Frequent activation of TAZ in human HB samples
First, we assessed the levels of TAZ protein in a collection of human HB specimens (n = 32) by IHC (Figure 1). In non-tumorous surrounding liver, cytoplasmic and nuclear positive staining for TAZ was limited to biliary cells, whereas hepatocytes showed either absent or faint cytoplasmic immunolabeling for this protein, in accordance with previous data . An equivalent staining pattern was detected for the TAZ paralog, YAP, as well as for NOTCH2, a TAZ target. As expected, β-catenin protein was expressed in the membrane of hepatocytes as well as in the membrane and cytoplasm of biliary cells. Noticeably, strong cytoplasmic and nuclear immunoreactivity for TAZ was detected in most HB specimens (22/32; 68.7%). The frequency was lower than that found for nuclear β-catenin (27/32, 84.4%) and the TAZ paralog YAP (26/32, 87.5%) but higher than nuclear NOTCH2 (19/32; 59.4%) in the same HB collection. Concomitant nuclear immunoreactivity for TAZ and β-catenin was detected in 18 of 22 TAZ-positive tumors (81.8%), while 15 of 22 (71.4%) human HB with TAZ activation displayed positive nuclear immunolabeling for YAP and NOTCH2. No significant association was found between the levels of TAZ, β-catenin, YAP, and NOTCH2 with clinicopathologic features of the patients, including age, gender, histology subtype, recurrence, or lung metastasis (data not shown).
Overall, the present data unravel the frequent activation of TAZ in human HB, which is frequently associated with induction/overexpression of YAP, β-catenin, and NOTCH2.
Constitutively activated TAZ (TAZS89A) cooperates with oncogenic β-catenin to promote HB development in mice
To elucidate the functional role of TAZ along HB pathogenesis, we investigated whether activated TAZ can either induce liver carcinogenesis or cooperate with other oncogenes to promote HB formation in mice. For this purpose, we hydrodynamically transfected the constitutively activated form of TAZ (TAZS89A) and the N-terminal deleted activated mutant form of β-catenin (∆N90-β-Catenin), alone or in combination, in the mouse liver (Figure 2A). TAZS89A is a mutant form allowing TAZ to escape phosphorylation-mediated degradation and, thus, leading to TAZ unrestrained activity . Overexpression of TAZS89A alone did not lead to liver tumor formation in mice. Indeed, 21 weeks post hydrodynamic injection, all mice appeared to be healthy. Accordingly, upon dissection, no tumor nodule was detected. Subsequent histological evaluation revealed that the mouse liver was completely normal, lacking any histological alteration (Figure 2B,C). As previous studies showed that overexpression of ∆N90-β-catenin alone does not lead to liver tumor development in mice but cooperates with other oncogenes for cancer formation [24-26], this β-catenin mutant form was co-injected with TAZS89A in the mouse liver (TAZS89A/∆N90-β-catenin mice) (Figure 2A). Noticeably, concomitant overexpression of TAZS89A with ∆N90-β-catenin resulted in rapid liver tumor formation in TAZS89A/∆N90-β-catenin mice. By 6 weeks post injection, 100% of the injected mice developed high liver tumor burden with palpable abdominal mass and were euthanized based on our IACUC protocol (Figure 2B,D,E).
Microscopically, small cluster of cells could be visualized as early as ~1.5 weeks post injection in the liver of TAZS89A/∆N90-β-catenin mice (Figure 3A). Positive immunoreactivity for Myc Tagged-∆N90-β-catenin and TAZ antibodies confirmed that tumor cells originated from the injected plasmids (Figure 3B,C), while immunolabeling for Ki67 implied active cell proliferation (Figure 3D). The first tumor nodules were visible in TAZS89A/∆N90-β-catenin mice by 3 weeks post injection (Figure 3E). Tumors quickly occupied nearly completely the liver parenchyma by 6 weeks post injection (Figure 3F).
