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.
Esophageal adenocarcinomas (EACs) are poorly responsive to chemotherapeutics. This study aimed to determine the levels of Aurora kinas A (AURKA) and the therapeutic potential of MLN8237, an investigational AURKA inhibitor, alone and in combination with Cisplatin. Using quantitative real time polymerase chain reaction we detected frequent AURKA gene amplification (15/34, 44%) and mRNA overexpression (37/44, 84%) in EACs (p<0.01). Immunohistochemistry analysis demonstrated overexpression of AURKA in more than two-thirds of EACs tissue samples (92/132, 70%) (p<0.001). Using FLO-1, OE19 and OE33 EAC cell lines, with constitutive AURKA overexpression and mutant-p53, we observed inhibition of colony formation with a single treatment of 0.5μM MLN8237 (p<0.05). This effect was further enhanced in combination with 2.5μM Cisplatin (p<0.001). 24hrs after treatment with the MLN8237 or MLN8237 and Cisplatin, cell cycle analyses demonstrated a sharp increase in the percentage of polyploid cells (p<0.001). This was followed by an increase in the percentage of cells in the sub-G1-phase at 72hrs, concordant with the occurrence of cell death (p<0.001). Western blot analysis demonstrated higher induction of TAp73β, PUMA, NOXA, cleaved caspase 3 and cleaved PARP with the combined treatment, as compared to a single agent treatment. Using xenograft models, we demonstrated an enhanced anti-tumor role for the MLN8237 and Cisplatin combination, as compared to single agent treatments (p<0.001). In conclusion, this study demonstrates frequent overexpression of AURKA and suggests that MLN8237 could be an effective anti-tumor agent, which can be combined with CDDP for a better therapeutic outcome in EACs.
FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past. Using a new generation electron bombardment-charge-coupled device camera coupled to an image splitter, we demonstrate that BRET can be used to image protein interactions in plant and animal cells and in tissues; even subcellular imaging is possible. We have applied this technology to image two different protein interactions: (i) dimerization of the developmental regulator, COP1, in plant seedlings; and (ii) CCAAT/ enhancer binding protein ␣ (C/EBP␣) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations.C/EBP ͉ COP1 ͉ FRET ͉ luminescence ͉ fluorescence I nteractions among proteins are key to the performance of their cellular activity. Identifying the partners with which a protein associates has been a major approach, in addition to genetics, toward discovering the key components of a biological pathway. Moreover, quantifying and localizing these protein interactions are crucial to the ultimate understanding of how proteins accomplish their raison d'etre. Many different methods are available for measuring protein interactions (1), but one of the most useful is based on Förster resonance energy transfer (2). In particular, FRET is a well established resonance energy technique for monitoring protein interactions (3). When two fluorophores (the ''donor'' and the ''acceptor'') with overlapping emission/absorption spectra are within Ϸ50 Å of one another and other conditions are met, the donor fluorophore is able to transfer its excited-state energy to the acceptor fluorophore. FRET can act as a ''molecular yardstick'' when two fluorophores of overlapping emission/absorption spectra are within a radius of Ϸ50 Å and other conditions are met. Therefore, if appropriate fluorophores are linked to proteins that interact with each other, the proximity of those proteins can be detected by FRET in living cells in which the fusion proteins are produced endogenously (3). Thus, the presence or absence of FRET acts as a molecular yardstick.However, because FRET demands that the donor fluorophore be excited by light, the practical usefulness of FRET can be limited by the concomitant consequences of that irradiation: photobleaching, autofluorescence, and direct excitation of the acceptor fluorophore. Furthermore, some tissues can be damaged by the excitation light or might be directly photoresponsive (e.g., retina and most plant tissues) so that a photobiologically regulated interaction can be disturbed by FRET. For these reasons, we ...
Background & Aims Chronic inflammation contributes to the pathogenesis of gastric tumorigenesis. The Aurora kinase A gene (AURKA) is frequently amplified and overexpressed in gastrointestinal cancers. We investigated the roles of AURKA in inflammation and gastric tumorigenesis. Methods We used quantitative real-time reverse transcription PCR, immunofluorescence, immunohistochemistry, luciferase reporter, immunoblot, co-immunoprecipitation, and in vitro kinase assays to analyze AGS and MKN28 gastric cancer cells. We also analyzed Tff1−/− mice, growth of tumor xenografts, and human tissues. Results We correlated increased expression of AURKA with increased levels of tumor necrosis factor-α and inflammation in the gastric mucosa of Tff1−/− mice (r = 0.62; P=.0001). MLN8237, an investigational small-molecule selective inhibitor of AURKA, reduced nuclear staining of NFκBp65 in human gastric cancer samples and mouse epithelial cells, suppressed NFκB reporter activity, and reduced the expression of NFκB target genes that regulate inflammation and cell survival. Inhibition of AURKA also reduced growth of xenograft tumors from human gastric cancer cells in mice and reversed the development of gastric tumors in Tff1−/− mice. AURKA was found to regulate NFκB activity by binding directly and phosphorylating IκBα in cells. Premalignant and malignant lesions from the gastric mucuosa of patients had increased levels of AURKA protein and nuclear NFκB, compared with healthy gastric tissue. Conclusions In analyses of gastric cancer cell lines, human tissue samples, and mouse models, we found AURKA to be upregulated during chronic inflammation to promote activation of NFκB and tumorigenesis. AURKA inhibitors might be developed as therapeutic agents for gastric cancer.
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