Trefoil factor 1 (TFF1) is a tumor suppressor gene that encodes a peptide belonging to the trefoil factor family of protease-resistant peptides. Although TFF1 expression is frequently lost in gastric carcinomas, the tumorigenic pathways this affects have not been determined. Here we show that Tff1-knockout mice exhibit age-dependent carcinogenic histological changes in the pyloric antrum of the gastric mucosa, progressing from gastritis to hyperplasia, low-grade dysplasia, high-grade dysplasia, and ultimately malignant adenocarcinoma. The histology and molecular signatures of gastric lesions in the Tff1-knockout mice were consistent with an inflammatory phenotype. In vivo, ex-vivo, and in vitro studies showed that TFF1 expression suppressed TNF-α-mediated NF-κB activation through the TNF receptor 1 (TNFR1)/IκB kinase (IKK) pathway. Consistent with these mouse data, human gastric tissue samples displayed a progressive decrease in TFF1 expression and an increase in NF-κB activation along the multi-step carcinogenesis cascade. Collectively, these results provide evidence that loss of TFF1 leads to activation of IKK complex-regulated NF-κB transcription factors and is an important event in shaping the NF-κB-mediated inflammatory response during the progression to gastric tumorigenesis.

Organic anion transporting polypeptide 1B3 (OATP1B3, SLCO1B3) is normally expressed in hepatocytes. In this study, we showed frequent overexpression of OATP1B3 in colorectal adenocarcinomas. Quantitative reverse transcription-PCR analysis of 17 colon tumors indicated tumoral overexpression of OATP1B3 by f100-fold, compared with 20 normal colon samples (P < 0.0001). Using immunohistochemistry on a tissue microarray containing 93 evaluable colon tumor specimens, we detected immunostaining of OATP1B3 in 75 colon adenocarcinomas (81%) and no immunostaining in normal samples. To determine the functional effects of OATP1B3 expression on drug-induced apoptosis, we used camptothecin and oxaliplatin on a panel of colorectal cancer cell lines stably overexpressing OATP1B3. The results indicated that OATP1B3 overexpression enhanced cell survival in RKO, and HCT116 p53+/+ cells that harbor wild-type p53 but not in Caco-2 and HCT116 p53À/À cells that lack p53, compared with the respective empty vector controls (P < 0.01). The terminal deoxynucleotidyl transferase-mediated nick-end labeling assay confirmed that HCT116 p53+/+ cells overexpressing OATP1B3 had significantly lower apoptotic levels compared with empty vector control (P < 0.001). The overexpression of OATP1B3 reduced the transcriptional activity of p53, with subsequent reductions in transcript and protein levels of its downstream transcription targets (P21WAF1 and PUMA). Overexpression of a point mutation (G583E) variant of OATP1B3 lacking transport activity did not confer an antiapoptotic effect or affect p53 transcriptional activity, suggesting that the antiapoptotic effect of OATP1B3 may be associated with its transport activity. Taken together, our results suggest that OATP1B3 overexpression in colorectal cancer cells may provide a survival advantage by altering p53-dependent pathways. [Cancer Res 2008;68(24):10315-23]

Aurora kinase A (AURKA) is located at 20q13, a region that is frequently amplified in gastric cancer. In this study, we have investigated the role of AURKA in regulating GSK-3β and β-catenin/TCF complex in gastric cancer cells. Our results demonstrate a significant increase in the phosphorylation of GSK-3β at Ser 9 following the over-expression of AURKA in AGS cells. The immunoprecipitation with antibodies specific for AURKA and GSK-3β indicated that the two proteins coexist in the same protein complex. The recombinant human AURKA protein phosphorylated the GSK-3β protein at Ser 9 in a concentration dependent manner, in vitro. The phosphorylation of β-catenin (Ser33/37/Thr41) by GSK-3β is known to target β-catenin towards degradation. In line with our findings, the increase in phospho-GSK-3β level was accompanied by a significant decrease in β-catenin phosphorylation (Ser33/37/Thr41) and accumulation of β-catenin protein. The knockdown of AURKA reversed the phosphorylation of GSK-3β and the β-catenin protein levels. The immunofluorescence analysis demonstrated co-localization of AURKA and GSK-3β proteins and a significant increase in the nuclear β-catenin levels in cells overexpressing AURKA. The β-catenin/TCF transcription activity was measured using the pTopFlash and its mutant pFopFlash luciferase reporter vectors. Indeed, AURKA over-expression led to a significant increase in the pTopFlash reporter activity, whereas kinase dead AURKA mutant (D274A) had no effect. Consistent with these findings, we detected a significant mRNA up-regulation of several direct targets of the β-catenin/TCF transcription complex (cyclin D1, c-MYC, c-MYC binding protein, CLDN1, FGF18, and VEGF), and a two-fold increase in the proliferation rate in AURKA over-expressing cells. We conclude that the AURKA/GSK-3β interaction plays an important role in regulating β-catenin, underscoring a novel oncogenic potential for AURKA in gastric tumorigenesis.

