The tight junction (TJ) protein claudin-4 is aberrantly upregulated in gastric cancer, but its clinical significance and the molecular mechanisms underlying claudin-4 overexpression in gastric cancer remain unclear. Here, we investigated its roles and epigenetic mechanisms regulating CLDN4 expression in gastric cancer. We show that increased membranous expression of claudin-4 in gastric carcinoma is associated with better patient prognosis, whereas cytoplasmic claudin-4 expression did not show a significant association with prognosis. Consistent with the correlation of increased membranous claudin-4 with favorable clinicopathological factors, claudin-4 overexpression inhibited the migration and invasion of gastric cancer cells; in contrast, it did not affect cell growth. Claudin-4 expression also increased the barrier function of TJs. Claudin-4 upregulation was strongly correlated with DNA hypomethylation in both gastric tissues and gastric cancer cells. Moreover, CLDN4 expression was repressed in normal gastric tissues in association with bivalent histone modifications, and loss of repressive histone methylations and gain of active histone modifications were associated with CLDN4 overexpression in gastric cancer cells. Interestingly, CLDN4 repression could be markedly derepressed by combined treatments that simultaneously target both histone modifications and DNA demethylation in CLDN4-hypermethylated cells, whereas concomitant changes in histone methylations and acetylations are required for CLDN4 induction in CLDN4-repressed cells with low DNA methylation. Taken together, this study reveals that membranous claudin-4 expression is associated with gastric cancer progression and that it is an independent positive prognosis marker in gastric carcinoma. Furthermore, our findings suggest that epigenetic derepression may be a possible mechanism underlying CLDN4 overexpression in gastric cancer and that claudin-4 may have potential as a promising target for the treatment of gastric cancer.

Transducer of ErbB-2.1 (Tob1), a tumor suppressor protein, is inactivated in a variety of cancers including stomach cancer. However, the role of Tob1 in gastric carcinogenesis remains elusive. The present study aimed to investigate whether Tob1 could inhibit gastric cancer progression in vitro, and to elucidate its underlying molecular mechanisms. We found differential expression of Tob1 in human gastric cancer (MKN28, AGS and MKN1) cells. The overexpression of Tob1 induced apoptosis in MKN28 and AGS cells, which was associated with sub-G1 arrest, activation of caspase-3, induction of Bax, inhibition of Bcl-2 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, Tob1 inhibited proliferation, migration and invasion, which were reversed in MKN1 and AGS cells transfected with Tob1 siRNA. Overexpression of Tob1 in MKN28 and AGS cells induced the expression of Smad4, leading to the increased expression and the promoter activity of p15, which was diminished by silencing of Tob1 using specific siRNA. Tob1 decreased the phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β) in MKN28 and AGS cells, resulting in the reduced protein expression and the transcriptional activity of β-catenin, which in turn decreased the expression of cyclin D1, cyclin-dependent kinase-4 (CDK4), urokinase plasminogen activator receptor (uPAR) and peroxisome proliferator and activator receptor-δ (PPARδ). Conversely, silencing of Tob1 induced the phosphorylation of Akt and GSK-3β, and increased the expression of β-catenin and its target genes. Collectively, our study demonstrates that the overexpression of Tob1 inhibits gastric cancer progression by activating Smad4- and inhibiting β-catenin-mediated signaling pathways.

