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Reduced DNA Damage And Genetic Instability in Mcl-1 Dhep /TNFR1 –/– and TAK1/Casp8 Dhep Mice and Intercrossings (A) Staining for gH2AX (black) and cleaved Casp3 (red), double-positive hepatocytes (black/red arrows). Scale bar, 50 mm. (B) IF staining for gH2AX and Ki67 in wild-type, Mcl-1 Dhep , and Mcl-1 Dhep /TNFR1 À/À mice, as well as TAK1 Dhep , TAK1/Casp8 Dhep , and TAK1 Dhep /RIPK3 À/À mice. Arrowheads indicate cells with positive IF staining. Scale bar, 10 mm. (C) Quantification of Ki67 + and Ki67 + /gH2AX + hepatocytes (n = 4 mice per group, n = 5 for Mcl-1 Dhep mice). (D) Rate of AI in wild-type, Mcl-1 Dhep , and Mcl-1 Dhep /TNFR1 À/À mice, TAK1 Dhep , TAK1 Dhep /RIPK3 À/À , and TAK1/Casp8 Dhep mice (TaqMan copy number assay, each square represents one area of microdissected liver tissue, lines indicate different areas of the same liver; red, AI; black, no AI). Mcl-1 Dhep mice and intercrossings at 2 months; TAK1 Dhep mice and intercrossings at 6 weeks of age. In (C), data are presented as mean ± SEM. Statistical significance was calculated using ANOVA with Bonferroni correction (C), or Fisher's exact test (D). *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S5.
Source publication
Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic...
Contexts in source publication
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... they suggest that persistently increased hepatocyte apoptosis, resulting in regenerative proliferation and high DNA replica- tion rate, determines hepatocarcinogenesis. This hypothesis is underpinned by stochastic considerations ( Figure S4). ...
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... for gH2AX and cleaved caspase-3 revealed that hepatocytes from 6-to 8-week-old Mcl-1 Dhep mice as well as TAK1 Dhep mice which were positive for gH2AX were mostly negative for cleaved caspase-3. Thus, gH2AX-positivity was unlikely to be a consequence of apoptosis of individual hepato- cytes ( Figure 4A). Immunofluorescence (IF) staining for gH2AX (legend continued on next page) and Ki67 of livers from 6-to 8-week-old mice revealed virtually no gH2AX + hepatocytes in wild-type livers, whereas Mcl-1 Dhep and TAK1 Dhep hepatocytes displayed the typical nuclear staining pattern and substantial gH2AX/Ki67 double positivity ($10% and 20%, respectively; Figures 4B and 4C). ...
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... contrast to wild-type mice, livers of Mcl-1 Dhep , TAK1 Dhep , and TAK1 Dhep /RIPK3 À/À mice showed widespread allelic imbal- ances (AI) at CFS, demonstrating genetic instability in hyper- apoptotic and hyper-proliferative mouse livers. Of note, although higher compared with wild-type mice, AI rates were much lower in Mcl-1 Dhep /TNFR1 À/À mice and TAK1 Dhep /Casp8 Dhep mice ( Figure 4D). ...
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... findings in human CLD and murine CLD models, under- pinned by stochastic considerations (Figure S4), argue that persistently increased hepatocyte apoptosis resulting in regen- erative proliferation and high DNA replication rate (independent of etiology) is a decisive determinant of hepatocarcinogenesis. This is in line with a recent report on the carcinogenic effect of replication errors stochastically occurring in highly proliferative stem cells (Tomasetti and Vogelstein, 2015). ...
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... DNA damage in a hyper-apoptotic environment such as in CLD patients, Mcl-1 Dhep , or TAK1 Dhep mice, is provoked by constantly enhanced regeneration causing replication stress. Taking into account that caspase-8 deficiency in TAK1 Dhep mice abolished apoptosis and nearly normalized proliferative levels ( Figure 4C; Vucur et al., 2013), it is obvious that TAK1/Casp8 Dhep mice are not tumor prone. ...
