Protein phosphatase 2A promotes hepatocellular carcinogenesis in the diethylnitrosamine mouse model through inhibition of p53

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most HCCs develop in cirrhotic livers. Alcoholic liver disease, chronic hepatitis B and chronic hepatitis C are the most common underlying liver diseases. Hepatitis C virus (HCV)-specific mechanisms that contribute to HCC are presently unknown. Transgenic expression of HCV proteins in the mouse liver induces an overexpression of the protein phosphatase 2A catalytic subunit (PP2Ac). We have previously reported that HCV-induced PP2Ac overexpression modulates histone methylation and acetylation and inhibits DNA damage repair. In this study, we analyze tumor formation and gene expression using HCV transgenic mice that overexpress PP2Ac and liver tissues from patients with HCC. We demonstrate that PP2Ac overexpression interferes with p53-induced apoptosis. Injection of the carcinogen, diethylnitrosamine, induced significantly more and larger liver tumors in HCV transgenic mice that overexpress PP2Ac compared with control mice. In human liver biopsies from patients with HCC, PP2Ac expression was significantly higher in HCC tissue compared with non-tumorous liver tissue from the same patients. Our findings demonstrate an important role of PP2Ac overexpression in liver carcinogenesis and provide insights into the molecular pathogenesis of HCV-induced HC

Genomic evolutionary trajectory of metastatic squamous cell carcinoma of the lung.

Background The extent of inter- and intratumoral genomic heterogeneity and the clonal evolution of metastatic squamous cell carcinoma of the lung (LUSC) are poorly understood. Genomic studies of LUSC are challenged by their low tumor cell content. We sought to define the genomic landscape and evolutionary trajectories of metastatic LUSC combining nuclei-flow sorting and whole exome sequencing. Methods Five patients with primary LUSC and six matched metastases were investigated. Tumor nuclei were sorted based on ploidy and expression of cytokeratin to enrich for tumor cells for whole exome sequencing. Results Flow-sorting increased the mean tumor purity from 26% (range, 12-50%) to 73% (range, 42-93%). Overall, primary LUSCs and their matched metastases shared a median of 79% (range, 67-85%) of copy number aberrations (CNAs) and 74% (range, 65-94%) of non-synonymous mutations, including in tumor suppressor genes such as TP53. Furthermore, the ploidy of the tumors remained unchanged between primary and metastasis in 4/5 patients over time. We found differences in the mutational signatures of shared mutations compared to the private mutations in the primary or metastasis. Conclusions Our results demonstrate a close genomic relationship between primary LUSCs and their matched metastases, suggesting late dissemination of the metastases from the primary tumors during tumor evolution

Expression of Ano1 and Ca²⁺ activated Cl⁻ currents in HNSCC cells.

<p>A) Western blots of Ano1 expressed in the three different human cell lines CAL-27, CAL-33, and BHY. B) Current/voltage relationships of whole cell currents activated by 100 µM ATP (filled circles) in CAL-27, CAL-33, and BHY cells. Application of the K⁺ channel inhibitors Ba²⁺ (5 mM) and TEA⁺ (10 mM) (grey circles) did not significantly change currents, suggesting a dominant role Cl⁻ currents. C) Summary of the calculated ATP-activated whole cell conductances. Mean ± SEM, (number of experiments).</p

Analysis of the 11q13 (Ano1 and CCND1) genomic amplification and protein expression in 365 HNSCCs.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043265#pone-0043265-t001" target="_blank"><b>Table 1</b></a><b> Legend.</b> A) Overview of the prevalence of genomic amplification of <i>Ano1</i> and Ano1 expression in HNSCCs from different tumor locations.</p><p>B)Genomic amplification of the 11q13 locus is associated with the presence of Ano1 protein expression.</p><p>C)Strong correlation between the genomic amplification of <i>Ano1</i> and <i>CCND1</i>.</p

<i>Ano1 causes cell migration.</i>

<p>A) Summary of BrdU incorporation in BHY cells after various treatments, shown as % of BrdU incorporation, when compared to cells treated with scrambled RNA (red dashed line). B) Representative images of wound healing in a scratch assay with BHY cells, treated with scrambled RNA (upper panels) or after Ano1-knockdown with siRNA-Ano1 (lower panels). Yellow arrowheads indicate edges of the damage. C) Time course for the wound healing process (closure of the scratch) for control cells (scrambled RNA) and after Ano1-knockdown (si-Ano1). D) Migration of BHY and CAL-33 cells measured continuously as cell index using the xCELLigence system. Inhibition of migration by blockade of Ano1 with AO1 (10 µM). E) Cell Proliferation measured continuously as cell index using the xCELLigence system. AO1 (10 µM) has no effect on proliferation. Experiments were performed at least in triplicates. Mean ± SEM, (number of experiments). ^#indicates significant difference (p<0.05, ANOVA).</p

Comprehensive analysis of Ano1 protein expression in normal tissues.

<p>#Mucosa and mucous glands were negative.</p><p>Summary of normal tissues with detectable Ano1 protein expression. Most normal tissues do not express Ano1. Only gall bladder, stomach and duodenum express Ano1 (A). Apical positivity (B) and Ano1-positive Cajal cells (C) were detected in several types of tissues. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043265#pone.0043265.s005" target="_blank">Table S5</a> for complete listing.).</p

Comprehensive analysis of Ano1 protein expression in human tumors.

*<p>These tumors only showed unspecific cytoplasmic Ano1 staining and were thus classified as negative.</p><p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043265#pone-0043265-t002" target="_blank"><b>Table 2</b></a><b> Legend.</b> Summary of human tumors with detectable Ano1 protein expression. Most tumors do not express Ano1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043265#pone.0043265.s004" target="_blank">Table S4</a> for complete listing).</p

Ano1 supports regulatory volume decrease.

<p>A) Original recordings of the calcein fluorescence in BHY cells, treated with scrambled RNA or siRNA-Ano1, before and after stimulation with 100 µM ATP. ATP induces transient cell shrinkage that was not affected by siRNA-knockdown of Ano1. B) Summary of the ATP-induced changes in calcein fluorescence in BHY and CAL-33 cells treated with scrambled RNA or siRNA-Ano1. C) Original recordings of calcein fluorescence in BHY and CAL-33 cells treated with scrambled RNA (blue) or siRNA-Ano1 (red) and effect of a hypotonic (Hypo; 200 mosmol/l) bath solution. D) Summary of the hypotonicity-induced changes in calcein fluorescence in BHY and CAL-33 cells treated with scrambled RNA or siRNA-Ano1. Mean ± SEM, (number of experiments). *indicates significant difference (p<0.05, paired t-test). ^#indicates significant difference (p<0.05, paired t-test).</p