ-CAMTA1 in driving the formation of EHE-like vascular tumors, suggesting that constitutive activation of TAZ underlies the pathological features of EHE. We show that TAZ-CAMTA1 initiates an angiogenic and regenerative-like transcriptional program in endothelial cells, and disruption of the TAZ-CAMTA1-TEAD interaction or ectopic expression of a dominant negative TEAD in vivo inhibits TAZ-CAMTA1-mediated transformation. Our study provides the first genetic model of a TAZ fusion oncoprotein driving its associated human cancer, pinpointing TAZ-CAMTA1 as the key driver and a valid therapeutic target of EHE.

Keywords: CAMTA1; Hippo pathway; TAZ; YAP; cancer; endothelial cells; epithelioid hemangioendothelioma; gene fusion; vascular anomalies; vascular malformations.

Figure 1.

TAZ-CAMTA1 expression in endothelial cells drives the formation of EHE-like vascular tumors in the lungs of mice. (A) Structure of TAZ, CAMTA1, and the resulting TAZ-CAMTA1 fusion protein formed from the t(1;3)(p36;q25) chromosomal translocation. TAZ-CAMTA1 contains the N terminus of TAZ with its TEAD-binding domain (TBD), WW domain, and three LATS1/2 phosphorylation sites, and the C terminus of CAMTA1 with its transcription activation domain (TAD), TIG domain, ankyrin repeats (ANK), IQ motifs, and a nuclear localization signal (NLS). TAZ-CAMTA1 has lost the TAD of TAZ, one of TAZ's LATS1/2 phosphorylation sites, and the CG-1 domain of CAMTA1. (B) Schematics of the Cdh5-tTA and TRE-TAZ-CAMTA1 alleles for endothelial-specific expression of TAZ-CAMTA1. (C) Schematic for induction of TAZ-CAMTA1 expression after birth. (D) Survival curve for Ctrl (single transgenic Cdh5-tTA or TRE-TAZ-CAMTA1 mice, n = 14, median survival = undefined) or TAZ-CAMTA1^iEC mice (Cdh5-tTA;TRE-TAZ-CAMTA1, n = 20, median survival = 39.5 d). (****) P < 0.0001, Mantel-Cox test. (E–I) Representative images showing H&E (E,F), CD31 (G), CD34 (H), and Erg (I) immunohistochemistry of a tumor found within a vessel of the lungs of 7-wk-old TAZ-CAMTA1^iEC mice. Black arrows in E point to the tumor, and blue arrows in F show plump spindled and epithelioid cells, while green arrows show cytoplasmic vacuoles, some with red blood cells. Red arrows in G– I show positive staining for the respective markers. Scale bars: E, 100 µm; F––I, 35 µm.

Figure 2.

