Senescence and immortality in hepatocellular carcinoma

Cataloged from PDF version of article.Cellular senescence is a process leading to terminal growth arrest with characteristic morphological features. This process is mediated by telomere-dependent, oncogene-induced and ROS-induced pathways, but persistent DNA damage is the most common cause. Senescence arrest is mediated by p16(INK4a)- and p21(Cip1)-dependent pathways both leading to retinoblastoma protein (pRb) activation. p53 plays a relay role between DNA damage sensing and p21(Cip1) activation. pRb arrests the cell cycle by recruiting proliferation genes to facultative heterochromatin for permanent silencing. Replicative senescence that occurs in hepatocytes in culture and in liver cirrhosis is associated with lack of telomerase activity and results in telomere shortening. Hepatocellular carcinoma (HCC) cells display inactivating mutations of p53 and epigenetic silencing of p16(INK4a). Moreover, they re-express telomerase reverse transcriptase required for telomere maintenance. Thus, senescence bypass and cellular immortality is likely to contribute significantly to HCC development. Oncogene-induced senescence in premalignant lesions and reversible immortality of cancer cells including HCC offer new potentials for tumor prevention and treatment. (C) 2008 Elsevier Ireland Ltd. All rights reserved

Genome-Wide Transcriptional Reorganization Associated with Senescence-to-Immortality Switch during Human Hepatocellular Carcinogenesis

Cataloged from PDF version of article.Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become "immortal") by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC) development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15-gene hepatocellular immortality signature test that discriminated HCC from cirrhosis with high accuracy. Our findings demonstrate that senescence bypass plays a central role in hepatocellular carcinogenesis engendering systematic changes in the transcription of genes regulating DNA repair, proliferation, differentiation and metabolism

Transcriptional Control in Cardiac Progenitors: Tbx1 Interacts with the BAF Chromatin Remodeling Complex and Regulates Wnt5a

Mutations of the Wnt5a gene, encoding a ligand of the non-canonical Wnt pathway, and the Ror2 gene, encoding its receptor, have been found in patients with cardiac outflow tract defects. We found that Wnt5a is expressed in the second heart field (SHF), a population of cardiac progenitor cells destined to populate the cardiac outflow tract and the right ventricle. Because of cardiac phenotype similarities between Wnt5a and Tbx1 mutant mice, we tested potential interactions between the two genes. We found a strong genetic interaction in vivo and determined that the loss of both genes caused severe hypoplasia of SHF–dependent segments of the heart. We demonstrated that Wnt5a is a transcriptional target of Tbx1 and explored the mechanisms of gene regulation. Tbx1 occupies T-box binding elements within the Wnt5a gene and interacts with the Baf60a/Smarcd1 subunit of a chromatin remodeling complex. It also interacts with the Setd7 histone H3K4 monomethyltransferase. Tbx1 enhances Baf60a occupation at the Wnt5a gene and enhances its H3K4 monomethylation status. Finally, we show that Baf60a is required for Tbx1–driven regulation of target genes. These data suggest a model in which Tbx1 interacts with, and probably recruits a specific subunit of, the BAF complex as well as histone methylases to activate or enhance transcription. We speculate that this may be a general mechanism of T-box function and that Baf60a is a key component of the transcriptional control in cardiac progenitors

Promoter methylation of Wnt5a is associated with microsatellite instability and BRAF V600E mutation in two large populations of colorectal cancer patients

BACKGROUND: In colorectal cancer (CRC), tumour microsatellite instability (MSI) status and CpG island methylator phenotype (CIMP) status are indicators of patient outcome, but the molecular events that give rise to these outcomes remain largely unknown. Wnt5a is a critical regulator of non-canonical Wnt activity and promoter hypermethylation of this gene has emerging prognostic roles in CRC; however the frequency and prognostic significance of this epigenetic event have not been explored in the context of colorectal tumour subtype. Consequently, we investigated the frequency and prognostic significance of Wnt5a methylation in a large cohort of MSI-stratified CRCs. METHODS: Methylation was quantified in a large cohort of 1232 colorectal carcinomas from two clinically distinct populations from Canada. Associations were examined between methylation status and clinicopathlogical features, including tumour MSI status, BRAF V600E mutation, and patient survival. RESULTS: In Ontario, Wnt5a methylation was strongly associated with MSI tumours after adjustment for age, sex, and tumour location (odds ratio (OR)=4.2, 95% confidence interval (CI)=2.4-7.4, P<10(-6)) and with BRAF V600E mutation, a marker of CIMP (OR=12.3, 95% CI=6.9-21.7, P<10(-17)), but was not associated with patient survival. Concordant results were obtained in Newfoundland. CONCLUSION: Methylation of Wnt5a is associated with distinct tumour subtypes, strengthening the evidence of an epigenetic-mediated Wnt bias in CRC

The Ability to Generate Senescent Progeny as a Mechanism Underlying Breast Cancer Cell Heterogeneity

Background Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, "normal-like", and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity. Methodology/Principal Findings A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21Cip1 correlated with senescence in these cell lines. p21Cip1 knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and "normal-like" tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice. Conclusions/Significance Luminal A and "normal-like" breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential. © 2010 Mumcuoglu et al

