Toshinari Minamoto

Jun NH2-Terminal Kinase Phosphorylation of p53 on Thr-81 Is Important for p53 Stabilization and Transcriptional Activities in Response to Stress

ABSTRACT The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.

Collagens in human atherosclerosis. Immunohistochemical analysis using collagen type-specific antibodies.

This study represents a systematic analysis of the distribution of collagen types in human atherosclerotic lesions. Formalin-fixed, paraffin-embedded aortic tissues of 40 lesions from 16 different individuals ranging in age from 1 month to 84 years were examined immunohistochemically using antibodies to type I, III, IV, V, and VI collagens. Preembedding immunoelectron microscopy was used to simultaneously localize type V and VI collagens within the lesions. Localization of type III collagen was very similar to that of type I, and type VI collagen appeared together with these two types of collagen in the thickened intimas of all stages of the lesion. Type V collagen was not detected in either fatty streaks or the mild intimal thickening of the aortas of children. With advancing age and lesion progression, the immunoreactivity with anti-type V collagen antibody became more intense. Type IV collagen was detected in the basement membrane region of intimal cells. In advanced lesions thick deposits of type IV collagen were found around the elongated smooth muscle cells. Using immunoelectron microscopy, type V collagen was found to be localized to cross-banded collagen fibers, and type VI collagen was found to be localized to beaded filaments present throughout the interstitium of the thickened intima. These findings suggest that collagens preserve the pathophysiological and functional integrity of the vascular wall by providing mechanical support as well as assuring the proper interaction of cells during the formation of atherosclerotic lesions.

CRD-BP mediates stabilization of βTrCP1 and c- myc mRNA in response to β-catenin signalling

Although constitutive activation of β-catenin/Tcf signalling is implicated in the development of human cancers, the mechanisms by which the β-catenin/Tcf pathway promotes tumorigenesis are incompletely understood. Messenger RNA turnover has a major function in regulating gene expression and is responsive to developmental and environmental signals. mRNA decay rates are dictated by cis-acting elements within the mRNA and by trans-acting factors, such as RNA-binding proteins (reviewed in refs 2, 3). Here we show that β-catenin stabilizes the mRNA encoding the F-box protein βTrCP1, and identify the RNA-binding protein CRD-BP (coding region determinant-binding protein) as a previously unknown target of β-catenin/Tcf transcription factor. CRD-BP binds to the coding region of βTrCP1 mRNA. Overexpression of CRD-BP stabilizes βTrCP1 mRNA and elevates βTrCP1 levels (both in cells and in vivo), resulting in the activation of the Skp1-Cullin1-F-box protein (SCF)βTrCP E3 ubiquitin ligase and in accelerated turnover of its substrates including IκB and β-catenin. CRD-BP is essential for the induction of both βTrCP1 and c-Myc by β-catenin signalling in colorectal cancer cells. High levels of CRD-BP that are found in primary human colorectal tumours exhibiting active β-catenin/Tcf signalling implicates CRD-BP induction in the upregulation of βTrCP1, in the activation of dimeric transcription factor NF-κB and in the suppression of apoptosis in these cancers.

Wnt/β-Catenin Signaling Induces the Expression and Activity of βTrCP Ubiquitin Ligase Receptor

Abstract β-transducing repeat-containing protein (βTrCP) targets the ubiquitination and subsequent degradation of both β-catenin and IκB, thereby playing an important role in β-catenin/Tcf and NF-κB-dependent signaling. Here evidence is presented that β-catenin/Tcf signaling elevates the expression of βTrCP mRNA and protein in a Tcf-dependent manner, which does not require βTrCP transcription. Induction of βTrCP expression by the β-catenin/Tcf pathway results in an accelerated degradation of the wild-type β-catenin, suggesting that the negative feedback loop regulation may control the β-catenin/Tcf pathway. This signaling also upregulated NF-κB transactivation without affecting the activity of IκB kinase, thereby establishing that the maintenance of the βTrCP level is important for coordination between β-catenin/Tcf and NF-κB signaling.