Histologically, most of the lesions consisted of small cells with prominent nuclei, similar to those described in YAPS127A/β-catenin mice [9, 12], and resembling human fetal or crowded fetal HB (Figure 3G,H). Nonetheless, a second, distinct tumor entity was appreciable in the liver parenchyma of TAZS89A/∆N90-β-catenin mice. Indeed, besides the “pure” hepatoblast-like lesions, many tumors displayed a mixed phenotype, consisting of an epithelial (hepatoblast) and a mesenchymal component, with the latter being characterized primarily by cells with spindle morphology (Figure 3J,K). To the best of our knowledge, this is the first reported mouse model showing features of epithelial and mesenchymal differentiation. Following this interesting observation, we sought to determine the molecular characteristics of the two tumor entities by using IHC (Figure 4). As expected, both types of lesions displayed immunoreactivity for Myc Tagged-∆N90-β-catenin and TAZ antibodies, implying their origin from the injected constructs. Increased levels of the injected constructs were confirmed by Western blot analysis (Supplementary Figure 1). In addition, immunoreactivity for hepatocellular markers (hepatocyte nuclear factor 4 alpha or HNF-4α, forkhead box A1/A2 or FOXA1/A2, CCAAT Enhancer Binding Protein Alpha or CEBPA, and glutamine synthetase or GLUL) was significantly more pronounced in hepatoblast-like lesions. Furthermore, levels of the progenitor markers CD44v6, NANOG, CD10, and EPCAM as well as the cholangioblastic markers cytokeratin 7 and 19 (CK7 and CK19) were higher in the epithelial lesions. Levels of the GATA binding factor 4 (GATA4), whose expression is frequently upregulated in human HB with mesenchymal features, were strongly induced in both tumor entities. The mesenchymal part of the lesions, on the other hand, showed higher levels of stromal and angiogenesis-related markers, such as ERG, CD34, alpha-smooth muscle actin (α-SMA), and CD56/NCAM1, whereas immunoreactivity for the epithelial marker E-Cadherin (CDH1) was lost in the same mesenchymal lesions (Supplementary Figure 2).
Furthermore, by real-time RT-PCR, we found that livers of TAZS89A/∆N90-β-catenin mice exhibited significantly higher levels than wild-type livers of glypican 3 (Gpc3), alpha-fetoprotein (Afp), Cited1, Dlk1, and Gata4 genes (Supplementary Figure 3), whose upregulation in human HB is well established [27, 28] . As expected, mRNA expression of β-catenin (glutamine synthetase or Glul, Axin2, and TBX3) and TAZ (connective tissue growth factor or CTGF, Cysteine-Rich Heparin-Binding Protein 61 or Cyr61, Jagged 1, Notch1, Notch2, and Hes1) canonical targets was more elevated in TAZS89A/∆N90-β-catenin livers when compared with the wild-type counterpart (Supplementary Figure 4).
TAZS89A/∆N90-β-catenin induced hepatoblastoma requires an intact TAZ interaction with TEAD transcription factors
TAZ is a transcriptional activator and it interacts with TEAD DNA binding proteins to induce downstream gene expression. Nonetheless, TAZ is also known to have functions independent of its binding to TEAD factors [10, 11, 29]. Thus, we sought to investigate whether TAZS89A/∆N90-β-catenin induced HB development requires an intact TAZ/TEAD interaction. To address this issue, we generated the TAZS89AS51A plasmid for hydrodynamic transfection. The S51A mutation prevents TAZ from binding to TEAD proteins . Therefore, we co-expressed TAZS89AS51A and ∆N90-β-catenin plasmids into the mouse liver (Figure 5A). All TAZS89AS51A/∆N90-β-catenin mice appeared to be healthy and were harvested at 5-8 weeks post injection (Figure 5B). Grossly, no tumors nodules were detected on the mouse liver. Subsequent histological analysis revealed that the liver tissues of TAZS89AS51A/∆N90-β-catenin mice was completely normal, and the liver weight/body weight ratio of these mice was equivalent of that of wild-type, un-injected liver (Figure 5C,D). Altogether, the present data demonstrate that TEAD proteins are required for TAZ driven HB development in mice.
Commonalities and differences between TAZS89A/∆N90-β-catenin and YAPS127A/∆N90-β-catenin lesions
Previously, we reported that the activated form of Yap (YAPS127A), the other oncogenic transducer of the Hippo signaling, cooperates with ∆N90-β-catenin to induce HB formation in vivo . Thus, we compared the liver tumors developed in TAZS89A/∆N90-β-catenin and YAPS127A/∆N90-β-catenin mice. Histologically, “pure” hepatoblast-like lesions were undistinguishable between the two models. However, mesenchymal lesions were absent in YAPS127A/∆N90-β-catenin mice (Supplementary Figure 5A). At the cellular level, the rates of proliferation, apoptosis, and angiogenesis (microvessel density; MVD) were compared between the epithelial lesions from TAZS89A/∆N90-β-catenin and YAPS127A/∆N90-β-catenin mice. No significant differences were observed in the rates of proliferation and MVD between the two mouse models, whereas apoptosis was significantly lower in TAZS89A/∆N90-β-catenin mice (Supplementary Figure 5B-D).
