Jinxiong Wei

Regulation of p53 Tumor Suppressor by Helicobacter pylori in Gastric Epithelial Cells

BACKGROUND & AIMS Infection with the gastric mucosal pathogen Helicobacter pylori is the strongest identified risk factor for distal gastric cancer. These bacteria colonize a significant part of the worlds population. We investigated the molecular mechanisms of p53 regulation in H pylori-infected cells. METHODS Mongolian gerbils were challenged with H pylori and their gastric tissues were analyzed by immunohistochemistry and immunoblotting with p53 antibodies. Gastric epithelial cells were co-cultured with H pylori and the regulation of p53 was assessed by real-time polymerase chain reaction, immunoblotting, immunofluorescence, and cell survival assays. Short hairpin RNA and dominant-negative mutants were used to inhibit activities of Human Double Minute 2 (HDM2) and AKT1 proteins. RESULTS We found that in addition to previously reported up-regulation of p53, H pylori can also negatively regulate p53 by increasing ubiquitination and proteasomal degradation via activation of the serine/threonine kinase AKT1, which phosphorylates and activates the ubiquitin ligase HDM2. These effects were mediated by the bacterial virulence factor CagA; ectopic expression of CagA in gastric epithelial cells increased phosphorylation of HDM2 along with the ubiquitination and proteasomal degradation of p53. The decrease in p53 levels increased survival of gastric epithelial cells that had sustained DNA damage. CONCLUSIONS H pylori is able to inhibit the tumor suppressor p53. H pylori activates AKT1, resulting in phosphorylation and activation of HDM2 and subsequent degradation of p53 in gastric epithelial cells. H pylori-induced dysregulation of p53 is a potential mechanism by which the microorganism increases the risk of gastric cancer in infected individuals.

Interaction of Helicobacter pylori with gastric epithelial cells is mediated by the p53 protein family

BACKGROUND & AIMS Although the p53 tumor suppressor has been extensively studied, many critical questions remain unanswered about the biological functions of p53 homologs, p73 and p63. Accumulating evidence suggests that both p73 and p63 play important roles in regulation of apoptosis, cell differentiation, and therapeutic drug sensitivity. METHODS Gastric epithelial cells were cocultured with Helicobacter pylori, and the roles of p63 and p73 proteins were assessed by luciferase reporter, real-time polymerase chain reaction, immunoblotting, and cell survival assays. Short hairpin RNA and dominant-negative mutants were used to inhibit activity of p73 and p63 isoforms. Human and murine gastric tissues were analyzed by immunohistochemistry with p73 and p63 antibodies and modified Steiners silver method. RESULTS Interaction of H pylori with gastric epithelial cells leads to robust up-regulation of p73 protein in vitro and in vivo in human gastritis specimens and H pylori-infected mice. The p73 increase resulted in up-regulation of pro-apoptotic genes, NOXA, PUMA, and FAS receptor in gastric epithelial cells. Down-regulation of p73 activity suppressed cell death and Fas receptor induced by H pylori. Bacterial virulence factors within the cag pathogenicity island, c-Abl tyrosine kinase, and interaction with p63 isoforms control the activity of p73. CONCLUSION Our findings implicate p73 in H pylori-induced apoptosis and more generally suggest that the p53 family may play a role in the epithelial cell response to H pylori infection.

ΔNp73α regulates MDR1 expression by inhibiting p53 function

The p73 protein is a transcription factor and member of the p53 protein family that expresses as a complex variety of isoforms. ΔNp73α is an N-terminally truncated isoform of p73. We found that ΔNp73 protein is upregulated in human gastric carcinoma suggesting that ΔNp73 may play an oncogenic role in these tumors. Although it has been shown that ΔNp73α inhibits apoptosis and counteracts the effect of chemotherapeutic drugs, the underlying mechanism by which this p73 isoform contributes to chemotherapeutic drug response remains to be explored. We found that ΔNp73α upregulates MDR1 mRNA and p-glycoprotein (p-gp), which is involved in chemotherapeutic drug transport. This p-gp upregulation was accompanied by increased p-gp functional activity in gastric cancer cells. Our data suggest that upregulation of MDR1 by ΔNp73α is mediated by interaction with p53 at the MDR1 promoter.

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.—Zaika, E., Wei, J., Yin, D., Andl, C., Moll, U., El‐Rifai, W., Zaika, A. I. p73 protein regulates DNA damage repair. FASEB J. 25, 4406–4414 (2011). www.fasebj.org

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. Mol Cancer Ther; 9(3); 693–705

Characterization of ΔNp73 expression and regulation in gastric and esophageal tumors

p73 is a member of the p53 protein family. Although the tumor suppressor function of p53 is clearly defined, the role of p73 in tumorigenesis is still a matter of debate. A complex pattern of expression of p73 isoforms makes it difficult to unambiguously interpret the experimental results. Previously, we along with others have found that the N-terminally truncated isoform of p73, ΔNp73, has potent anti-apoptotic and oncogenic properties in vitro and in vivo. In this study, we analyzed, for the first time, the regulation of ΔNp73 in a large number of gastric, gastroesophageal junction and esophageal tumors. We found that expression of ΔNp73 mRNA and protein is increased in these neoplasms. Furthermore, the upregulation of the ΔNp73 protein is significantly associated with poor patient survival. Oncogenic properties of ΔNp73 were further confirmed by finding that ΔNp73 facilitates anchorage-independent growth of gastric epithelial cells in soft agar. As little is currently known about the regulation of ΔNp73 transcription, we investigated the alternative p73 gene promoter that mediates the ΔNp73 expression. Analyzing the ΔNp73 promoter in silico as well as by using chromatin immunoprecipitation, site-directed mutagenesis and deletion analyses, we identified the evolutionary conserved region within the ΔNp73 promoter that contains binding sites for HIC1 (hypermethylated in cancer) protein. We found that HIC1 negatively regulates ΔNp73 transcription in mucosal epithelial cells. This leads to a decrease in ΔNp73 protein levels and may normally control the oncogenic potential of the ΔNp73 isoform.

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.

Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

Gastroesophageal reflux disease (GERD) is the main etiological factor behind the recent rapid increase in the incidence of esophageal adenocarcinoma. During reflux, esophageal cells are exposed to bile at low pH resulting in cellular damage and inflammation, which are known to facilitate cancer development. In this study, we investigated the regulation of p73 isoform, ΔNp73α, in the reflux condition. Previous studies have reported that ΔNp73 exhibits anti-apoptotic and oncogenic properties through inhibition of p53 and p73 proteins. We found that direct exposure of esophageal cells to bile acids in an acidic environment alters the phosphorylation of ΔNp73, its subcellular localization and increases ΔNp73 protein levels. Upregulation of ΔNp73 was also observed in esophageal tissues collected from patients with GERD and Barrett’s metaplasia, a precancerous lesion in the esophagus associated with gastric reflux. c-Abl, p38 MAPK, and IKK protein kinases were identified to interact in the regulation of ΔNp73. Their inhibition with chemotherapeutic agents and siRNA suppresses ΔNp73. We also found that pro-inflammatory cytokines, IL-1β and TNFα, are potent inducers of ΔNp73α, which further enhance the bile acids/acid effect. Combined, our studies provide evidence that gastroesophageal reflux alters the regulation of oncogenic ΔNp73 isoform that may facilitate tumorigenic transformation of esophageal metaplastic epithelium.

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.