BACKGROUNDUpper gastrointestinal adenocarcinomas are a common cause of cancer‐related deaths. In this study, the authors investigated the prevalence and biological significance of Aurora Kinase A (AURKA) overexpression in upper gastrointestinal adenocarcinomas.METHODSQuantitative real‐time polymerase chain reaction (qRT‐PCR) and immunohistochemical staining on tumor tissue microarrays (TMA) were used to study the expression of AURKA in upper gastrointestinal adenocarcinomas. To investigate the biological and signaling impact of AURKA, the authors used multiple in vitro assays that included 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT), TUNEL (terminal deoxynucleotidyl transferase–mediated nick‐end labeling), cytochrome C release, flow cytometry, luciferase reporter, and Western blot analysis.RESULTSFrequent overexpression of AURKA transcript in upper gastrointestinal adenocarcinomas was detected compared with normal samples (47%; P = .001). The immunohistochemical analysis of 130 tumors demonstrated moderate‐to‐strong immunostaining of AURKA in >50% of upper gastrointestinal adenocarcinomas. By using camptothecin as a drug‐induced apoptosis in vitro model, the authors demonstrated that the expression of AURKA provided protection against apoptosis to gastrointestinal cancer cells (AGS and RKO) (P = .006) and RIE‐1 primary intestinal epithelial cells (P = .001). The AURKA overexpression mediated an increase in phosphorylation of AKTSer473 with an increase in HDM2 level. The shRNA‐knockdown of AKT in AURKA‐overexpressing cells reversed this effect and showed a significant increase in the p53 protein level, indicating a possible nexus of AURKA/AKT/p53. Indeed, overexpression of AURKA led to a remarkable reduction in the transcription activity of p53, with subsequent reductions in transcript and protein levels of its downstream proapoptotic transcription targets (p21, BAX, NOXA, and PUMA).CONCLUSIONSStudy results indicated that AURKA provides potent antiapoptotic properties to gastrointestinal cells by regulating levels of p53 through the AKT/HDM2 axis. Cancer 2008. ©2008 American Cancer Society.

We investigated the role of Aurora kinase A (AURKA) in regulating p73-dependent apoptosis using the p53-deficient cancer cell lines H1299, TE7, and HCT116p53 À/À . Overexpression of AURKA led to down-regulation of the TAp73-induced activation of the p53/p73-dependent luciferase reporter plasmid (pG13-luc). The reduction in the TAp73 transcription activity was confirmed by measuring the activity of luciferase reporters for p21/WAF1, and PUMA. The siRNA knockdown of endogenous AURKA reversed these effects and Western blot analysis showed a significant increase in the protein level of TAp73 and its downstream transcription targets, PUMA, NOXA, and p21/WAF1. The coexpression of AURKA together with TAp73 inhibited the activation of the pG13-luc, PUMA-luc, and p21/WAF1-luc reporter plasmids with reduction in the protein levels of TAp73 transcription targets. Treatment with AURKA-selective small molecule inhibitor MLN8054 led to a significant increase in the activities of pG13-luc, PUMA-luc, and p21/WAF1-luc reporter plasmids. This effect was accompanied by a significant increase in the mRNA and protein levels of several TAp73 transcription targets: p21/WAF1, PUMA, and NOXA. Flow cytometry cell cycle analysis, after MLN8054 treatment, showed more than a 2-fold increase in cell death. The apoptotic outcome was corroborated by showing an increase in cleaved caspase-3 protein levels by Western blot. Using terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling assay, we showed that the expression of dominantnegative mutant TAp73 expression plasmid (p73DD) counteracted the MLN8054-induced cell death. Taken together, our results indicate that AURKA regulates TAp73-dependent apoptosis and highlight the potential of the AURKA inhibitor MLN8054 in treating cancers that are defective in p53 signaling. [Cancer Res 2008;68(21):8998-9004]