TAZ-CAMTA1 is required for tumor progression and maintenance. (A) Schematic for removal of TAZ-CAMTA1 transgene expression after tumor formation. (B) Survival curve for mice that were maintained on doxycycline water (median survival = undefined, n = 6) or normal water (median survival = 16 d, n = 5) after P40. (**) P < 0.01, Mantel-Cox test. (C–H) H&E (C–E) and CD31 immunostaining (F–H) of Ctrl (C,F), TAZ-CAMTA1^iEC (D,G), and TAZ-CAMTA1^iEC mice given doxycycline for 2 wk (E,H). All P40 TAZ-CAMTA1^iEC mice (n = 5) exhibited tumors, while no P40 Ctrl mice (n = 5) or P54 TAZ-CAMTA1^iEC mice (receiving 2 wk of doxycycline after P40, n = 3) had any lung tumors. Black arrows point to examples of tumors within blood vessels. Scale bar, 50 µm. (I) Quantification of tumors observed per 40× field of view in P40 Ctrl (0 ± 0, n = 5), P40 TAZ-CAMTA1^iEC (2.5 ± 0.7, n = 5, [**] P = 0.01), P54 Ctrl (0 ± 0, n = 3), or P54 TAZ-CAMTA1^iEC mice (0 ± 0, n = 3, P > 0.05). (J) Quantification of noncapillary vessels affected in P40 Ctrl (0% ± 0, n = 5), P40 TAZ-CAMTA1^iEC (51.3% ± 8.9, n = 5, [***] P < 0.001), P54 Ctrl (0% ± 0, n = 3), or P54 TAZ-CAMTA1^iEC mice (0% ± 0, n = 3, P > 0.05). (K) Quantification of independent tumors observed per vessel in P40 Ctr (0 ± 0, n = 5), P40 TAZ-CAMTA1^iEC (2.1 ± 0.3, n = 5, [****] P < 0.0001), P54 Ctrl (0 ± 0, n = 3), or P54 TAZ-CAMTA1^iEC mice (0 ± 0, n = 3, P > 0.05). (L–N) Endomucin (Emcn) and Ki67 immunofluorescence of P40 and P54 TAZ-CAMTA1^iEC vessels. P40 TAZ-CAMTA1^iEC (14.4 ± 2.6, n = 3) vessels had significantly more Ki67 positivity than Ctrl (1.8 ± 0.2, n = 3, [**] P < 0.01), while P54 TAZ-CAMTA1^iEC (1.4 ± 1.4, n = 3) vessels had no difference in Ki67 positivity compared with control (0.8 ± 0.8, n = 3, P > 0.05). White arrows point to Ki67⁺Emcn⁺ endothelial cells. Scale bars, 25 µm. All data are presented as mean ± S.E.M, and P-values were calculated with unpaired t-tests.

Figure 3.

TAZ-CAMTA1 is highly tumorigenic in endothelial cells. (A–C) Ctrl (−Dox) (3.3 colonies ± 0.7, n = 3) or TAZ-CAMTA1-expressing (+Dox) (338 colonies ± 27.1, n = 3) MS1 cells grown on soft agar. TAZ-CAMTA1 expression significantly increased the colony forming ability of MS1 cells on soft agar. (***) P < 0.001. (D,E) Representative images of tumors established in nude mice from Ctrl (−Dox) or TAZ-CAMTA1-expressing (+Dox) MS1 cells. Scale bar, 1 cm. Red arrows point to tumors. (F) Growth curve of tumors formed from Ctrl (13.4 ± 8.9, n = 5) or TAZ-CAMTA1-expressing (213.8 ± 76.7, n = 5) MS1 cells. (*) P < 0.05. Values represent final time point, day 100. All values are mean ± SEM, and P-value was calculated with an unpaired t-test. (G–L) Representative H&E (G,H), CD31 (I,J), and FLAG (K,L) immunohistochemistry of tumors derived from Ctrl (−Dox) (G,I,K) or TAZ-CAMTA1-expressing (+Dox) (H,J,L) MS1 cells. Red arrows point to positive immunostaining. Scale bars,100 µm. (M–O) Representative H&E (M), CD31 (N), and FLAG (O) immunohistochemistry of a lung metastasis identified in a nude mouse injected subcutaneously with MS1 cells expressing TAZ-CAMTA1. Red arrows point to positive immunostaining. Scale bars,100µm.

Figure 4.

TAZ-CAMTA1 drives a proliferative endothelial cell population with a YAP/TAZ target gene signature. (A) Schematic for isolation of lung ECs for scRNA-seq. (B) t-SNE plot of clusters of cells observed from scRNA-seq. (C) t-SNE plots showing expression of various marker genes. Red arrows point to cluster showing positive expression. (D) Volcano plot of 806 differentially expressed genes in TAZ-CAMTA1-induced proliferative subpopulation. Wilcoxon rank sum test, adjusted P-value < 0.05. Genes in red have a log(FC) > 1 and genes in blue have a log(FC) < 1. Some pertinent genes and their corresponding points are marked. (E) t-SNE plots of markers from proliferative subpopulation. (F) Immunostaining validation of differentially expressed genes identified from scRNA-seq analysis. White arrows point to positive staining of Pdgfa, Cryab, or S100a6 in P40 TAZ-CAMTA1^iEC but not Ctrl lung vessels. Scale bars, 25 µm. (G) GSEA plot showing normalized enrichment score (NES) of YAP-conserved signature in the proliferative subpopulation, NES = 1.8. P < 0.01, FDR = 0.1. (H) t-SNE plots of canonical YAP/TAZ target genes. Red arrows point to the proliferative cluster showing high expression of these genes.