Citations
... Inhibition of Ripk1 or Casp8 leads to chromosomal instability [39]. During radiation or chemical-induced DNA damage, Casp8 also forms a DNA damage sensor complex with Fadd/c-FLIP/Ripk1, orchestrating replication-associated DNA damage and H2AX phosphorylation [40]. DNA damage-triggered activation of nuclear Casp8 also overcomes the p53-dependent G2/M checkpoint through the cleavage of USP28, leading to drug-resistance [41]. ...
... In addition, the myelodysplastic phenotype observed in Casp8 −/− mice is also independent of Ripk3 because it can also be observed in some Casp8 −/− Ripk3 −/− mice. We believe that such a myelodysplastic phenotype might be associated with the role of Casp8 in the regulation of mitosis, chromosomal stability and DNA damage repair [40,42]. Future study needs to determine whether there are increased genetic mutations and chromosomal abnormalities in Casp8 −/− HSPCs. ...
Myelodysplastic syndromes (MDS) are a heterogeneous group of pre-leukemic hematopoietic disorders characterized by cytopenia in peripheral blood due to ineffective hematopoiesis and normo- or hypercellularity and morphologic dysplasia in bone marrow (BM). An inflammatory BM microenvironment and programmed cell death of hematopoietic stem/progenitor cells (HSPCs) are thought to be the major causes of ineffective hematopoiesis in MDS. Pyroptosis, apoptosis and necroptosis (collectively, PANoptosis) are observed in BM tissues of MDS patients, suggesting an important role of PANoptosis in MDS pathogenesis. Caspase 8 (Casp8) is a master regulator of PANoptosis, which is downregulated in HSPCs from most MDS patients and abnormally spliced in HSPCs from MDS patients with SRSF2 mutation. To study the role of PANoptosis in hematopoiesis, we generated inducible Casp8 knockout mice ( Casp8 −/− ). Mx1-Cre-Casp8 −/− mice died of BM failure within 10 days of polyI:C injections due to depletion of HSPCs. Rosa-ERT2Cre-Casp8 −/− mice are healthy without significant changes in BM hematopoiesis within the first 1.5 months after Casp8 deletion. Such mice developed BM failure upon infection or low dose polyI:C/LPS injections due to the hypersensitivity of Casp8 −/− HSPCs to infection or inflammation-induced necroptosis which can be prevented by Ripk3 deletion. However, impaired self-renewal capacity of Casp8 −/− HSPCs cannot be rescued by Ripk3 deletion due to activation of Ripk1-Tbk1 signaling. Most importantly, mice transplanted with Casp8 −/− BM cells developed MDS-like disease within 4 months of transplantation as demonstrated by anemia, thrombocytopenia and myelodysplasia. Our study suggests an essential role for a balance in Casp8, Ripk3-Mlkl and Ripk1-Tbk1 activities in the regulation of survival and self-renewal of HSPCs, the disruption of which induces inflammation and BM failure, resulting in MDS-like disease.
... Des travaux de recherche antérieurs avaient d'ailleurs permis de montrer, dans des modèles murins de NASH, que le stress oxydant dans les hépatocytes en division active la réponse aux lésions de l'ADN (DNA damage-response) [14,15]. La prolifération compensatoire des hépatocytes et la réponse aux dommages de l'ADN semblent être déterminants pour le développement du cancer du foie chez les personnes atteintes d'une maladie hépatique chronique, notamment dans un contexte de NAFLD [16]. Nous avons cherché à déterminer les principaux évènements moléculaires qui sous-tendent l'activation de la réponse aux lésions de l'ADN au cours de la NAFLD. ...
... Chronic liver injury triggers an inflammatory response, leading to the death of hepatocytes and the accumulation of extracellular matrix, resulting in the formation of scar tissue that underlies liver fibrosis [2][3][4]. Studies have shown that liver fibrosis can affect the reserve function and regenerative capacity of the liver [5][6][7]. Furthermore, liver fibrosis often progresses to liver cirrhosis, liver failure, and liver cancer, seriously affecting the prognosis and life quality of patients [8]. ...
Background
Non-coding RNAs play important roles in liver regeneration; however, their functions and mechanisms of action in the regeneration of fibrotic liver have not been elucidated. We aimed to clarify the expression patterns and regulatory functions of lncRNAs, circRNAs, miRNAs, and mRNAs in the proliferative phase of fibrotic liver regeneration.