At the molecular level, TAZS89A/∆N90-β-catenin and YAPS127A/∆N90-β-catenin tumors expressed Afp and Gpc3 genes at similar levels. In addition, YAPS127A/∆N90-β-catenin mouse malignant lesions expressed EpCam at higher levels, whereas TAZS89A/∆N90-β-catenin tumors displayed higher levels of HB markers Dlk1 and Cited1 as well as of the Glul hepatocellular marker. TAZS89A/∆N90-β-catenin tumors also exhibited the highest mRNA expression of the Gata4 transcription factor (Supplementary Figure 6). By immunohistochemistry, we found that levels of hepatocellular markers HNF-4α, FOXA2, FOXA1, CEBPA (the last two not shown), were equivalent in the two models. In contrast, GLUL protein was expressed homogeneously in TAZS89A/∆N90-β-catenin malignant lesions, whereas only patchy immunoreactivity for this protein was detected in YAPS127A/∆N90-β-catenin corresponding lesions. No differences were detected in the levels of the progenitor markers CD44v6, and CD10 as well as the cholangioblast markers CK7 and CK19 in the two models, whereas staining intensity for EPCAM was significantly stronger and more homogeneous in YAPS127A/∆N90-β-catenin tumors (Supplementary Figure 7), in accordance with real-time RT-PCR data.
Finally, we determined the levels of two transcription factors, whose role in liver cancer is becoming more and more relevant, namely nuclear factor erythroid 2 like 2 (NFE2L2/NRF2) and heat shock factor transcription factor 1 (HSF1) in TAZS89A/∆N90-β-catenin and YAPS127A/∆N90-β-catenin tumors[30-32]. Levels of NRF2, its regulator Kelch like ECH associated protein 1 (Keap1), and those of its target NAD(P)H quinone dehydrogenase 1 (Nqo1), were upregulated in both mouse models, without statistical differences between the two cohorts, when compared with wild-type mice (Supplementary Figure 8). In striking contrast, a robust overexpression of HSF1 was observed only in TAZS89A/∆N90-β-catenin mouse lesions both via RT-PCR and IHC (Supplementary Figure 8). Altogether, the present data indicate the existence of common and distinct features between TAZS89A/∆N90-β-catenin and YAPS127A/∆N90-β-catenin tumors.
HSF1 is dispensable along TAZS89A/∆N90-β-catenin dependent hepatoblastoma development
Following the interesting finding of upregulation of HSF1 exclusively in TAZS89A/∆N90-β-catenin tumors, we assessed the levels of HSF1 in human HB specimens by IHC to determine a possible role of HSF1 in this tumor type. Of note, HSF1 was ubiquitously (32/32, 100%) upregulated and localized in the nucleus of HB cells in the collection examined (Supplementary Figure 9), whereas low/absent immunoreactivity for HSF1 was detected in corresponding surrounding non-tumorous livers. Subsequently, the effect of HSF1 inactivation was investigated in the TAZS89A/∆N90-β-catenin mouse model. Specifically, to achieve this goal, we co-injected TAZS89A and ∆N90-β-catenin plasmids with a dominant negative form of HSF1 (dnHSF1) in the mouse liver via hydrodynamic gene delivery (these mice will be referred to as TAZS89A/∆N90-β-catenin/dnHSF1) (Figure 6A). This approach was found to be highly effective in completely suppressing liver carcinogenesis driven by either AKT or c-Myc protooncogene in mice [33, 34]. Unexpectedly, HSF1 inactivation neither affected HB development (Figure 6B) nor changed the histopathological features of TAZS89A/∆N90-β-catenin mice (Figure 6C). At the microscopical level, in fact, both epithelial and mesenchymal features were retained by HB lesions developed in TAZS89A/∆N90-β-catenin/dnHSF1 mice (Figure 6C). Furthermore, no significant differences were detected in liver/body weight ratio between TAZS89A/∆N90-β-catenin and TAZS89A/∆N90-β-catenin/dnHSF1 mice (Figure 6D).
Taken together, the present data indicate that TAZS89A/∆N90-β-catenin HB development does not primarily depend on HSF1 in mice.