Figure 5.

TAZ^S4A phenocopies TAZ-CAMTA1, and TAZ-CAMTA1 requires the TEAD family of transcription factors to drive tumorigenesis. (A–D) Representative images of Ctrl (1.0 ± 0.1, n = 3), TAZ (0.7 ± 0.1, n = 3), or TAZ^S4A-expressing (318.6 ± 11.3, n = 3) MS1 cells growing in soft agar. Only TAZ^S4A is sufficient to promote anchorage-independent growth. (****) P < 0.0001, one-way ANOVA with post-hoc Tukey test. (E) Schematic for generation of endothelial-specificTAZ^S4A (TAZ^iEC) transgenic mice. (F) Survival curve for Ctrl (median survival = undefined, n = 10) and TAZ^iEC (median survival = 45 days, n = 9) mice. (****) P < 0.0001, Mantel-Cox test. (G) Representative H&E of tumor located in noncapillary vessel of a P35 TAZ^iEC mouse. Scale bar, 35 µm. (H) Schematic for generation of TRE-TEAD2DN;Cdh5-tTA (T-DN^iEC) and TRE-TEAD2DN;TRE-TAZ-CAMTA1;Cdh5-tTA (TC^iEC;T-DN^iEC) transgenic mice. (I) Survival curve of T-DN^iEC mice (median survival = undefined, n = 7), TAZ-CAMTA1^iEC mice (median survival = 45 d, n = 5), and TEAD2DN^iEC, TAZ-CAMTA1^iEC mice (median survival = undefined, n = 7). (***) P < 0.001, Mantel-Cox test. (J) Representative H&E of a vessel in a TC^iEC;T-DN^iEC mouse showing the absence of any vascular tumors. Scale bar, 100 µm. None of the TC^iEC;T-DN^iEC mice examined (n = 3) showed lung tumors at P40.

Figure 6.

TAZ-CAMTA1 and TAZ^S4A drive a gene program associated with angiogenesis and regeneration. (A) Number of differentially expressed genes (DEG; FDR < 0.01) in MS1 cells expressing TAZ^S4A or TAZ-CAMTA1 as compared with control MS1 cells. Overlapped region indicates genes expressed in both populations. Hypergeometric test P-value and Jaccard index are indicated. (BC) Volcano plot of DEGs of TAZ^S4A or TAZ-CAMTA1. Red (overexpressed) and blue (underexpressed) are genes with −log₁₀(p) > 25. (D,E) Normalized enrichment scores (NES) for selected oncogenic pathways in MS1 cells expressing TAZ^S4A or TAZ-CAMTA1 as determined by GSEA. Red indicates positive enrichment and blue indicates negative enrichment. (F) qPCR for Vegfa expression in control MS1 cells (1.0 ± 0.0, n = 3) and MS1 cells expressing TAZ^S4A (3.1 ± 0.1, n = 3) or TAZ-CAMTA1 (4.9 ± 0.1, n = 3). (***) P < 0.001. one-way ANOVA with post-hoc Tukey test. All data are mean ± SEM. (G) Enrichment of an angiogenesis gene signature in TAZ-CAMTA1-expressing MS1 cells, NES = 2.6. (**) P < 0.01, FDR < 0.01. (H) Heat map showing expression of selected regenerative endothelial cell markers in TAZ^S4A- or TAZ-CAMTA1-expressing MS1 cells.