Sfrp5 Modulates Both Wnt and BMP Signaling and Regulates Gastrointestinal Organogensis in the Zebrafish, Danio rerio

Sfrp5 belongs to the family of secreted frizzled related proteins (Sfrp), secreted inhibitors of Wingless-MMTV Integration Site (Wnt) signaling, which play an important role in cancer and development. We selected sfrp5 because of its compelling expression profile in the developing endoderm in zebrafish, Danio rerio. In this study, overexpression of sfrp5 in embryos results in defects in both convergent extension (CE) by inhibition of non-canonical Wnt signaling and defects in dorsoventral patterning by inhibition of Tolloid-mediated proteolysis of the BMP inhibitor Chordin. From 25 hours post fertilization (hpf) to 3 days post fertilization (dpf), both overexpression and knockdown of Sfrp5 decrease the size of the endoderm, significantly reducing liver cell number. At 3 dpf, insulin-positive endodermal cells fail to coalesce into a single pancreatic islet. We show that Sfrp5 inhibits both canonical and non-canonical Wnt signaling during embryonic and endodermal development, resulting in endodermal abnormalities. © 2013 Stuckenholz et al

Aflatoxin genotoxicity is associated with a defective DNA damage response bypassing p53 activation

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths. Aflatoxins, which may play a causative role in 5-28% of HCCs worldwide, are activated in liver cells and induce principally G→T mutations, including the TP53 codon 249(G→T) hotspot mutation. The DNA damage checkpoint response acts as an antitumour mechanism against genotoxic agents, but its role in aflatoxin-induced DNA damage is unknown. Aim: We studied the DNA damage checkpoint response of human cells to aflatoxin B1 (AFB1). Methods and results: The treatment of HepG2 hepatoma cells with mutation-inducing doses (3-5μmol/l) of AFB1 induced DNA adducts, 8-hydroxyguanine lesions and DNA strand breaks that lasted several days. Persistent phospho-H2AX and 53BP1 foci were also detected, but cell growth was not affected. AFB1-exposed HepG2 cells formed phospho-H2AX and 53BP1 foci, but failed to phosphorylate both Chk1 and Chk2. Huh7 hepatoma and HCT116 colorectal cancer cell lines also exhibited a similarly incomplete checkpoint response. p53 phosphorylation also failed, and AFB1-exposed cells did not show p53-dependent G1 arrest or a sustained G2/M arrest. These observations contrasted sharply with the fully functional DNA damage response of cells to Adriamycin. Cotreatment of cells with AFB1 did not inhibit p53 and p21Cip1 accumulation induced by Adriamycin. Thus, the deficient checkpoint response to AFB1 was not due to an inhibitory effect, but could be explained by an inefficient activation. Conclusion: Genotoxic doses of AFB1 induce an incomplete and inefficient checkpoint response in human cells. This defective response may contribute to the mutagenic and carcinogenic potencies of aflatoxins. © 2011 John Wiley &amp; Sons A/S

Aflatoxin genotoxicity is associated with a defective DNA damage response bypassing p53 activation

Cataloged from PDF version of article.Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths. Aflatoxins, which may play a causative role in 5-28% of HCCs worldwide, are activated in liver cells and induce principally G→T mutations, including the TP53 codon 249(G→T) hotspot mutation. The DNA damage checkpoint response acts as an antitumour mechanism against genotoxic agents, but its role in aflatoxin-induced DNA damage is unknown. Aim: We studied the DNA damage checkpoint response of human cells to aflatoxin B1 (AFB1). Methods and results: The treatment of HepG2 hepatoma cells with mutation-inducing doses (3-5μmol/l) of AFB1 induced DNA adducts, 8-hydroxyguanine lesions and DNA strand breaks that lasted several days. Persistent phospho-H2AX and 53BP1 foci were also detected, but cell growth was not affected. AFB1-exposed HepG2 cells formed phospho-H2AX and 53BP1 foci, but failed to phosphorylate both Chk1 and Chk2. Huh7 hepatoma and HCT116 colorectal cancer cell lines also exhibited a similarly incomplete checkpoint response. p53 phosphorylation also failed, and AFB1-exposed cells did not show p53-dependent G1 arrest or a sustained G2/M arrest. These observations contrasted sharply with the fully functional DNA damage response of cells to Adriamycin. Cotreatment of cells with AFB1 did not inhibit p53 and p21Cip1 accumulation induced by Adriamycin. Thus, the deficient checkpoint response to AFB1 was not due to an inhibitory effect, but could be explained by an inefficient activation. Conclusion: Genotoxic doses of AFB1 induce an incomplete and inefficient checkpoint response in human cells. This defective response may contribute to the mutagenic and carcinogenic potencies of aflatoxins. © 2011 John Wiley & Sons A/S

Genome-Wide Transcriptional Reorganization Associated with Senescence-to-Immortality Switch during Human Hepatocellular Carcinogenesis

Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become "immortal") by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC) development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15-gene hepatocellular immortality signature test that discriminated HCC from cirrhosis with high accuracy. Our findings demonstrate that senescence bypass plays a central role in hepatocellular carcinogenesis engendering systematic changes in the transcription of genes regulating DNA repair, proliferation, differentiation and metabolism. © 2013 Yildiz et al