Human papillomavirus (HPV) types 16 and 18 are the major etiologic factors in the development of cervical epithelial neoplasia. Our study was designed to validate antiviral short interfering RNA (siRNA) targeting the E6 and E7 oncogenes as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP) in cervical carcinoma. Specifically, the therapeutic efficacy of combination of CDDP and E6/E7-specific siRNA was assessed in an in vivo cervical cancer xenograft models. The combination of CDDP and E6/E7-specific siRNA had greater efficacy than the combination of CDDP and E6-specific siRNA especially in terms of inducing cellular senescence. Through in vitro and in vivo experiments, the mechanism of synergy between these two treatments was revealed, demonstrating that the combination of E6/E7-specific siRNA and CDDP therapy was significantly superior to either modality alone. In vitro, long-term exposure of HeLa cells to the combination of CDDP and E6/E7-specific siRNA induced apoptosis and cellular senescence. In vivo, E6/E7-specific siRNA potentiated the antitumor efficacy of CDDP via induction of apoptosis, senescence and antiangiogenesis. Our results suggest that E6/E7-specific siRNA may be an effective sensitizer of CDDP chemotherapy in cervical cancer.Cervical cancer is one of the most common types of gynecological malignancies worldwide. Infection with high-risk human papillomavirus (HPV), such as types 16 and 18, is a major cause of cervical cancer. The E6 and E7 proteins encoded by HPV play a critical role in cervical carcinoma. The E6 viral oncoprotein binds to wild-type tumor protein p53 (TP53), while E7 binds to the retinoblastoma (RB) family of tumor suppressor proteins and disrupts RB/E2F complexes, thereby driving cell division.1,2 E6 oncoprotein forms a complex with ubiquitin protein ligase E3A (UBE3A), also known as E6-associated protein. The E6/UBE3A complex ubiquitinates TP53, causing accelerated degradation of TP53 and disruption of TP53-mediated cellular response to oncogenic addiction and DNA damage.Although most of the HPV-associated cervical carcinomas, unlike many other cancers, carry the wild-type TP53 gene, the levels of TP53 protein in these carcinomas remain remarkably low because the protein is constantly targeted for degradation by E6.3,4 Thus, E6 and the E6/UBE3A complex may be suitable targets for cervical cancer therapy.Recently, antiviral RNA interference (RNAi) has been developed as a novel therapeutic strategy and has entered clinical trials.5 RNAi targeting E6 or E6/E7 promotes the accumulation of TP53 and/or hypo-phosphorylated RB Key words: human papillomavirus, cisplatin (CDDP) chemotherapy, HPV E6/E7 siRNA, chemosensitizer, cervical carcinoma Abbreviations: Cisplatin (CDDP): cis-diaminedichloroplatinum II; FCFM: fibered confocal fluorescence microscopy; GFP: green fluorescent protein; HPF: high power field; HPV: human papillomavirus; RNAi: RNA interference; SA-b-Gal assay: senescence-associated b-galactosidase assay; shRNA: short hairpin RNA; siRNA: short ...

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SMAD4 has been suggested to inhibit the activity of the WNT/b-catenin signaling pathway in cancer. However, the mechanism by which SMAD4 antagonizes WNT/b-catenin signaling in cancer remains largely unknown. Aurora A kinase (AURKA), which is frequently overexpressed in cancer, increases the transcriptional activity of b-catenin/T-cell factor (TCF) complex by stabilizing b-catenin through the inhibition of GSK-3b. Here, SMAD4 modulated AURKA in a TGFb-independent manner. Overexpression of SMAD4 significantly suppressed AURKA function, including colony formation, migration, and invasion of cell lines. In addition, SMAD4 bound to AURKA induced degradation of AURKA by the proteasome. A luciferase activity assay revealed that the transcriptional activity of the b-catenin/TCF complex was elevated by AURKA, but decreased by SMAD4 overexpression. Moreover, target gene analysis showed that SMAD4 abrogated the AURKA-mediated increase of b-catenin target genes. However, this inhibitory effect of SMAD4 was abolished by overexpression of AURKA or silencing of AURKA in SMAD4-overexpressed cells. Meanwhile, the SMAD4-mediated repression of AURKA and b-catenin was independent of TGFb signaling because blockage of TGFbR1 or restoration of TGFb signaling did not prevent suppression of AURKA and b-catenin signaling by SMAD4. These results indicate that the tumor-suppressive function of SMAD4 is mediated by downregulation of b-catenin transcriptional activity via AURKA degradation in a TGFb-independent manner.