Figure 7.

TAZ-CAMTA1 is still susceptible to regulation by the Hippo pathway. (A) Gene ontology plot of TAZ-CAMTA1-binding proteins identified by mass spectrometry. (B) Spectral counts of Hippo pathway-related proteins identified in anti-FLAG immunoprecipitates from 293T cells expressing either empty vector (EV) or 2×-FLAG-TAZ-CAMTA1 (TC). (C–E) Immunostaining of transfected FLAG-tagged TAZ-CAMTA1 in 293A cells (C) or 293A LATS1/2 KO cells (D). 293A LATS1/2 KO (81.3% ± 6.4%, n = 6) cells had significantly more TAZ-CAMTA1 localized to the nucleus than 293A cells (47.8% ± 8.3%, n = 6). (**) P < 0.01, unpaired t-test. (F–H) Immunostaining of transfected FLAG-tagged TAZ-CAMTA1 or TAZ-CAMTA1^S3A in 293T cells. TAZ-CAMTA1 (70.3% ± 4.4%, n = 10) was significantly more localized to the cytoplasm than TAZ-CAMTA1^S3A (35.0% ± 4.9%, n = 10). (****) P < 0.0001, unpaired t-test. (I,J,K) qPCR for expression of Amotl2 (I), Ankrd1 (J), and Ctgf (K) in MS1 cells stably expressing empty vector (Ctrl), TAZ-CAMTA1 (TC), or TAZ-CAMTA1^S3A (TC^3A). Cells expressing TAZ-CAMTA1^S3A had significantly increased expression of Amotl2 (Ctrl: 1.0 ± 0.1, n = 3; TC: 1.4 ± 0.1, n = 3; TC^3A: 4.3 ± 0.1, n = 3), Ankrd1 (Ctrl: 1.0 ± 0.1, n = 3; TC: 0.9 ± 0.0, n = 3; TC^3A: 2.3 ± 0.1, n = 3), and Ctgf (Ctrl: 1.0 ± 0.3, n = 3; TC: 1.0 ± 0.0, n = 3; TC^3A: 1.5 ± 0.0, n = 3). (*) P < 0.05, (**) P < 0.01, (****) P < 0.0001, one-way ANOVA with post-hoc Tukey test. (L–N) Immunostaining of doxycycline-induced FLAG-tagged TAZ-CAMTA1 in MS1 cells left untreated or treated with 1 µM latrunculin B (LatB) for 1 h. LatB-treated MS1 cells (45.5% ± 6.3%, n = 10) had more TAZ-CAMTA1 localized to the cytoplasm than untreated cells (5.7% ± 1.8%, n = 10). (****) P < 0.0001, unpaired t-test. (O) qPCR for expression of Cyr61 in MS1 cells without (−Dox) or with TAZ-CAMTA1 induction (+Dox), and untreated or treated with 5 µM simvastatin for 24 h. TAZ-CAMTA1-expressing cells had significantly reduced Cyr61 (untreated: 3.6 ± 0.2, n = 3, treated: 1.9 ± 0.1, n =3, [**] P < 0.01) after treatment with simvastatin. P-values were obtained by an unpaired t-test of the +Dox group treated or untreated. (P–R) Representative images showing soft agar growth of TAZ-CAMTA1-induced MS1 cells (TC^iEC) in the absence or presence of 1 µM simvastatin (+Sim). Simvastatin significantly reduced the colony formation of TC^iEC cells (Ctrl: 0.7 colonies ± 0.3, n = 3; TC^iEC: 322.7 colonies ± 55.4, n = 3; TC^iEC + Sim: 2.0 colonies ± 1.2, n = 3). (***) P < 0.001, one-way ANOVA with post-hoc Tukey test. All data are mean ± SEM. Scale bars, 25 µm.