Methods
Based on a mouse model of liver fibrosis with 70% hepatectomy, whole-transcriptome profiling was performed using high-throughput sequencing on samples collected at 0, 12, 24, 48, and 72 h after hepatectomy. Hub genes were selected by weighted gene co-expression network analysis and subjected to enrichment analysis. Integrated analysis was performed to reveal the interactions of differentially expressed (DE) lncRNAs, circRNAs, miRNAs, and mRNAs, and to construct lncRNA–mRNA cis- and trans-regulatory networks and lncRNA/circRNA–miRNA–mRNA ceRNA regulatory networks. Real-Time quantitative PCR was used to validate part of the ceRNA network.
Results
A total of 1,329 lncRNAs, 48 circRNAs, 167 miRNAs, and 6,458 mRNAs were differentially expressed, including 812 hub genes. Based on these DE RNAs, we examined several mechanisms of ncRNA regulatory networks, including lncRNA cis and trans interactions, circRNA parental genes, and ceRNA pathways. We constructed a cis-regulatory core network consisting of 64 lncRNA–mRNA pairs (53 DE lncRNAs and 58 hub genes), a trans-regulatory core network consisting of 103 lncRNA–mRNA pairs (18 DE lncRNAs and 85 hub genes), a lncRNA–miRNA–mRNA ceRNA core regulatory network (20 DE lncRNAs, 12 DE miRNAs, and 33 mRNAs), and a circRNA–miRNA–mRNA ceRNA core regulatory network (5 DE circRNAs, 5 DE miRNAs, and 39 mRNAs).
Conclusions
These results reveal the expression patterns of lncRNAs, circRNAs, miRNAs, and mRNAs in the proliferative phase of fibrotic liver regeneration, as well as core regulatory networks of mRNAs and non-coding RNAs underlying liver regeneration. The findings provide insights into molecular mechanisms that may be useful in developing new therapeutic approaches to ameliorate diseases that are characterized by liver fibrosis, which would be beneficial for the prevention of liver failure and treatment of liver cancer.
... One of the first indications that Caspase-8 plays a role in regulating the cell cycle suggesting a scaffolding role for Caspase-8 in cancer, came from Boege et al., who uncovered Caspase-8 as a key component of DNA damage sensing in cancer [67]. Specifically, Boege et al. revealed that, in hepatocellular carcinoma (HCC), Caspase-8 has a non-apoptotic scaffolding role that is essential for DNA damage sensing and subsequent H2AX phosphorylation. ...
... According to Boege et al., and similarly to other non-canonical roles of Caspase-8 [46,49], its ability to influence DNA sensing is independent of its catalytic activity. Indeed, the authors demonstrated that Caspase-8 influences the capacity of cells in DNA damage recognition by forming a complex with RIPK1/FADD/cFLIP [67]. They show in vivo that mice harboring a Caspase-8 deletion in hepatocytes fail to activate the DNA damage response (DDR) pathway and to phosphorylate the histone H2AX, even upon detection of DNA damage [67]. ...
... Indeed, the authors demonstrated that Caspase-8 influences the capacity of cells in DNA damage recognition by forming a complex with RIPK1/FADD/cFLIP [67]. They show in vivo that mice harboring a Caspase-8 deletion in hepatocytes fail to activate the DNA damage response (DDR) pathway and to phosphorylate the histone H2AX, even upon detection of DNA damage [67]. ...
Simple Summary
Caspase-8 is a protease mediating the activation of the extrinsic apoptotic process that leads to programmed cellular death. Evasion of apoptosis is one of the key hallmarks of cancer and Caspase-8 has commonly been associated with an antitumor protective role. However, observations that several solid tumor types inconsistently display aberrantly high levels of Caspase-8 has fueled studies that challenge this dogma. In this review, we summarize the current state of the art on how tumors benefit from high levels of Caspase-8 expression. In addition, we discuss the mechanisms through which tumors are able to alter the function of Caspase-8 and turn a protective protein into an ally. Specifically, we focus on the role played by tyrosine kinases in inhibiting the enzymatic role of Caspase-8 and remodulating Caspase-8 function in cancer through tyrosine phosphorylation.
