Anna Vilgelm

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

BACKGROUND & AIMSnInfection 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.nnnMETHODSnMongolian 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.nnnRESULTSnWe 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.nnnCONCLUSIONSnH 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.

Therapeutic prospects for p73 and p63: Rising from the shadow of p53

The p53 protein family consists of three transcription factors: p53, p63, and p73. These proteins share significant structural and functional similarities and each has unique biological functions as well. Although the role of p53 in cellular stress is extensively studied, many questions remain about p63 and p73. In this review we summarize current data on functional interactions within the p53 family, their regulation and roles in response to genotoxic stress. We also discuss the significance of p73 and p63 for cancer therapy and outline novel approaches in development of therapeutic drugs that specifically target the p53 family.

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

BACKGROUND & AIMSnAlthough 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.nnnMETHODSnGastric 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.nnnRESULTSnInteraction 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.nnnCONCLUSIONnOur 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.

Targeting aurora kinases limits tumour growth through DNA damage-mediated senescence and blockade of NF-κB impairs this drug-induced senescence

Oncogene‐induced senescence can provide a protective mechanism against tumour progression. However, production of cytokines and growth factors by senescent cells may contribute to tumour development. Thus, it is unclear whether induction of senescence represents a viable therapeutic approach. Here, using a mouse model with orthotopic implantation of metastatic melanoma tumours taken from 19 patients, we observed that targeting aurora kinases with MLN8054/MLN8237 impaired mitosis, induced senescence and markedly blocked proliferation in patient tumour implants. Importantly, when a subset of tumour‐bearing mice were monitored for tumour progression after pausing MLN8054 treatment, 50% of the tumours did not progress over a 12‐month period. Mechanistic analyses revealed that inhibition of aurora kinases induced polyploidy and the ATM/Chk2 DNA damage response, which mediated senescence and a NF‐κB‐related, senescence‐associated secretory phenotype (SASP). Blockade of IKKβ/NF‐κB led to reversal of MLN8237‐induced senescence and SASP. Results demonstrate that removal of senescent tumour cells by infiltrating myeloid cells is crucial for inhibition of tumour re‐growth. Altogether, these data demonstrate that induction of senescence, coupled with immune surveillance, can limit melanoma growth.

RAF265 inhibits the growth of advanced human melanoma tumors.

Purpose: The purpose of this preclinical study was to determine the effectiveness of RAF265, a multikinase inhibitor, for treatment of human metastatic melanoma and to characterize traits associated with drug response. Experimental Design: Advanced metastatic melanoma tumors from 34 patients were orthotopically implanted to nude mice. Tumors that grew in mice (17 of 34) were evaluated for response to RAF265 (40 mg/kg, every day) over 30 days. The relation between patient characteristics, gene mutation profile, global gene expression profile, and RAF265 effects on tumor growth, mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation, proliferation, and apoptosis markers was evaluated. Results: Nine of the 17 tumors that successfully implanted (53%) were mutant BRAF (BRAFV600E/K), whereas eight of 17 (47%) tumors were BRAF wild type (BRAFWT). Tumor implants from 7 of 17 patients (41%) responded to RAF265 treatment with more than 50% reduction in tumor growth. Five of the 7 (71%) responders were BRAFWT, of which 1 carried c-KITL576P and another N-RASQ61R mutation, while only 2 (29%) of the responding tumors were BRAFV600E/K. Gene expression microarray data from nonimplanted tumors revealed that responders exhibited enriched expression of genes involved in cell growth, proliferation, development, cell signaling, gene expression, and cancer pathways. Although response to RAF265 did not correlate with pERK1/2 reduction, RAF265 responders did exhibit reduced pMEK1, reduced proliferation based upon reduced Ki-67, cyclin D1 and polo-like kinase1 levels, and induction of the apoptosis mediator BCL2-like 11. Conclusions: Orthotopic implants of patient tumors in mice may predict prognosis and treatment response for melanoma patients. A subpopulation of human melanoma tumors responds to RAF265 and can be characterized by gene mutation and gene expression profiles. Clin Cancer Res; 18(8); 2184–98. ©2012 AACR.

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.

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.

The gastrin gene promoter is regulated by p73 isoforms in tumor cells

p73, a new p53 family member, is a transcription factor that is increasingly recognized in cancer research as an important player in tumorigenesis as well as in chemotherapeutic drug sensitivity. Despite the substantial structural and functional similarities to p53, accumulating evidence suggests that p53 and p73 may differently regulate their transcriptional targets. In this study, we have investigated the role of p73 in regulation of the gastrin gene promoter. Gastrin is a peptide hormone and an important factor in determining the progression of a number of human malignancies. Our results show that p73 can bind to the gastrin promoter. This leads to transcriptional upregulation of gastrin mRNA. We also found that the levels of gastrin and p73 transcripts correlate in primary gastric tumors. Taken together, our results demonstrate a novel mechanism for regulation of gastrin gene transcription and support a concept that p53 and p73 may have different biological roles in tumors.

Coordinated interaction of multifunctional members of the p53 family determines many key processes in multicellular organisms

For the first time, p53 was found in complex with the viral large T-antigen in cells transformed with the small DNA virus SV40. p53 cDNA was cloned in the early 1980s, and the full-length p53 gene was cloned soon afterwards. The p53 family is comprised of three genes—TP53, TP63, and TP73—each of which is expressed as a set of structurally and functionally different isoforms. All of them intensely interact with each other, forming a united functional network of proteins. The review discusses the evolution of the p53 family and the significance of all its members in embryo development, reproduction, regeneration, regulation of aging and lifespan, and defense against cancer. Special attention is paid to the role of poorly studied members of the p53 family, p63 and p73, in carcinogenesis and tumor progression. Different isoforms of these proteins might exert opposite effects on these processes.