Loss of endogenous YAP does not affect TAZS89A/∆N90-β-catenin driven hepatoblastoma formation
Next, we asked whether activated YAP and TAZ have redundant and/or distinct roles in HB formation. First, we evaluated the levels of YAP, the TAZ paralog, in HB lesions from TAZS89A/∆N90-β-catenin mice. We found that, while total protein levels of YAP in TAZS89A/∆N90-β-catenin lesions were equivalent to those in wild-type livers (Supplementary Figure 1), nuclear accumulation of YAP (a sign of its activation) was detected in TAZS89A/∆N90-β-catenin HB lesions (Figure 7). Subsequently, we investigated whether TAZS89A/∆N90-β-catenin overexpression was able to promote HB development in mice in the absence of endogenous YAP protein. For this purpose, we applied conditional Yap KO (Yapflox/loxf) mice. Specifically, we hydrodynamically injected TAZS89A/∆N90-β-catenin plasmids together with pCMV-Cre plasmids into Yapflox/flox mice (TAZS89A/∆N90-β-catenin/Cre). As the control, TAZS89A/∆N90-β-catenin plasmids were mixed with pCMV empty vector and co-injected into Yapflox/flox mice (TAZS89A/∆N90-β-catenin/pCMV) (Figure 7A). We found that both cohorts of mice developed liver tumors and were required to be euthanized by 10 to 12 weeks post injection (Fig. 7B and 7D). Histologically, HB lesions were detected in TAZS89A/∆N90-β-catenin/pCMV and TAZS89A/∆N90-β-catenin/Cre injected mice, consisting of both the epithelial and the mesenchymal component (Fig. 7D). Importantly, while cytoplasmic and nuclear staining of YAP could be detected in TAZS89A/∆N90-β-catenin/pCMV HB cells, no YAP expression was found in TAZS89A/∆N90-β-catenin/Cre HB cells. Instead, positive YAP staining was readily observed in the surrounding normal liver tissues as well as stromal cells within the HB lesions (Fig. 7D). Loss of YAP expression in TAZS89A/∆N90-β-catenin/Cre tumor tissues was further validated by Western blotting (Fig. 7C).
Overall, the present data indicate that activated TAZ induces HB formation in the absence of the endogenous YAP.
Canonical Notch signaling is not required for TAZS89A/∆N90-β-catenin HB development in vivo
Mounting evidence indicate the Notch pathway as a downstream effector of the Hippo cascade in liver cancer [35-37]. In addition, NOTCH2 has been found to be consistently overexpressed in human HB . Furthermore, our present data indicate the upregulation of members of the Notch pathway (Jag1, Notch1, Notch2, and Hes1) in the lesions of TAZS89A/∆N90-β-catenin mice. Thus, we investigated whether the canonical Notch signaling is required for TAZS89A/∆N90-β-catenin driven HB formation in vivo. For this purpose, we hydrodynamically injected TAZS89A/∆N90-β-catenin plasmids together with the dominant negative form of RBP-J (dnRBP-J; these mice will be referred to as TAZS89A/∆N90-β-catenin/dnRBP-J) that has been shown to effectively inhibit the canonical Notch cascade (Figure 8A) [37, 39]. Additional mice were injected with TAZS89A/∆N90-β-catenin with pT3 empty vector as control (TAZS89A/∆N90-β-catenin/pT3). We found that all mice develop high liver tumor burden and were required to be euthanized around 6 to 9 weeks post injection (Figure 8B-D). Histologically, HB lesions could be appreciated in both cohorts of mice. However, overexpression of dnRBP-J in the livers of TAZS89A/∆N90-β-catenin mice led to the disappearance of the mesenchymal compartment of HB lesions (Figure 8C), indicating that the Notch pathway might contribute to the differentiation toward a mesenchymal phenotype of TAZS89A/∆N90-β-catenin HB lesions.
Overall, our study indicates that Notch signaling is activated in TAZS89A/∆N90-β-catenin tissues and influences the tumor cell differentiation. However, Notch activation is dispensable in TAZS89A/∆N90-β-catenin driven HB development.
TAZ cooperates with β-catenin and YAP for the growth of human HB cells in vitro
The findings in human and mouse HB specimens support a major role of TAZ in HB formation. To further investigate the relevance of TAZ activation in this tumor type, we examined whether TAZ expression is required for human HB cell growth in vitro. Thus, we silenced TAZ in two human HB cell lines (HepG2 and Hep293TT cells) using specific siRNA against TAZ (Supplementary Figure 10, 11). Of note, TAZ knockdown resulted in a decrease of cell proliferation and induction of apoptosis in the two HB cell lines. These anti-growth effects were increased when the two cell lines were concomitantly subjected to TAZ and β-catenin silencing. At the molecular level, suppression of TAZ did not affect β-catenin and YAP mRNA and protein levels, whereas β-catenin knockdown resulted in downregulation of TAZ and YAP.
Finally, we assessed whether TAZ and YAP have redundant roles in human HB cells. For this purpose, we silenced TAZ and YAP genes using specific siRNAs, either alone or in combination, in HepG2 and Hep293TT cell lines (Supplementary Figure 12, 13). We found that concomitant knockdown of TAZ and YAP resulted in a significantly more pronounced growth restraint when compared with the silencing of either TAZ or YAP alone. At the molecular level, knockdown of TAZ did not affect the levels of YAP, whereas silencing of YAP triggered upregulation of TAZ. These findings suggest the existence of a compensatory mechanism triggering upregulation of TAZ when YAP is suppressed in HB cells.
Altogether, the present data indicate that TAZ acts in concert with β-catenin and YAP to induce HB growth in vitro.