Protein adduction causes non-mutational inhibition of p53 tumor suppressor.

p53 is a key tumor suppressor that is frequently mutated in human tumors. In this study, we investigated how p53 is regulated in precancerous lesions prior to mutations in the p53 gene. Analyzing esophageal cells in conditions of genotoxic stress that promotes development of esophageal adenocarcinoma, we find that p53 protein is adducted with reactive isolevuglandins (isoLGs), products of lipid peroxidation. Modification of p53 protein with isoLGs diminishes its acetylation and binding to the promoters of p53 target genes causing modulation of p53-dependent transcription. It also leads to accumulation of adducted p53 protein in intracellular amyloid-like aggregates that can be inhibited by isoLG scavenger 2-HOBA in vitro and in vivo. Taken together, our studies reveal a posttranslational modification of p53 protein that causes molecular aggregation of p53 protein and its non-mutational inactivation in conditions of DNA damage that may play an important role in human tumorigenesis.

Helicobacter pylori pathogen inhibits cellular responses to oncogenic stress and apoptosis.

Helicobacter pylori (H. pylori) is a common gastric pathogen that infects approximately half of the world's population. Infection with H. pylori can lead to diverse pathological conditions, including chronic gastritis, peptic ulcer disease, and cancer. The latter is the most severe consequence of H. pylori infection. According to epidemiological studies, gastric infection with H. pylori is the strongest known risk factor for non-cardia gastric cancer (GC), which remains one of the leading causes of cancer-related deaths worldwide. However, it still remains to be poorly understood how host-microbe interactions result in cancer development in the human stomach. Here we focus on the H. pylori bacterial factors that affect the host ubiquitin proteasome system. We investigated E3 ubiquitin ligases SIVA1 and ULF that regulate p14ARF (p19ARF in mice) tumor suppressor. ARF plays a key role in regulation of the oncogenic stress response and is frequently inhibited during GC progression. Expression of ARF, SIVA1 and ULF proteins were investigated in gastroids, H. pylori-infected mice and human gastric tissues. The role of the H. pylori type IV secretion system was assessed using various H. pylori isogenic mutants. Our studies demonstrated that H. pylori infection results in induction of ULF, decrease in SIVA1 protein levels, and subsequent ubiquitination and degradation of p14ARF tumor suppressor. Bacterial CagA protein was found to sequentially bind to SIVA1 and ULF proteins. This process is regulated by CagA protein phosphorylation at the EPIYA motifs. Downregulation of ARF protein leads to inhibition of cellular apoptosis and oncogenic stress response that may promote gastric carcinogenesis.

Role of Bacterial and Viral Pathogens in Gastric Carcinogenesis.

Gastric cancer (GC) is one of the deadliest malignancies worldwide. In contrast to many other tumor types, gastric carcinogenesis is tightly linked to infectious events. Infections with Helicobacter pylori (H. pylori) bacterium and Epstein-Barr virus (EBV) are the two most investigated risk factors for GC. These pathogens infect more than half of the world's population. Fortunately, only a small fraction of infected individuals develops GC, suggesting high complexity of tumorigenic processes in the human stomach. Recent studies suggest that the multifaceted interplay between microbial, environmental, and host genetic factors underlies gastric tumorigenesis. Many aspects of these interactions still remain unclear. In this review, we update on recent discoveries, focusing on the roles of various gastric pathogens and gastric microbiome in tumorigenesis.

Bacterial CagA protein compromises tumor suppressor mechanisms in gastric epithelial cells.

Approximately half of the world's population is infected with the stomach pathogen Helicobacter pylori. Infection with H. pylori is the main risk factor for distal gastric cancer. Bacterial virulence factors, such as the oncoprotein CagA, augment cancer risk. Yet despite high infection rates, only a fraction of H. pylori-infected individuals develop gastric cancer. This raises the question of defining the specific host and bacterial factors responsible for gastric tumorigenesis. To investigate the tumorigenic determinants, we analyzed gastric tissues from human subjects and animals infected with H. pylori bacteria harboring different CagA status. For laboratory studies, well-defined H. pylori strain B128 and its cancerogenic derivative strain 7.13, as well as various bacterial isogenic mutants were employed. We found that H. pylori compromises key tumor suppressor mechanisms: the host stress and apoptotic responses. Our studies showed that CagA induces phosphorylation of XIAP E3 ubiquitin ligase, which enhances ubiquitination and proteasomal degradation of the host proapoptotic factor Siva1. This process is mediated by the PI3K/Akt pathway. Inhibition of Siva1 by H. pylori increases survival of human cells with damaged DNA. It occurs in a strain-specific manner and is associated with the ability to induce gastric tumor.

