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Characterization of hepatoblastoma in S45Yeb-catenin-S127A-YAP1 and S33Yeb-catenin-S127A-YAP1 mice by analysis of liver lysates. Western blot analysis using lysates from the same groups of mice shows increased levels of b-catenin targets glutamine synthetase (GS), cyclin D1, and regucalcin, as well as YAP1 targets survivin, Cyr61, and jagged 1, compared with wild-type (WT) livers from age-matched mice. Glyceraldehyde-3ephosphate dehydrogenase (GAPDH) verified comparable loading. Molecular weights are indicated to the right of each blot. Arrows indicate the correct molecular weight band.
Source publication
Hepatoblastoma (HB), the most common pediatric primary liver neoplasm, shows nuclear localization of β-catenin and yes-associated protein 1 (YAP1) in almost 80% of the cases. Co-expression of constitutively active S127A-YAP1 and ΔN90 deletion-mutant β-catenin (YAP1–ΔN90-β-catenin) causes HB in mice. Because heterogeneity in downstream signaling is...
Contexts in source publication
Context 1
... tumors were strongly positive for the b-catenin target cyclin D1 compared with normal liver (Figure 2). Likewise, YAP1 targets, including Cyr61, survivin, and jagged 1, were significantly increased, and regucalcin was modestly increased, with expression of activated b-catenin/YAP1 (Figure 3). As expected, activation of b-catenin and YAP1 targets, such as cyclin D1, which regulates cell cycle progression, resulted in marked proliferation in HB from becatenin-YAP1 mice, as indicated by Ki-67 positive staining (Figure 2). ...
Context 2
... tumors were strongly positive for the b-catenin target cyclin D1 compared with normal liver (Figure 2). Likewise, YAP1 targets, including Cyr61, survivin, and jagged 1, were significantly increased, and regucalcin was modestly increased, with expression of activated b-catenin/YAP1 (Figure 3). As expected, activation of b-catenin and YAP1 targets, such as cyclin D1, which regulates cell cycle progression, resulted in marked proliferation in HB from becatenin-YAP1 mice, as indicated by Ki-67 positive staining (Figure 2). ...
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Citations
... The Hippo signaling pathway plays a critical role in liver organogenesis and cancer [18][19][20] . The dysregulated downstream effector molecule of Hippo signaling, YAP1, is involved in hepatoblastoma tumorigenesis [21][22][23][24][25] . Combination of the activated form of YAP1 (YAP S127A ) with either hepatoblastoma relevant NFE2L2 mutant or CTNNB1 mutant promotes liver tumorigenesis although either alone is unable to transform normal liver cells in these mouse models 26 . ...
... Previous approaches have been applied to establish hepatoblastoma models, including xenograft implantation [107][108][109][110][111][112][113][114][115] , generation of transgenic mice 29,55,116 and hydrodynamic tail vein injection of oncogenes 22,24 , and each of these models has its pros and cons 35 . Additionally, most of these models recapitulate the well-differentiated fetal type of hepatoblastoma, which usually has a good clinical outcome even without chemotherapy administration 117 . ...
A lack of relevant genetic models and cell lines hampers our understanding of hepatoblastoma pathogenesis and the development of new therapies for this neoplasm. Here, we report an improved MYC-driven hepatoblastoma-like murine model that recapitulates the pathological features of embryonal type of hepatoblastoma, with transcriptomics resembling the high-risk gene signatures of the human disease. Single-cell RNA-sequencing and spatial transcriptomics identify distinct subpopulations of hepatoblastoma cells. After deriving cell lines from the mouse model, we map cancer dependency genes using CRISPR-Cas9 screening and identify druggable targets shared with human hepatoblastoma (e.g., CDK7, CDK9, PRMT1, PRMT5). Our screen also reveals oncogenes and tumor suppressor genes in hepatoblastoma that engage multiple, druggable cancer signaling pathways. Chemotherapy is critical for human hepatoblastoma treatment. A genetic mapping of doxorubicin response by CRISPR-Cas9 screening identifies modifiers whose loss-of-function synergizes with (e.g., PRKDC) or antagonizes (e.g., apoptosis genes) the effect of chemotherapy. The combination of PRKDC inhibition and doxorubicin-based chemotherapy greatly enhances therapeutic efficacy. These studies provide a set of resources including disease models suitable for identifying and validating potential therapeutic targets in human high-risk hepatoblastoma.
