Induction of Chromosome Instability by Activation of Yes-Associated Protein and Forkhead Box M1 in Liver Cancer

doi:10.1053/j.gastro.2017.02.018

Gastroenterology

Volume 152, Issue 8, June 2017, Pages 2037-2051.e22

https://doi.org/10.1053/j.gastro.2017.02.018 Get rights and content

Background & Aims

Many different types of cancer cells have chromosome instability. The hippo pathway leads to phosphorylation of the transcriptional activator yes-associated protein 1 (YAP1, YAP), which regulates proliferation and has been associated with the development of liver cancer. We investigated the effects of hippo signaling via YAP on chromosome stability and hepatocarcinogenesis in humans and mice.

Methods

We analyzed transcriptome data from 242 patients with hepatocellular carcinoma (HCC) to search for gene signatures associated with chromosomal instability (CIN); we investigated associations with overall survival time and cancer recurrence using Kaplan–Meier curves. We analyzed changes in expression of these signature genes, at mRNA and protein levels, after small interfering RNA–mediated silencing of YAP in Sk-Hep1, SNU182, HepG2, or pancreatic cancer cells, as well as incubation with thiostrepton (an inhibitor of forkhead box M1 [FOXM1]) or verteporfin (inhibitor of the interaction between YAP and TEA domain transcription factor 4 [TEAD4]). We performed co-immunoprecipitation and chromatin immunoprecipitation experiments. We collected liver tissues from mice that express a constitutively active form of YAP (YAP^S127A) and analyzed gene expression signatures and histomorphologic parameters associated with chromosomal instability. Mice were given injections of thiostrepton and livers were collected and analyzed by immunoblotting, immunohistochemistry, histology, and real-time polymerase chain reaction. We performed immunohistochemical analyses on tissue microarrays of 105 HCCs and 7 nontumor liver tissues.

Results

Gene expression patterns associated with chromosome instability, called CIN25 and CIN70, were detected in HCCs from patients with shorter survival time or early cancer recurrence. TEAD4 and YAP were required for CIN25 and CIN70 signature expression via induction and binding of FOXM1. Disrupting the interaction between YAP and TEAD4 with verteporfin, or inhibiting FOXM1 with thiostrepton, reduced the chromosome instability gene expression patterns. Hyperplastic livers and tumors from YAP^S127A mice had increased CIN25 and CIN70 gene expression patterns, aneuploidy, and defects in mitosis. Injection of YAP^S127A mice with thiostrepton reduced liver overgrowth and signs of chromosomal instability. In human HCC tissues, high levels of nuclear YAP correlated with increased chromosome instability gene expression patterns and aneuploidy.

Conclusions

By analyzing cell lines, genetically modified mice, and HCC tissues, we found that YAP cooperates with FOXM1 to contribute to chromosome instability. Agents that disrupt this pathway might be developed as treatments for liver cancer. Transcriptome data are available in the Gene Expression Omnibus public database (accession numbers: GSE32597 and GSE73396).

Section snippets

Materials

Antibodies used for Western immunoblotting or immunohistochemical staining as well as corresponding dilutions, primer, and small interfering RNA (siRNA) sequences are listed in Supplementary Table 1, Supplementary Table 2, Supplementary Table 3, Supplementary Table 4, Supplementary Table 5. A detailed description of the in vitro analyses as well as protocols for mouse work, sample preparation, real-time polymerase chain reaction (PCR), Western immunoblotting, expression profiling, luciferase

Presence of CIN25 Signature in HCC and Pancreatic Cancer Patients Correlates With Poor Survival and Cancer Recurrence

The relevance of CIN in liver cancer is discussed controversially and to date no data for the existence of CIN signatures in HCC exists. Therefore, the occurrence of the CIN25 signature, which represents the top 25 genes with the highest correlation to total functional aneuploidy, was tested by using transcriptome data of 242 human HCC patients.3, 17 Data were clustered into groups with low (32%, cluster 1), moderate (35%, cluster 2), and high (33%, cluster 3) expression of the CIN signature