From genetics to signaling pathways: molecular pathogenesis of esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) has one of the fastest rising incidence rates in the U.S. and many other Western countries. One of the unique risk factors for EAC is gastroesophageal reflux disease (GERD), a chronic digestive condition in which acidic contents from the stomach, frequently mixed with duodenal bile, enter the esophagus resulting in esophageal tissue injury. At the cellular level, progression to EAC is underlined by continuous DNA damage caused by reflux and chronic inflammatory factors that increase the mutation rate and promote genomic instability. Despite recent successes in cancer diagnostics and treatment, EAC remains a poorly treatable disease. Recent research has shed new light on molecular alterations underlying progression to EAC and revealed novel treatment options. This review focuses on the genetic and molecular studies of EAC. The molecular changes that occur during the transformation of normal Barrett's esophagus to esophageal adenocarcinoma are also discussed.

Helicobacter pylori pathogen regulates p14ARF tumor suppressor and autophagy in gastric epithelial cells.

Infection with Helicobacter pylori is one of the strongest risk factors for development of gastric cancer. Although these bacteria infect approximately half of the world's population, only a small fraction of infected individuals develops gastric malignancies. Interactions between host and bacterial virulence factors are complex and interrelated, making it difficult to elucidate specific processes associated with H. pylori-induced tumorigenesis. In this study, we found that H. pylori inhibits p14ARF tumor suppressor by inducing its degradation. This effect was found to be strain-specific. Downregulation of p14ARF induced by H. pylori leads to inhibition of autophagy in a p53-independent manner in infected cells. We identified TRIP12 protein as E3 ubiquitin ligase that is upregulated by H. pylori, inducing ubiquitination and subsequent degradation of p14ARF protein. Using isogenic H. pylori mutants, we found that induction of TRIP12 is mediated by bacterial virulence factor CagA. Increased expression of TRIP12 protein was found in infected gastric epithelial cells in vitro and human gastric mucosa of H. pylori-infected individuals. In conclusion, our data demonstrate a new mechanism of ARF inhibition that may affect host-bacteria interactions and facilitate tumorigenic transformation in the stomach.

Activation of NADPH oxidases leads to DNA damage in esophageal cells.

Gastroesophageal reflux disease (GERD) is the strongest known risk factor for esophageal adenocarcinoma. In the center of tumorigenic events caused by GERD is repeated damage of esophageal tissues by the refluxate. In this study, we focused on a genotoxic aspect of exposure of esophageal cells to acidic bile reflux (BA/A). Analyzing cells generated from patients with Barrett's esophagus and human esophageal specimens, we found that BA/A cause significant DNA damage that is mediated by reactive-oxygen species. ROS originate from mitochondria and NADPH oxidases. We specifically identified NOX1 and NOX2 enzymes to be responsible for ROS generation. Inhibition of NOX2 and NOX1 with siRNA or chemical inhibitors significantly suppresses ROS production and DNA damage induced by BA/A. Mechanistically, our data showed that exposure of esophageal cells to acidic bile salts induces phosphorylation of the p47phox subunit of NOX2 and its translocation to the cellular membrane. This process is mediated by protein kinase C, which is activated by BA/A. Taken together, our studies suggest that inhibition of ROS induced by reflux can be a useful strategy for preventing DNA damage and decreasing the risk of tumorigenic transformation caused by GERD.

Prevention of DNA damage in Barrett's esophageal cells exposed to acidic bile salts.

Esophageal adenocarcinoma (EA) is one of the fastest rising tumors in the USA. The major risk factor for EA is gastroesophageal reflux disease (GERD). During GERD, esophageal cells are exposed to refluxate which contains gastric acid frequently mixed with duodenal bile. This may lead to mucosal injury and Barrett's metaplasia (BE) that are important factors contributing to development of EA. In this study, we investigated DNA damage in BE cells exposed to acidic bile salts and explored for potential protective strategies. Exposure of BE cells to acidic bile salts led to significant DNA damage, which in turn, was due to generation of reactive oxygen species (ROS). We found that acidic bile salts induce a rapid increase in superoxide radicals and hydrogen peroxide, which were determined using electron paramagnetic resonance spectroscopy and Amplex Red assay. Analyzing a panel of natural antioxidants, we identified apocynin to be the most effective in protecting esophageal cells from DNA damage induced by acidic bile salts. Mechanistic analyses showed that apocynin inhibited ROS generation and increases the DNA repair capacity of BE cells. We identified BRCA1 and p73 proteins as apocynin targets. Downregulation of p73 inhibited the protective effect of apocynin. Taken together, our results suggest potential application of natural compounds such as apocynin for prevention of reflux-induced DNA damage and GERD-associated tumorigenesis.

Activin a signaling regulates cell invasion and proliferation in esophageal adenocarcinoma.