Frequent and characteristic K-ras activation and absence of p53 protein accumulation in aberrant crypt foci of the colon

BACKGROUND/AIMS The relationship of aberrant crypt foci (ACF) to colorectal carcinogenesis is still controversial. Histological examination and analyses of K-ras mutations and p53 gene expression were performed to characterize ACF. METHODS ACF were identified microscopically in grossly normal colorectal mucosa. The ACF were separated into two pieces, one for histological and immunohistochemical examinations and the other for molecular analysis. K-ras mutations in codons 12 and 13 were analyzed by polymerase chain reaction amplification, followed by restriction fragment length polymorphism and sequencing analyses. Intranuclear p53 protein was immunostained by the avidin-biotin complex method. RESULTS Histologically, elongation and apical branching of the crypts in ACF were striking. K-ras mutations were detected in 58% of ACF (33 of 57; 46% [26 of 57] in codon 12, and 12% [7 of 57] in codon 13) and in 44% of adenocarcinomas (11 of 25; all in codon 12). In ACF, GAT mutations (12 of 26) were as frequent as GTT mutations (11 of 26) in codon 12, although GTT mutations in codon 12 were predominant in adenocarcinomas (10 of 11). No accumulation of p53 protein was detected in any ACF, although it was detected in 52% (13 of 25) of the colorectal carcinomas. CONCLUSIONS ACF do not seem histologically to be neoplasms, although genetically they are monoclonal lesions. K-ras mutation is critical in the formation of ACF, but p53 alteration could play a causal role in tumor progression.

Superficial-type adenomas and adenocarcinomas of the colon and rectum: A comparative morphological study

BACKGROUND/AIMS It has been uncertain whether colorectal carcinomas preferentially arise on preexisting adenomas or de novo. However, from a morphological viewpoint, it seems unlikely that pedunculated or exophytic malignant polyps progress to the deeply ulcerated advanced carcinomas usually found clinically. METHODS The morphological features of 26 nonpolypoid, superficial-type colorectal tumors (17 adenomas and 9 adenocarcinomas) were compared to clarify the developmental route of colorectal carcinomas. RESULTS The adenomas and adenocarcinomas were very similar in size and gross appearance; however, examination of the surface appearances of unsectioned tumors by dissecting microscopy was helpful for distinguishing the two. Histologically, no adenomatous tissue was found in any case of superficial-type adenocarcinoma. Five of the nine adenocarcinomas, even including those of small size, invaded the submucosal layer, and two showed lymph node metastasis. CONCLUSIONS These findings suggest that superficial-type adenocarcinomas show rapid growth and aggressive behavior. We suggest that this type of carcinoma may not progress by the adenoma-to-carcinoma pathway but that it may arise from a very small superficial-type adenoma.

Helicobacter pylori infection promotes methylation and silencing of trefoil factor 2, leading to gastric tumor development in mice and humans.

BACKGROUND & AIMS Trefoil factors (TFFs) regulate mucosal repair and suppress tumor formation in the stomach. Tff1 deficiency results in gastric cancer, whereas Tff2 deficiency increases gastric inflammation. TFF2 expression is frequently lost in gastric neoplasms, but the nature of the silencing mechanism and associated impact on tumorigenesis have not been determined. METHODS We investigated the epigenetic silencing of TFF2 in gastric biopsy specimens from individuals with Helicobacter pylori-positive gastritis, intestinal metaplasia, gastric cancer, and disease-free controls. TFF2 function and methylation were manipulated in gastric cancer cell lines. The effects of Tff2 deficiency on tumor growth were investigated in the gp130(F/F) mouse model of gastric cancer. RESULTS In human tissue samples, DNA methylation at the TFF2 promoter began at the time of H pylori infection and increased throughout gastric tumor progression. TFF2 methylation levels were inversely correlated with TFF2 messenger RNA levels and could be used to discriminate between disease-free controls, H pylori-infected, and tumor tissues. Genome demethylation restored TFF2 expression in gastric cancer cell lines, so TFF2 silencing requires methylation. In Tff2-deficient gp130(F/F)/Tff2(-/-) mice, proliferation of mucosal cells and release of T helper cell type-1 (Th-1) 1 cytokines increased, whereas expression of gastric tumor suppressor genes and Th-2 cytokines were reduced, compared with gp130(F/F)controls. The fundus of gp130(F/F)/Tff2(-/-) mice displayed glandular atrophy and metaplasia, indicating accelerated preneoplasia. Experimental H pylori infection in wild-type mice reduced antral expression of Tff2 by increased promoter methylation. CONCLUSIONS TFF2 negatively regulates preneoplastic progression and subsequent tumor development in the stomach, a role that is subverted by promoter methylation during H pylori infection.