Abstract
Caspase-8 is a cysteine-aspartic acid protease that has been identified as an initiator caspase that plays an essential role in the extrinsic apoptotic pathway. Evasion of apoptosis is a hallmark of cancer and Caspase-8 expression is silenced in some tumors, consistent with its central role in apoptosis. However, in the past years, several studies reported an increased expression of Caspase-8 levels in many tumors and consistently identified novel “non-canonical” non-apoptotic functions of Caspase-8 that overall promote cancer progression and sustain therapy resistance. These reports point to the ability of cancer cells to rewire Caspase-8 function in cancer and raise the question of which are the signaling pathways aberrantly activated in cancer that may contribute to the hijack of Caspase-8 activity. In this regard, tyrosine kinases are among the first oncogenes ever identified and genomic, transcriptomic and proteomic studies indeed show that they represent a class of signaling molecules constitutively activated in most of the tumors. Here, we aim to review and discuss the role of Caspase-8 in cancer and its interplay with Src and other tyrosine kinases.
... BCL2 is reported to contribute to the development and homeostasis of the mouse epidermis [1238]. Along similar lines, MCL1 and BCL-X L play roles in the development and homeostasis of several tissues including the myocardium [1239,1240], the CNS [148, [1241][1242][1243][1244][1245][1246][1247][1248], the hepatic parenchyma [298,845,[1249][1250][1251], vascular endothelium [1252], thymic epithelium [1253], as well as the intestinal [1254], mammary [1255,1256], lung [1257] and renal [277] epithelium. ...
... However, both hepatic and intestinal carcinogenesis involve a robust inflammatory component that is exacerbated by tissue damage and cell death [1263]. Moreover, MCL1deficient tissues show an increased cell turnover, which results in elevated level of replicative stress and genetic instability, potentially promoting carcinogenesis [845,1254]. Also, when many cells die, progenitors get mobilized and must divide extensively. ...
... Hyperactivation of CASP8 in the context of RIPK1 and TNF receptor-associated factor 2 (TRAF2) deficiency has indeed been implicated in the development of hepatocellular carcinoma [842], although such effects may be independent of apoptosis induction [843,844]. In contrast, recent studies show a tumor-suppressive function of CASP8 in the liver and certain other tissues [845][846][847][848]. In particular, there is evidence of a role of CAPS8 in early tumorigenesis (but not tumor progression) exerted by modulating the DNA damage response [845] or the level of chromosomal instability (CIN) [846]. ...
Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.
... In many studies, in accordance with our study, it was determined that DEN application caused an increase in liver enzyme activity. [24][25][26][27][28][29] The erythropoietin (EPO) hormone increases in conditions where oxygen intake is reduced, such as hypoxia, chronic obstructive pulmonary disease (COPD) and climbing to high altitudes. [30] In our study, the amount of EPO was tried to be measured, but could not be measured because it was outside the limits that can be measured by the device. ...
Background: Currently, with the development of technology, the use of many chemicals especially Diethylnitrosamine (DEN) in agriculture and industry has increased. The polyphenolic compounds of Epigallocatechin gallate (EGCG) is the active ingredient of green tea. It has been reported that green tea has antioxidant effects. In this study, effects of low dose Epigallocatechin gallate (EGCG), against exposure of Diethylnitrosamine administered rats.
Methods: As a group, groups were divided into five groups of ten rats for the application as Control, Sham, DEN EGCG and DEN+EGCG. The parameters analyzed are hemogram, biochemical, peripheral smear and weight.
Results: DEN injection has significantly increased LDH, AST, ALT and ALP values, Which are signs of hepatocyte injuries. The number of WBCs increased in the EGCG group. In terms of HDL and TChol levels, the group in which DEN+EGCG were applied together was found to be the highest and TG and LDL levels were found to be lowest. The current study will be a comprehensive study demonstrating the effects of low-dose EGCG against DEN-administred rats.