... [7,8] Several studies of genomic mutations in patients with HBL revealed that HBL is a genomically simple disease with an average 1.9% mutations most commonly in four genes: CTNNB1 (β-catenin), nuclear factor, erythroid 2-like 2 (NFE2L2)/nuclear erythroid 2 p45-related factor 2 (NRF2), AT-rich interaction domain 1A, and in the promoter of the Telomerase Reverse Transcriptase gene. [9][10][11] Studies of the CTNNB1 and NFE2L2/NRF2 mutations demonstrated their significant roles in the development of HBL [11][12][13] ; however, it becomes clear that aggressive HBL carries additional carcinogenic alterations that activate additional cancerpromoting pathways. We recently identified short chromosomal regions (cancer-enhancing genomic regions or aggressive liver cancer domains [CEGRs/ALCDs]) that are located within over 200 oncogenes and that drive their expression. ...
Fibrolamellar hepatocellular carcinoma (FLC) is a disease that occurs in children and young adults. The development of FLC is associated with creation of a fusion oncoprotein DNAJB1-PKAc kinase, which activates multiple cancer-associated pathways. The aim of this study was to examine the role of human genomic regions, called cancer-enhancing genomic regions or aggressive liver cancer domains (CEGRs/ALCDs), in the development of FLC. Previous studies revealed that CEGRs/ALCDs are located in multiple oncogenes and cancer-associated genes, regularly silenced in normal tissues. Using the regulatory element locus intersection (RELI) algorithm, we searched a large compendium of chromatin immunoprecipitation-sequencing (ChIP) data sets and found that CEGRs/ALCDs contain regulatory elements in several human cancers outside of pediatric hepatic neoplasms. The RELI algorithm further identified components of the β-catenin-TCF7L2/TCF4 pathway, which interacts with CEGRs/ALCDs in several human cancers. Particularly, the RELI algorithm found interactions of transcription factors and chromatin remodelers with many genes that are activated in patients with FLC. We found that these FLC-specific genes contain CEGRs/ALCDs, and that the driver of FLC, fusion oncoprotein DNAJB1-PKAc, phosphorylates β-catenin at Ser675, resulting in an increase of β-catenin-TCF7L2/TCF4 complexes. These complexes increase a large family of CEGR/ALCD-dependent collagens and oncogenes. The DNAJB1-PKAc-β-catenin-CEGR/ALCD pathway is preserved in lung metastasis. The inhibition of β-catenin in FLC organoids inhibited the expression of CEGRs/ALCDs-dependent collagens and oncogenes, preventing the formation of the organoid's structure. Conclusion: This study provides a rationale for the development of β-catenin-based therapy for patients with FLC.
... Glutamine synthetase is a downstream target of the Wnt/Beta-catenin pathway. 11,12 Normal Glutamine synthetase staining is seen in a single layer of normal hepatocytes around the central vein. Cyclin D1 is a relatively less commonly used marker, which shows staining in fetal, embryonal and small-cell undifferentiated subtypes with variable staining in mesenchymal components. ...
ABSTRACT Objective: To assess the utility of four immunohistochemical stains in the categorization of hepatoblastoma. Study Design: Retrospective longitudinal study. Place and Duration of Study: Histopathology Department, Shaukat Khanum Memorial Cancer Hospital, Peshawar Pakistan, from 2016 to 2018. Methodology: We retrospectively reviewed 50 cases of hepatoblastoma from 2016-2018, from our hospital database and studied the pattern of four immunohistochemical stains (Beta-catenin, Glutamine synthetase, Heppar 1 and Cyclin D1) in various subtypes. Results: Ten out of fifty cases were mixed epithelial and mesenchymal hepatoblastomas, and forty were pure epithelial. In the pure epithelial category, the fetal subtype showed Heppar 1 and Cyclin D1 positivity in twenty-eight cases, while Glutamine synthetase and Beta-catenin were positive in all the thirty cases. One case of embryonal subtype displayed negativity for Heppar 1; the rest of the immunohistochemical stains were positive. Ten tumours exhibiting mixed epithelial and mesenchy-mal morphology showed positivity for all the four immunohistochemical stains in the epithelial component. However, Heppar 1 and cyclin D1 were negative in the mesenchymal component in all the ten cases. Five cases (50%) showed Glutamine synthetase and Beta-catenin positivity in the mesenchymal component. Conclusion: All the four immunohistochemical stains, especially Beta-catenin and Glutamine synthetase, were efficient diagnostic tool, especially for tumours with complex or vague morphological features.