Long Interspersed Nuclear Element 1 Hypomethylation Is a Marker of Poor Prognosis in Stage IA Non–Small Cell Lung Cancer

Purpose: Global hypomethylation and the hypermethylation of gene promoter regions are common events in tumor DNA. The aim of this study was to evaluate the prognostic significance of both global hypomethylation and gene promoter hypermethylation in DNA from non–small cell lung cancer (NSCLC). Experimental Design: Genomic DNA was obtained from the tumor tissue of 379 NSCLC patients who underwent surgery. Methylation levels were measured by real-time PCR following bisulfite modification of DNA and were correlated with clinicopathologic parameters and patient prognosis. Methylation of long interspersed nuclear element 1 (LINE-1) was used as a surrogate marker for global methylation. Hypermethylation of the APC, CDH13, and RASSF1 promoter regions was also evaluated. Results: Tumor tissue showed significantly higher CDH13 and RASSF1 methylation levels compared with normal lung tissue, but lower LINE-1 methylation levels. APC, RASSF1, and LINE-1 methylation levels were significant prognostic factors in univariate analysis of an initial cohort of 234 cases. APC and LINE-1 methylation remained significant prognostic factors in multivariate analysis that included age, gender, smoking history, histologic type, and pathologic stage. LINE-1 methylation showed marginally significant prognostic value in stage IA and IB disease. Expansion of the study cohort to 364 cases revealed that LINE-1 methylation had significant prognostic value for stage IA NSCLC patients in multivariate analysis. Conclusions: LINE-1 hypomethylation was an independent marker of poor prognosis in stage IA NSCLC. Validation of this finding in additional tumor cohorts could have clinical relevance for the management of early-stage NSCLC. Clin Cancer Res; 16(8); 2418–26. ©2010 AACR.

Inhibition of GSK‐3β activity attenuates proliferation of human colon cancer cells in rodents

The authors’ recent discovery that glycogen synthase kinase‐3β (GSK‐3β) participates in colon cancer cells’ survival and proliferation prompted us to investigate whether GSK‐3β inhibition alters proliferation of colon cancer cells in vivo. Groups of four or five athymic mice (Balb/c, nu/nu) with subcutaneous xenografts of SW480 human colon cancer cells were treated with dimethyl sulfoxide (DMSO) or different doses (1, 2 and 5 mg/kg body weight) of either small‐molecule GSK‐3β inhibitor (SB‐216763 and AR‐A014418) by intraperitoneal injection three times per week for 5 weeks. Compared with DMSO (a diluent of the GSK‐3β inhibitors) as a control, either GSK‐3β inhibitor significantly inhibited proliferation of cancer cell xenografts in the rodents in a dose‐dependent manner. Histochemical and immunohistochemical analysis of tumor xenografts demonstrated a significant, dose‐dependent decrease in fractions of proliferating cells and an increase in the incidence of apoptosis of cancer cells in mice treated with either GSK‐3β inhibitor. No adverse events or effects were observed in the rodents during the course of treatment, except for rare lethal accidents due to intraperitoneal injection. Morphological examination showed no apparent pathologic changes in major organs including the lungs, liver, pancreas, kidneys, spleen and large bowel of rodents treated with DMSO and the GSK‐3β inhibitors. The results indicate that the GSK‐3β inhibitors would be a novel class of therapeutic agent for colon cancer. (Cancer Sci 2007; 98: 1388–1393)

Inhibition of gastric carcinogenesis by the hormone gastrin is mediated by suppression of TFF1 epigenetic silencing.

BACKGROUND & AIMS Epigenetic alterations have been correlated with field cancerization in human patients, but evidence from experimental models that specific epigenetic changes can initiate cancer has been lacking. Although hormones have been associated with cancer risk, the mechanisms have not been determined. The peptide hormone gastrin exerts a suppressive effect on antral gastric carcinogenesis. METHODS N-methyl-N-nitrosourea (MNU)-dependent gastric cancer was investigated in hypergastrinemic (INS-GAS), gastrin-deficient (GAS(-/-)), Tff1-deficient (Tff1(+/-)), and wild-type (WT) mice. Epigenetic alterations of the trefoil factor 1 (TFF1) tumor suppressor gene were evaluated in vitro and in vivo. RESULTS Human intestinal-type gastric cancers in the antrum exhibited progressive TFF1 repression and promoter hypermethylation. Mice treated with MNU exhibited a field defect characterized by widespread Tff1 repression associated with histone H3 lysine 9 methylation and H3 deacetylation at the Tff1 promoter in epithelial cells. In MNU-induced advanced cancers, DNA methylation at the Tff1 promoter was observed. Tumor induction and Tff1 repression were increased in MNU-treated mice by Helicobacter infection. Hypergastrinemia suppressed MNU-dependent tumor initiation and progression in a manner that correlated with gene silencing and epigenetic alterations of Tff1. In contrast, homozygous gastrin-deficient and heterozygous Tff1-deficient mice showed enhanced MNU-dependent field defects and cancer initiation compared with WT mice. In gastric cancer cells, gastrin stimulation partially reversed the epigenetic silencing in the TFF1 promoter. CONCLUSIONS Initiation of antral gastric cancer is associated with progressive epigenetic silencing of TFF1, which can be suppressed by the hormone gastrin.