... This sets the basis for a transition from a chronically stressed liver to liver cancer development. 150 Notably, a strong link has been observed between hepatocyte apoptosis, increased alanine aminotransferase and aspartate aminotransferase levels, and DNA damage/instability (e.g. nuclear gH2Ax or p53BP1 positivity in hepatocytes). ...
... nuclear gH2Ax or p53BP1 positivity in hepatocytes). 150 Caspase 8, with its function as a scavenging protein, was also shown to be part of a protein complex including c-Flip and RipK1 that is involved in DNA damage control. Thus, hepatocytes losing caspase 8 expression not only survive longer due to impaired apoptosis but also accumulate DNA damageincreasing the risk of DNA instability and mutations. ...
... During progression this process is selected against, most likely because a strongly increased mutational load is disadvantageous for tumour growth. 150 In a recent publication by Donne et al., novel mechanisms underlying the activation of the DNA damage response during NAFLD were described. 151 The authors have shown that proliferating hepatocytes from NASH livers harbour replication stress, with a change in the replication fork's speed alongside ATR pathway activation causing DNA breaks. ...
The liver is the central metabolic organ of the body regulating energy and lipid metabolism and at the same time has potent immunological functions. Overwhelming the metabolic capacity of the liver by obesity and sedentary lifestyle leads to hepatic lipid accumulation, chronic necro-inflammation, enhanced mitochondrial/ER-stress and development of non-alcoholic fatty liver disease (NAFLD), with its pathologic form nonalcoholic steatohepatitis (NASH). Based on knowledge on pathophysiological mechanisms, specifically targeting metabolic diseases to prevent or slow down progression of NAFLD to liver cancer will become possible. Genetic/environmental factors contribute to development of NASH and liver cancer progression. The complex pathophysiology of NAFLD-NASH is reflected by environmental factors, particularly the gut microbiome and its metabolic products. NAFLD-associated HCC occurs in most of the cases in the context of a chronically inflamed liver and cirrhosis. Recognition of environmental alarmins or metabolites derived from the gut microbiota and the metabolically injured liver create a strong inflammatory milieu supported by innate and adaptive immunity. Several recent studies indicate that the chronic hepatic microenvironment of steatosis induces auto-aggressive CD8+CXCR6+PD1+ T cells secreting TNF and upregulating FasL to eliminate parenchymal and non-parenchymal cells in an antigen independent manner. This promotes chronic liver damage and a pro-tumorigenic environment. CD8+CXCR6+PD1+ T cells possess an exhausted, hyperactivated, resident phenotype and trigger NASH to HCC transition, and might be responsible for a less efficient treatment response to immune-check-point inhibitors - in particular atezolizumab/bevacizumab. Here, we provide an overview of NASH-related inflammation/pathogenesis focusing on new discoveries on the role of T cells in NASH-immunopathology and therapy response. This review discusses preventive measures to halt disease progression to liver cancer and therapeutic strategies to manage NASH-HCC patients.
... Secondly, GO enrichment showed that ZNF385A and ZNF346 co-expressed genes were significantly enriched in the inflammatory response, and GSEA analysis displayed that the high expression group was significantly enriched in the apoptosis pathway ( Figure 4D-G). Similarly, previous studies illustrated that a large number of hepatocyte deaths usually lead to chronic inflammation [26,27], and that sustained increased apoptosis leads to regenerative proliferation and a high DNA replication rate [28]. Thirdly, more inflammatory cells, including lymphocytes, macrophages, and neutrophils, were infiltrated in the ZNF385A and ZNF346 overexpression groups, and they were accompanied by many inflammatory cytokines that increased significantly ( Figures 6A,B and 7A,B). ...
