α-catenin interaction with YAP/FoxM1/TEAD-induced CEP55 supports liver cancer cell migration.

Adherens junctions (AJs) facilitate cell-cell contact and contribute to cellular communication as well as signaling under physiological and pathological conditions. Aberrant expression of AJ proteins is frequently observed in human cancers; however, how these factors contribute to tumorigenesis is poorly understood. In addition, for some factors such as α-catenin contradicting data has been described. In this study we aim to decipher how the AJ constituent α-catenin contributes to liver cancer formation. TCGA data was used to detect transcript changes in 23 human tumor types. For the detection of proteins, liver cancer tissue microarrays were analyzed by immunohistochemistry. Liver cancer cell lines (HLF, Hep3B, HepG2) were used for viability, proliferation, and migration analyses after RNAinterference-mediated gene silencing. To investigate the tumor initiating potential, vectors coding for α-catenin and myristoylated AKT were injected in mice by hydrodynamic gene delivery. A BioID assay combined with mass spectrometry was performed to identify α-catenin binding partners. Results were confirmed by proximity ligation and co-immunoprecipitation assays. Binding of transcriptional regulators at gene promoters was investigated using chromatin-immunoprecipitation. α-catenin mRNA was significantly reduced in many human malignancies (e.g., colon adenocarcinoma). In contrast, elevated α-catenin expression in other cancer entities was associated with poor clinical outcome (e.g., for hepatocellular carcinoma; HCC). In HCC cells, α-catenin was detectable at the membrane as well as cytoplasm where it supported tumor cell proliferation and migration. In vivo, α-catenin facilitated moderate oncogenic properties in conjunction with AKT overexpression. Cytokinesis regulator centrosomal protein 55 (CEP55) was identified as a novel α-catenin-binding protein in the cytoplasm of HCC cells. The physical interaction between α-catenin and CEP55 was associated with CEP55 stabilization. CEP55 was highly expressed in human HCC tissues and its overexpression correlated with poor overall survival and cancer recurrence. Next to the α-catenin-dependent protein stabilization, CEP55 was transcriptionally induced by a complex consisting of TEA domain transcription factors (TEADs), forkhead box M1 (FoxM1), and yes-associated protein (YAP). Surprisingly, CEP55 did not affect HCC cell proliferation but significantly supported migration in conjunction with α-catenin. Migration-supporting CEP55 is induced by two independent mechanisms in HCC cells: stabilization through interaction with the AJ protein α-catenin and transcriptional activation via the FoxM1/TEAD/YAP complex.

Tang Y ，Thiess L ，Weiler SME ，Tóth M ，Rose F ，Merker S ，Ruppert T ，Schirmacher P ，Breuhahn K ... -  《Cell Communication and Signaling》

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

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. 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 (YAPS127A) 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. 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 YAPS127A mice had increased CIN25 and CIN70 gene expression patterns, aneuploidy, and defects in mitosis. Injection of YAPS127A 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. 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).

Weiler SME ，Pinna F ，Wolf T ，Lutz T ，Geldiyev A ，Sticht C ，Knaub M ，Thomann S ，Bissinger M ，Wan S ，Rössler S ，Becker D ，Gretz N ，Lang H ，Bergmann F ，Ustiyan V ，Kalin TV ，Singer S ，Lee JS ，Marquardt JU ，Schirmacher P ，Kalinichenko VV ，Breuhahn K ... -  《-》

Yes-associated protein (YAP) induces a secretome phenotype and transcriptionally regulates plasminogen activator Inhibitor-1 (PAI-1) expression in hepatocarcinogenesis.

Marquard S ，Thomann S ，Weiler SME ，Bissinger M ，Lutz T ，Sticht C ，Tóth M ，de la Torre C ，Gretz N ，Straub BK ，Marquardt J ，Schirmacher P ，Breuhahn K ... -  《Cell Communication and Signaling》

[YAP induces chromosomal instability in liver cancer patients].

Weiler S 《-》

Overexpression of Rac GTPase Activating Protein 1 Contributes to Proliferation of Cancer Cells by Reducing Hippo Signaling to Promote Cytokinesis.

Agents designed to block or alter cytokinesis can kill or stop proliferation of cancer cells. We aimed to identify cytokinesis-related proteins that are overexpressed in hepatocellular carcinoma (HCC) cells and might be targeted to slow liver tumor growth. Using the Oncomine database, we compared the gene expression patterns in 16 cancer microarray datasets and assessed gene enrichment sets using gene ontology. We performed immunohistochemical analysis of an HCC tissue microarray and identified changes in protein levels that are associated with patient survival times. Candidate genes were overexpressed or knocked down with small hairpin RNAs in SMMC7721, MHCC97H, or HCCLM3 cell lines; we analyzed their proliferation, viability, and clone-formation ability and their growth as subcutaneous or orthotopic xenograft tumors in mice. We performed microarray analyses to identify alterations in signaling pathways and immunoblot and immunofluorescence assays to detect and localize proteins in tissues. Yeast 2-hybrid screens and mass spectrometry combined with co-immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co-immunoprecipitation and proximity ligation assays. Chromatin immunoprecipitation, promoter luciferase activity, and quantitative real-time polymerase chain reaction analyses were used to identify factors that regulate transcription of specific genes. The genes that were most frequently overexpressed in different types of cancer cells were involved in cell division processes. We identified 3 cytokinesis-regulatory proteins among the 10 genes most frequently overexpressed by all cancer cell types. Rac GTPase activating protein 1 (RACGAP1) was the cytokinesis-regulatory protein that was most highly overexpressed in multiple cancers. Increased expression of RACGAP1 in tumor tissues was associated with shorter survival times of patients with cancer. Knockdown of RACGAP1 in HCC cells induced cytokinesis failure and cell apoptosis. In microarray analyses, we found knockdown of RACGAP1 in SMMC7721 cells to reduce expression of genes regulated by yes-associated protein (YAP) and WW domain containing transcription regulator 1 (WWTR1 or TAZ). RACGAP1 reduced activation of the Hippo pathway in HCC cells by increasing activity of RhoA and polymerization of filamentous actin. Knockdown of YAP reduced phosphorylation of RACGAP1 and redistribution at the anaphase central spindle. We found transcription of the translocated promoter region, nuclear basket protein (TPR) to be regulated by YAP and coordinately expressed with RACGAP1 to promote proliferation of HCC cells. TPR redistributed upon nuclear envelope breakdown and formed complexes with RACGAP1 during mitosis. Knockdown of TPR in HCC cells reduced phosphorylation of RACGAP1 by aurora kinase B and impaired their redistribution at the central spindle during cytokinesis. STAT3 activated transcription of RACGAP in HCC cells. In an analysis of gene expression patterns of multiple tumor types, we found RACGAP1 to be frequently overexpressed, which is associated with shorter survival times of patients. RACGAP1 promotes proliferation of HCC cells by reducing activation of the Hippo and YAP pathways and promoting cytokinesis in coordination with TPR.

Yang XM ，Cao XY ，He P ，Li J ，Feng MX ，Zhang YL ，Zhang XL ，Wang YH ，Yang Q ，Zhu L ，Nie HZ ，Jiang SH ，Tian GA ，Zhang XX ，Liu Q ，Ji J ，Zhu X ，Xia Q ，Zhang ZG ... -  《-》