Discussion

The evolutionary conserved Hippo/YAP signaling pathway is a central regulator of organ size in development and regeneration.³² In the liver, inactivation of the Hippo kinase cascade or overexpression of its negatively regulated downstream effector YAP leads to tissue overgrowth and tumor initiation, classifying the Hippo signaling axis as a tumor-suppressor pathway and YAP as a hepatic oncogene.25, 33, 34 Different effector mechanisms for the tumor-initiating and promoting properties have been

Acknowledgments

The authors thank Heike Conrad and Elisabeth Specht-Delius for excellent technical assistance, J. Lorenzo Bermejo for help with biostatistics analyses, and T. R. Brummelkamp and F. D. Camargo for providing the Col1A1-YAP^S127A transgenic mice. Tissue samples were provided by the tissue bank of the National Center of Tumor Diseases (Heidelberg, Germany) in accordance with the regulations of the tissue bank and the approval of the Ethics Committee of Heidelberg University.

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YAP can facilitate the transcription of several genes involved in cholesterol metabolism (Wang et al., 2014). In turn, studies have shown that cholesterol level is associated with the cancer-promoting properties of YAP/TAZ (Lee et al., 2010; Tao et al., 2014; Weiler et al., 2017). For the Hippo/YAP signaling pathway in a variety of tumors, there are no direct inhibitors currently.
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Aneuploidy, extrachromosomal DNA, and massive chromosomal structural abnormalities caused by CIN underlie tumor heterogeneity, which enhances tumor environmental adaptability and promotes immune escape, drug resistance, and metastasis [13–15]. Studies indicates that CIN exists in cirrhotic liver, dysplastic nodules, and in more than 90% of HCC cases [16,17]. Therefore, CIN-associated genes may be a promising focus for depicting the progression of HCC and targeting CIN may be a promising therapy for HCC.
Hepatocellular carcinoma (HCC) is a type of cancer characterized by high heterogeneity and a complex multistep progression process. Significantly-altered biomarkers for HCC need to be identified. Differentially expressed genes and weighted gene co-expression network analyses were used to identify progression-related biomarkers. LASSO-Cox regression and random forest algorithms were used to construct the progression-related prognosis (PRP) score. Three chromosomal instability-associated genes (KIF20A, TOP2A, and TTK) have been identified as progression-related biomarkers. The robustness of the PRP scores were validated using four independent cohorts. Immune status was observed using the single-sample gene set enrichment analysis (ssGSEA). Comprehensive analysis showed that the patients with high PRP score had wider genomic alterations, more malignant phenotypes, and were in a state of immunosuppression. The diagnostic models constructed via logistic regression based on the three genes showed satisfactory performances in distinguishing HCC from cirrhotic tissues or dysplastic nodules. The nomogram combining PRP scores with clinical factors had a better performance in predicting prognosis than the tumor node metastasis classification (TNM) system. We further confirmed that KIF20A, TOP2A, and TTK were highly expressed in HCC tissues than in cirrhotic tissues. Downregulation of all three genes aggravated chromosomal instabilities in HCC and suppressed HCC cells viability both in vitro and in vivo. Overall, our study highlights the important roles of chromosomal instability-associated genes during the progression of HCC and their potential clinical diagnosis and prognostic value and provides promising new ideas for developing therapeutic strategies to improve the outcomes of HCC patients.
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Dysregulation of the Hippo pathway that promotes cell survival, proliferation and tumorigenesis, relays on the coordinated interactions of YAP with the factors that determine YAP translocation and the related transcriptional programming. Here, we demonstrate that ETV4, a transcriptional factor participating in various protumorigenic processes, enhances YAP-mediated transactivation and hepatocellular carcinoma (HCC) progression. Mechanistically, the enhancement of YAP activities is mediated by the interaction between ETV4 and YAP, which not only increases nuclear YAP accumulation but also directly augments the YAP/TEAD4-mediated transcriptional activation in tumor cells. Functionally, the interplay of ETV4 and YAP promotes growth of liver tumor cells, and activates the genes related to myeloid cell recruitment, including CXCL1 and CXCL5, leading to an enriched presence of myeloid-derived suppressive cells and macrophages but a decreased infiltration of T cells and NK cells in transplanted tumors. More importantly, the correlations between YAP activation, the altered immune cell distribution and ETV4 expression are observed in human HCCs. Therefore, our study reveals a functional interaction between ETV4 and YAP that contributes to HCC progression, and provides mechanistic insights into the regulation of nuclear YAP retention and transactivation.
YAP1 induces hyperglycemic stress-mediated cardiac hypertrophy and fibrosis in an AKT-FOXM1 dependent signaling pathway
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Cardiac disease is one of the most common complications associated with diabetes. Cardiac hypertrophy and fibrosis often lead to structural and functional abnormalities leading to risks of heart failure. Several regulatory molecules related to major signaling pathways have been found to overexpress in different tissues during diabetes which show very low level of expression in non-diabetic condition. YAP1 and FOXM1 are recently being reported to play important role in various hypertrophic and fibrotic disorders. But, very limited information is still known regarding their roles in cardiomyopathies especially in the context of diabetes and hyperglycemic stress. YAP1 is known to be associated with AKT- GSK3β signaling that is one of the important regulatory pathways in glucose and lipid metabolism. On the other hand, the expression of FOXM1 has been found to be significantly upregulated in adult lung tissue with induction of fibrosis but little is known about their role in cardiac diseases. In our study, YAP1 and FOXM1 have been found to overexpress in cardiac tissue under hyperglycemic condition leading to cardiomyocyte hypertrophy and increased fibrotic response. Further YAP1 inhibition has resulted in a reduced expression of FOXM1 pointing to a possible association of YAP1 and FOXM1 in high glucose-stressed cardiomyocyte. As mechanism we have found that YAP1 undergoes reduced ser127 phosphorylation as well as extensive O-GlcNAcylation mediated activation under hyperglycemia. Upregulated YAP1 further acts through increased AKT phosphorylation causing inhibition of GSK3β that in turn results in increased FOXM1 expression, leading to cardiomyocyte hypertrophy and fibrosis.