Hepatocellular carcinoma (HCC) has a high mortality rate worldwide, and there are still many problems in the early diagnosis, molecular targeted therapy, and immunotherapy. It is necessary to explore valuable diagnostic markers and new therapeutic targets in HCC. Zinc finger protein 385A (ZNF385A) and zinc finger protein 346 (ZNF346) represent a unique class of RNA-binding Cys2 His2 (C2H2) zinc finger proteins that are involved in the regulation of cell cycle and apoptosis, but little is known of their roles in HCC. Based on multiple databases and analysis tools, we explored the expression, clinical relation, prognostic value, possible biological function, and pathways of ZNF385A and ZNF346, and their relationship with immune infiltration. Our results revealed that ZNF385A and ZNF346 were highly expressed and were associated with poor prognosis in HCC. Hepatitis B virus (HBV) infection may lead to the overexpression of ZNF385A and ZNF346, which was accompanied by elevated apoptosis and chronic inflammation. Moreover, ZNF385A and ZNF346 were positively correlated with immune-suppressive cells, inflammatory cytokines, immune checkpoint genes, and poor immunotherapy efficacy. Finally, the knockdown of ZNF385A and ZNF346 was observed to negatively affect the proliferation and migration of HepG2 cells in vitro. In conclusion, ZNF385A and ZNF346 are promising candidate biomarkers for the diagnosis, prognosis, and response to immunotherapy in HCC, and this study may help to understand the tumor microenvironment (TME) of liver cancer, and to develop new therapeutic targets.
... Moreover, the activation Wnt signalling is mitigated by IL-36Ra or IL-36γ neutralising antibody, in which IL-36γ and IL-36Ra played the critical roles of in gut inflammation and tumorigenesis through Wnt/β-catenin [73]. MCL1, a member of the pro-survival BCL2 family, has been identified as having an essential role in the preservation of intestinal homeostasis and the prevention of carcinoma [74]. IEC-specific MCL1 deficiency shares hallmark features with IBD and is independent of microbe-induced chronic inflammation. ...
Colorectal cancer (CRC) represents one of the most common cancers worldwide, with a high mortality rate despite the decreasing incidence and new diagnostic and therapeutic strategies. CRC arises from both epidemiologic and molecular backgrounds. In addition to hereditary factor and genetic mutations, the strongly varying incidence of CRC is closely linked to chronic inflammatory disorders of the intestine and terrible dietary habits. The Wnt signalling pathway is a complex regulatory network that is implicated in many CRC physiological processes, including cancer occurrence, development, prognosis, invasion, and metastasis. It is currently believed to include classical Wnt/β-catenin, Wnt/PCP, and Wnt/Ca2+. In this review, we summarise the recent mechanisms and potential regulators of the three branches of the Wnt signalling pathway in CRC.
... Interestingly, monosomy of chromosome 16, where WWOX resides, was reported in mice as being protective against toxins 24 . In chronic liver disease models, WWOX loss was among the initial alterations preceding morphological visible neoplastic changes 42 . In HCC cell line studies, loss of WWOX copy numbers was observed and correlated well with absent or lower mRNA expression 18 . ...
Nonalcoholic steatohepatitis (NASH)-induced hepatocellular carcinoma (HCC) and its precursor, nonalcoholic fatty liver disease (NAFLD) are an unmet health issue due to widespread obesity. We assessed copy number changes of genes associated with hepatocarcinogenesis and oxidative pathways at a single-cell level. Eleven patients with NASH-HCC and 11 patients with NAFLD were included. Eight probes were analyzed using multiplex interphase fluorescence in situ hybridization (miFISH), single-cell imaging and phylogenetic tree modelling: Telomerase reverse transcriptase (TERT), C-Myc (MYC), hepatocyte growth factor receptor tyrosine kinase (MET), tumor protein 53 (TP53), cyclin D1 (CCND1), human epidermal growth factor receptor 2 (HER2), the fragile histidine triad gene (FHIT) and FRA16D oxidoreductase (WWOX). Each NASH-HCC tumor had up to 14 distinct clonal signal patterns indicating multiclonality, which correlated with high tumor grade. Changes frequently observed were TP53 losses, 45%; MYC gains, 36%; WWOX losses, 36%; and HER2 gains, 18%. Whole-genome duplications were frequent (82%) with aberrant tetraploid cells evolving from diploid ancestors. Non-tumorous NAFLD/NASH biopsies did not harbor clonal copy number changes. Fine mapping of NASH-HCC using single-cell multiplex FISH shows that branched tumor evolution involves genome duplication and that multiclonality increases with tumor grade. The loss of oxidoreductase WWOX and HER2 gains could be potentially associated with NASH-induced hepatocellular carcinoma.