... The Hippo signaling pathway plays a critical role in liver organogenesis and cancer [18][19][20] . The dysregulated downstream effector molecule of Hippo signaling, YAP1, is involved in hepatoblastoma tumorigenesis [21][22][23][24][25] . Hepatic developmental pathways may determine the differentiation capacity of mutated liver progenitor/stem cells, and differentiation status may determine the aggressiveness of hepatoblastoma 9 . ...
... Previous approaches have been applied to establish hepatoblastoma models, including xenograft implantation 92-100 , generation of transgenic mice 28,48,101 and hydrodynamic tail vein injection of oncogenes 22,24 , and each of these models has its pros and cons 34 . Additionally, most of these models develop the well-differentiated fetal type of hepatoblastoma, which usually has a good clinical outcome even without chemotherapy administration 102 . ...
A lack of relevant genetic models and cell lines hampers our understanding of hepatoblastoma pathogenesis and the development of new therapies for this neoplasm. We report a liver-specific MYC-driven hepatoblastoma murine model that faithfully recapitulates the pathological features of mixed fetal and embryonal hepatoblastoma, with transcriptomics resembling the high-risk gene signatures of the human disease. Single-cell RNA-sequencing and spatial transcriptomics identified distinct subpopulations of hepatoblastoma cells. After deriving cell lines from the mouse model, we mapped the cancer dependency genes using CRISPR-Cas9 screening and identified druggable targets shared with human hepatoblastoma (i.e., CDK7, CDK9, PRMT1, PRMT5). Our screen also revealed oncogenes and tumor suppressor genes in hepatoblastoma that engage multiple, druggable cancer signaling pathways. Chemotherapy is critical for human hepatoblastoma treatment. A genetic mapping of doxorubicin response by CRISPR-Cas9 screening identified modifiers whose loss-of-function synergizes with (e.g., PRKDC) or antagonizes (e.g., apoptosis genes) with the effect of chemotherapy. The combination of PRKDC inhibition and doxorubicin-based chemotherapy greatly enhances therapeutic efficacy. These studies have provided a useful set of resources including disease models suitable to identify and validate potential therapeutic targets in human high-risk hepatoblastoma.
... The Wnt/β-catenin pathway interacts with other signaling pathways, as shown for Hippo/YAP pathway (Min, 2019;Tao, 2014). In addition, an impact of the tumor microenvironment on glutamine metabolism has been recently proposed (Castegna and Menga 2018), suggesting that complex relationships exist in vivo that are not easily documented in vitro. ...
Purpose
Glutamine plays an important role in cell viability and growth of various tumors. For the fetal subtype of hepatoblastoma, growth inhibition through glutamine depletion was shown. We studied glutamine depletion in embryonal cell lines of hepatoblastoma carrying different mutations. Since asparagine synthetase was identified as a prognostic factor and potential therapeutic target in adult hepatocellular carcinoma, we investigated the expression of its gene ASNS and of the gene GLUL, encoding for glutamine synthetase, in hepatoblastoma specimens and cell lines and investigated the correlation with overall survival.
Methods
We correlated GLUL and ASNS expression with overall survival using publicly available microarray and clinical data. We examined GLUL and ASNS expression by RT-qPCR and by Western blot analysis in the embryonal cell lines Huh-6 and HepT1, and in five hepatoblastoma specimens. In the same cell lines, we investigated the effects of glutamine depletion. Hepatoblastoma biopsies were examined for histology and CTNNB1 mutations.
Results
High GLUL expression was associated with a higher median survival time. Independent of mutations and histology, hepatoblastoma samples showed strong GLUL expression and glutamine synthesis. Glutamine depletion resulted in the inhibition of proliferation and of cell viability in both embryonal hepatoblastoma cell lines. ASNS expression did not correlate with overall survival.