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: Conflicts of interest The authors disclose no conflicts.

: Funding This study was supported by a grant from the German Cancer Aid (Deutsche Krebshilfe/Dr Mildred Scheel Stiftung: DKH 111524 to K.B.), by grants from the German Cancer Aid (DKH 110989) and the Volkswagen Foundation (Lichtenberg program) (J.U.M.), and by a scholarship from Hartmut Hoffman-Berling International Graduate School (S.W.).

: Author names in bold designate shared co-first authorship.

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Original ResearchFull Report: Basic and Translational—LiverInduction of Chromosome Instability by Activation of Yes-Associated Protein and Forkhead Box M1 in Liver Cancer

Background & Aims

Methods

Results

Conclusions

Section snippets

Materials

Presence of CIN25 Signature in HCC and Pancreatic Cancer Patients Correlates With Poor Survival and Cancer Recurrence

Discussion

Acknowledgments

Am J Pathol

Cancer Cell

Mod Pathol

J Hepatol

Adv Cancer Res

Curr Biol

Cell

Chromosomal instability and cancer: a complex relationship with therapeutic potential

J Clin Invest

A mechanism linking extra centrosomes to chromosomal instability

Nature

A signature of chromosomal instability inferred from gene expression profiles predicts clinical outcome in multiple human cancers

Nat Genet

Examining the link between chromosomal instability and aneuploidy in human cells

J Cell Biol

Induction of aneuploidy by increasing chromosomal instability during dedifferentiation of hepatocellular carcinoma

Proc Natl Acad Sci U S A

Hepatocellular telomere shortening correlates with chromosomal instability and the development of human hepatoma

Hepatology

Telomere shortening and inactivation of cell cycle checkpoints characterize human hepatocarcinogenesis

Hepatology

Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP

Genes Dev

Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction

Nat Cell Biol

N-terminal truncation of Lats1 causes abnormal cell growth control and chromosomal instability

J Cell Sci

CDK1 phosphorylation of YAP promotes mitotic defects and cell motility and is essential for neoplastic transformation

Cancer Res

Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in human hepatocellular carcinoma

Gastroenterology

A unique metastasis gene signature enables prediction of tumor relapse in early-stage hepatocellular carcinoma patients

Cancer Res

TEAD mediates YAP-dependent gene induction and growth control

Genes Dev

YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cycle-promoting genes

Oncogene

Original Research
Full Report: Basic and Translational—Liver
Induction of Chromosome Instability by Activation of Yes-Associated Protein and Forkhead Box M1 in Liver Cancer