TGFß signaling has been implicated in the metaplasia from squamous epithelia to Barrett's esophagus and, ultimately, esophageal adenocarcinoma. The role of the family member Activin A in Barrett's tumorigenesis is less well established. As tumorigenesis is influenced by factors in the tumor microenvironment, such as fibroblasts and the extracellular matrix, we aimed to determine if epithelial cell-derived Activin affects initiation and progression differently than Activin signaling stimulation from a mimicked stromal source. Using Barrett's esophagus cells, CPB, and the esophageal adenocarcinoma cell lines OE33 and FLO-1, we showed that Activin reduces colony formation only in CPB cells. Epithelial cell overexpression of Activin increased cell migration and invasion in Boyden chamber assays in CPB and FLO-1 cells, which exhibited mesenchymal features such as the expression of the CD44 standard form, vimentin, and MT1-MMP. When grown in organotypic reconstructs, OE33 cells expressed E-cadherin and Keratin 8. As mesenchymal characteristics have been associated with the acquisition of stem cell-like features, we analyzed the expression and localization of SOX9, showing nuclear localization of SOX9 in esophageal CPB and FLO-1 cells.In conclusion, we show a role for autocrine Activin signaling in the regulation of colony formation, cell migration and invasion in Barrett's tumorigenesis.

Microbial Regulation of p53 Tumor Suppressor.

p53 tumor suppressor has been identified as a protein interacting with the large T antigen produced by simian vacuolating virus 40 (SV40). Subsequent research on p53 inhibition by SV40 and other tumor viruses has not only helped to gain a better understanding of viral biology, but also shaped our knowledge of human tumorigenesis. Recent studies have found, however, that inhibition of p53 is not strictly in the realm of viruses. Some bacterial pathogens also actively inhibit p53 protein and induce its degradation, resulting in alteration of cellular stress responses. This phenomenon was initially characterized in gastric epithelial cells infected with Helicobacter pylori, a bacterial pathogen that commonly infects the human stomach and is strongly linked to gastric cancer. Besides H. pylori, a number of other bacterial species were recently discovered to inhibit p53. These findings provide novel insights into host-bacteria interactions and tumorigenesis associated with bacterial infections.

Helicobacter pylori bacteria alter the p53 stress response via ERK-HDM2 pathway.

H. pylori infection is the strongest known risk factor for gastric cancer. Inhibition of host tumor suppressor mechanisms by the bacteria underlies the development of this disease. Among the tumor suppressors affected by H. pylori are p53 and E-cadherin, which inhibition has been shown to increase the risk of gastric cancer. In this report, we investigated the interaction between E-cadherin and p53 in H. pylori-infected cells. We found that downregulation of E-cadherin leads to cellular stress and activation of p53. In the setting of H. pylori infection, this mechanism, however, is disrupted. We found that although co-culture of gastric epithelial cells with H. pylori led to downregulation of E-cadherin and cellular stress, it resulted in inhibition of p53, which is mediated by intracellular Erk kinases and HDM2 protein induced by H. pylori. Experimental inhibition of HDM2/p53 interactions restored p53 activity, and decreased survival of infected cells. Collectively, our results revealed that regulation of p53 and E-cadherin is tightly linked through the p53 stress response mechanism that is inhibited by H. pylori via activation of Erk1/2-HDM2-p53 pathway leading to survival of damaged cells. This might be advantageous to the bacteria but may increase the cancer risk.

Bacterial CagA protein induces degradation of p53 protein in a p14ARF-dependent manner.

OBJECTIVE: Infection with Helicobacter pylori is the strongest known risk factor for adenocarcinoma of the stomach. Tumorigenic transformation of gastric epithelium induced by H. pylori is a highly complex process driven by an active interplay between bacterial virulence and host factors, many aspects of which remain obscure. In this work, we investigated the degradation of p53 tumour suppressor induced by H. pylori. DESIGN: Expression of p53 protein in gastric biopsies was assessed by immunohistochemistry. Gastric cells were co-cultured with H. pylori strains isolated from high-gastric risk and low-gastric risk areas and assessed for expression of p53, p14ARF and cytotoxin-associated gene A (CagA) by immunoblotting. siRNA was used to inhibit activities of ARF-BP1 and Human Double Minute 2 (HDM2) proteins. RESULTS: Our analysis demonstrated that H. pylori strains expressing high levels of CagA virulence factor and associated with a higher gastric cancer risk more strongly suppress p53 compared with low-risk strains in vivo and in vitro. We found that degradation of p53 induced by bacterial CagA protein is mediated by host HDM2 and ARF-BP1 E3 ubiquitin ligases, while the p14ARF protein counteracts H. pylori-induced signalling. CONCLUSIONS: Our results provide novel evidence that tumorigenicity associated with H. pylori infection is linked to inhibition of p53 protein by CagA. We propose a model in which CagA-induced degradation of p53 protein is determined by a relative level of p14ARF. In cells in which p14ARF levels were decreased due to hypermethylation or deletion of the p14ARF gene, H. pylori efficiently degraded p53, whereas p53 is protected in cells expressing high levels of p14ARF.

p73 protein regulates DNA damage repair.