Conclusion
Growth inhibition resulting from glutamine depletion, as described for the hepatoblastoma fetal subtype, is also detected in established embryonal hepatoblastoma cell lines carrying different mutations. At variance with adult hepatocellular carcinoma, in hepatoblastoma asparagine synthetase has no prognostic significance.
... Role of Yap in pathogenesis of hepatocellular carcinoma is well-documented Sylvester and Colnot, 2014;Yimlamai et al., 2015). Ectopic expression of S127 mutated Yap results in the development of hepatoblastoma in mice (Min et al., 2019). Role of Yap in liver regeneration after partial hepatectomy has also been demonstrated (Oh et al., 2018). ...
Overdose of acetaminophen (APAP) is the major cause of acute liver failure in the Western world with very limited treatment options. Previous studies from our groups and others have shown that timely activation of liver regeneration is a critical determinant of transplant free survival of APAP-induced acute liver failure (ALF) patients. Here we report that hepatocyte specific deletion of Yes associated protein (Yap), the downstream mediator of the Hippo Kinase signaling pathway results in faster recovery from APAP-induced acute liver injury (ALI). Initial studies performed with male C57BL/6J mice showed a rapid activation of Yap and its target genes within first 24 hours after APAP administration. Treatment of hepatocyte specific Yap knockout mice (Yap-KO) with 300 mg/kg APAP resulted in equal initial liver injury but a significantly accelerated recovery in Yap-KO mice. The recovery was accompanied by significantly rapid hepatocyte proliferation supported by faster activation of Wnt/β-catenin pathway. Furthermore, Yap-KO mice had significantly earlier and higher pro-regenerative inflammatory response following APAP overdose. Global gene expression analysis indicated that Yap-KO mice had a robust activation of transcription factors involved in response to ER stress (XBP1) and maintaining hepatocyte differentiation (HNF4α). In conclusion, these data indicate that inhibition of Yap in hepatocytes results in rapid recovery from APAP overdose due to an earlier activation of liver regeneration.
... Cellular studies demonstrated that YAP1 rapidly induces the expression of LATS2, thus forming a negative feedback loop that self-limits its activity . The YAP1 S127A mutant, which escapes negative regulation by LATS kinases, is more effective at inducing tumor and has been used in many cancer models (Zhang S. et al., 2017;Min et al., 2019;Smith et al., 2021). It is possible that, due to such a negative feedback mechanism, simple overexpression of wild-type YAP1 may not be sufficient to trigger long-lasting proliferative events, which may partially explain the relative lower frequencies of YAP1 amplification in human cancers. ...
The Hippo pathway is highly conserved from Drosophila to mammals. As a key regulator of cell proliferation, the Hippo pathway controls tissue homeostasis and has a major impact on tumorigenesis. The originally defined core components of the Hippo pathway in mammals include STK3/4, LATS1/2, YAP1/TAZ, TEAD, VGLL4, and NF2. However, for most of these genes, mutations and copy number variations are relatively uncommon in human cancer. Several other recently identified upstream and downstream regulators of Hippo signaling, including FAT1, SHANK2, Gq/11, and SWI/SNF complex, are more commonly dysregulated in human cancer at the genomic level. This review will discuss major genomic events in human cancer that enable cancer cells to escape the tumor-suppressive effects of Hippo signaling.
... The β-catenin-GS axis has been well studied in the liver tumor. GLUL gene is induced by activation of the β-catenin pathway in HCC and hepatoblastoma (HB) [29,30]. In addition, GS and β-catenin show a positive correlation in human HCC samples. ...
... The relationship between yes-associated protein 1 (YAP) and GS in liver cancer has also been studied [30,36]. YAP is a co-activator involved in the Hippo pathway. ...
... YAP induces the expression and activity of GS to reprogram nitrogen metabolism, including nucleotide biosynthesis, resulting in hepatomegaly and liver cancer cell growth. Furthermore, YAP1 is co-expressed with β-catenin, and YAP1/β-catenin mutants induce GS promoter activity in HB [30]. Together, these findings demonstrate that YAP cooperates with the anabolic demands of cell growth during tumorigenesis. ...