Although the p53 tumor suppressor is relatively well characterized, much less is known about the functions of other members of the p53 family, p73 and p63. Here, we present evidence that in specific pathological conditions caused by exposure of normal cells to bile acids in acidic conditions, p73 protein plays the predominant role in the DNA damage response. These pathological conditions frequently occur during gastric reflux in the human esophagus and are associated with progression to esophageal adenocarcinoma. We found that despite strong DNA damage induced by bile acid exposure, only p73 (but not p53 and p63) is selectively activated in a c-Abl kinase-dependent manner. The activated p73 protein induces DNA damage repair. Using a human DNA repair PCR array, we identified multiple DNA repair genes affected by p73. Two glycosylases involved in base excision repair, SMUG1 and MUTYH, were characterized and found to be transcriptionally regulated by p73 in DNA damage conditions. Using a surgical procedure in mice, which recapitulates bile acid exposure, we found that p73 deficiency is associated with increased DNA damage. These findings were further investigated with organotypic and traditional cell cultures. Collectively our studies demonstrate that p73 plays an important role in the regulation of DNA damage repair.

Interactions of the p53 protein family in cellular stress response in gastrointestinal tumors.

p53, p63, and p73 are members of the p53 protein family involved in regulation of cell cycle, apoptosis, differentiation, and other critical cellular processes. Here, we investigated the contribution of the entire p53 family in chemotherapeutic drug response in gastrointestinal tumors. Real-time PCR and immunohistochemistry revealed complexity and variability of expression profiles of the p53 protein family. Using colon and esophageal cancer cells, we found that the integral transcription activity of the entire p53 family, as measured by the reporter analysis, associated with response to drug treatment in studied cells. We also found that p53 and p73, as well as p63 and p73, bind simultaneously to the promoters of p53 target genes. Taken together, our results support the view that the p53 protein family functions as an interacting network of proteins and show that cellular responses to chemotherapeutic drug treatment are determined by the total activity of the entire p53 family rather than p53 alone.

p63 and p73 expression in extrahepatic bile duct carcinoma and their clinical significance.

p53 plays a pivotal role in the prevention of human tumor formation. p73 and p63 are new members of the p53 tumor suppressor family, which are becoming increasingly recognized as important players in human tumorigenesis. However, the roles of these proteins are not well elucidated in extrahepatic bile duct (EBD) carcinoma. We examined expressions of the p63 and p73 genes and proteins in normal biliary epithelia, biliary dysplasias, and EBD carcinomas using immunohistochemistry and RT-PCR analysis. p63 and p73 proteins were overexpressed in 26.3 and 41.0% of EBD carcinomas, respectively. p63 protein expression was more frequent in tumors with vascular invasion (P = 0.002) and distal location (P = 0.04), while p73 expression was more common in cancers with deeper tumor invasion (P = 0.04). Patients with tumors co-expressing both p63 and p73 were found to have a significantly worse overall survival rate compared to those with either p63 or p73 expression (P < 0.05) as determined in univariate and multivariate analyses. Our results strongly imply that the p53 family members have different functions in EBD carcinomas. Our data also indicate that interactions between p63 and p73 play an important role in tumorigenesis of EBD carcinoma.

p73 isoforms can induce T-cell factor-dependent transcription in gastrointestinal cells.

A new p53 family member, p73, and its isoform DeltaNp73 are increasingly recognized in cancer research as important players in tumorigenesis, as well as in chemotherapeutic drug sensitivity. Despite substantial structural similarities to p53, accumulating evidence suggests that p53 and p73 may play different roles in human tumorigenesis. In this study, we have investigated the role of p73 and DeltaNp73 in upper gastrointestinal tumorigenesis. Our results indicate that p73 and DeltaNp73 are frequently overexpressed in >60% of primary adenocarcinomas of the stomach and esophagus. We have demonstrated that this overexpression can lead to the suppression of p73 transcriptional and apoptotic activity in gastrointestinal cells. Moreover, it induces beta-catenin up-regulation and T-cell factor/lymphocyte enhancement factor-dependent transcription. Wild-type p53, but not mutant p53, can inhibit this effect. Our results demonstrate a novel mechanism for activation of beta-catenin in gastrointestinal tumors and support the concept that overexpression of p73 isoforms can play an important role in tumorigenesis.