The significance of glutamine in cancer metabolism has been extensively studied. Cancer cells consume an excessive amount of glutamine to facilitate rapid proliferation. Thus, glutamine depletion occurs in various cancer types, especially in poorly vascularized cancers. This makes glutamine synthetase (GS), the only enzyme responsible for de novo synthesizing glutamine, essential in cancer metabolism. In cancer, GS exhibits pro-tumoral features by synthesizing glutamine, supporting nucleotide synthesis. Furthermore, GS is highly expressed in the tumor microenvironment (TME) and provides glutamine to cancer cells, allowing cancer cells to maintain sufficient glutamine level for glutamine catabolism. Glutamine catabolism, the opposite reaction of glutamine synthesis by GS, is well known for supporting cancer cell proliferation via contributing biosynthesis of various essential molecules and energy production. Either glutamine anabolism or catabolism has a critical function in cancer metabolism depending on the complex nature and microenvironment of cancers. In this review, we focus on the role of GS in a variety of cancer types and microenvironments and highlight the mechanism of GS at the transcriptional and post-translational levels. Lastly, we discuss the therapeutic implications of targeting GS in cancer.
... Specifically, while overexpression of either YAP or TAZ was unable to promote liver malignant transformation, the combined overexpression of YAP with either constitutive active β-catenin (β-catenin/Δ90) or β-catenin point mutants (S33Y or S45Y) caused HB development in mice. 66,68 A subsequent study showed that YAP withdrawal triggers HB regression in YAP/β-catenin mice, identifying YAP as a potential therapeutic target for HB patients. 69 Moreover, overexpression of β-catenin combined with TAZ induces HB development in mice, in a TEAD4-dependent manner. ...
Hepatoblastoma (HB) is the predominant primary liver tumor in children. While the prognosis is favorable when the tumor can be resected, the outcome is dismal for patients with progressed HB. Therefore, a better understanding of the molecular mechanisms responsible for HB is imperative for early detection and effective treatment. Sequencing analysis of human HB specimens unraveled the pivotal role of Wnt/β-catenin pathway activation in this disease. Nonetheless, β-catenin activation alone does not suffice to induce HB, implying the need for additional alterations. Perturbations of several pathways, including Hippo, Hedgehog, NRF2/KEAP1, HGF/c-Met, NK-1R/SP, and PI3K/AKT/mTOR cascades and aberrant activation of c-MYC, n-MYC, and EZH2 proto-oncogenes, have been identified in HB, although their role requires additional investigation. Here, we summarize the current knowledge on HB molecular pathogenesis, the relevance of the preclinical findings for the human disease, and the innovative therapeutic strategies that could be beneficial for the treatment of HB patients.
... Elevated expression of hTERT (human telomerase reverse transcriptase) and c-MYC appear to be predictive of a poor prognosis because both of them play crucial roles in the activation of Wnt/β-catenin signaling in aggressive phenotypes of HB [34,35]. With further study of HB pathogenicity genes, researchers have found that methylation and polymorphism of genes, microRNAs and lncRNAs are also related to the carcinogenesis of HB [15,[36][37][38]. However, there has been no prior study about the association of LIN28A polymorphisms with HB susceptibility. ...
Hepatoblastoma (HB) is the most prevalent primary hepatic cancer in children aged 6 months to 3 years. LIN28A is recurrently mutated in various diseases, and critically involved in tumorigenesis. However, a limited number of studies have examined the involvement of LIN28A polymorphisms in HB risk. We used the TaqMan assay to genotype four LIN28A polymorphisms (rs3811464 G>A, rs3811463 T>C, rs34787247 G>A, and rs11247957 G>A) in 275 Chinese children with HB and 1018 cancer-free controls from five medical centers in China. Their association with HB risk was evaluated on the basis of odds ratio (OR) and corresponding 95% confidence interval (CI). Overall, no significant associations were found in single locus and combine analysis. Interestingly, in the stratified analysis, we found that subjects with 1-3 risk genotypes were more likely to develop HB in patients ≥17 months of age (adjusted OR=1.76, 95% CI=1.04-2.98, P=0.034). The rs3811464 GA/AA genotypes were associated with decrease HB risk in patients with clinical stage III+IV disease (adjusted OR=0.50, 95% CI=0.26-0.96, P=0.038). Our results suggest that the LIN28A polymorphisms have a weak association with HB susceptibility in the Chinese children. LIN28A rs3811464 G>A may decrease HB risk in stage III+IV patients which need further validations with larger samples and different ethnicities.
