Moreover, induction of DTX1 in AGS cells by geldanamycin conferred susceptibility of those cells to TRAIL-induced apoptosis

Moreover, induction of DTX1 in AGS cells by geldanamycin conferred susceptibility of those cells to TRAIL-induced apoptosis. cells to TRAIL-induced apoptosis. Our results reveal a tumor-suppressive role for DTX1 and suggest a new approach to increasing TRAIL efficacy by raising DTX1 levels in gastric cancer therapy. DTX1 also enhanced c-FLIP degradation and FasL-induced and K02288 TRAIL-induced apoptosis in T cells, suggesting that DTX1 constitutes one of the physiological mechanisms regulating c-FLIP stability. Introduction Gastric cancer cells are characterized by their resistance to apoptosis induction by death receptors. Gastric cancer has one of the worlds leading cancer mortality rates, with a poor 5-year survival rate1C3. Advanced stages of gastric cancer show local invasion, peritoneal dissemination, and hepatic or para-aortic lymph node metastasis. Surgery remains the curative therapy, but is limited to non-metastatic gastric cancer. The efficacy of chemotherapy for gastric cancer is poor due to multidrug resistance (MDR). Therefore, K02288 identification of novel biomarkers and development of new therapeutics for gastric cancer are one of the demanding priorities. The Death receptor (DR) agonist TRAIL has been explored for its efficacy to induce apoptosis in different types of cancers4C6, including gastric cancer7,8. Like other death receptors, engagement of TRAIL receptors (DR4 and DR5) by TRAIL results in the formation of death-inducing signaling complexes (DISC) containing FADD and procaspase-89C12. Procaspase-8 undergoes autoproteolytic cleavage to generate active caspase-8 at DISC, leading to activation of downstream caspases and irreversible cell damage. Cellular FLICE-inhibitory protein (c-FLIP) is a master anti-apoptotic factor that suppresses death receptor-induced apoptosis by interfering with the processing of procaspase-8 at DISC9C15. c-FLIP also inhibits necrosis and autophagy16C18. c-FLIP is partly accountable for the failure of TRAIL receptor agonists in clinical attempts to treat cancers4,19, so it is a target for cancer therapy19C21. Expression of c-FLIP is induced by activation signaling, including NF-B22, Akt, and ERK13,19,22C24. K02288 K02288 Levels of c-FLIP protein are subjected to regulation by two ubiquitin E3 ligases, ITCH and CBL, through the promotion of polyubiquitination and subsequent proteosomal degradation of c-FLIP25,26. TRAIL receptors and the downstream effector caspase-8 are intact in gastric cancer cells27,28. However, gastric cancers are generally resistant to TRAIL-induced cell death, and induction of TRAIL-mediated cytotoxicity always requires co-stimulation with a sensitizing reagent. c-FLIP is upregulated in gastric cancer and is associated with metastasis and tumor progression29,30. As in other types of cancer, c-FLIP contributes to the resistance to TRAIL-induced apoptosis in gastric cancer31C34. We have previously shown that enhances the susceptibility to TRAIL-induced apoptosis in gastric cancer cells by downregulation of c-FLIP34. Deltex (DTX) is a target of Notch, and is composed of Notch-binding WWE domains at the N-terminus, followed by a proline-rich motif, and a C-terminal RING finger domain35,36. DTX1 confers ligand-independent activation of Notch by directing the ubiquitination and endosomal entry of Notch37,38. Similar to the E3 ligases Itch and Cbl-b39, DTX1 is a target of NFAT and is involved in T cell tolerance40,41. We recently found that DTX1 promotes the degradation of PKC and PLC- in a way similar to ITCH and Cbl-b42. In the present study, we show that DTX1 is specifically downregulated in gastric cancer and is critical for the resistance of gastric cancer cells to TRAIL-induced cell death. DTX1 binds to c-FLIP and promotes degradation of c-FLIP through the endosome-lysosomal pathway. Re-introduction of DTX1 into gastric cancer cells increased TRAIL-induced apoptosis and also reduced c-FLIP. In addition, a treatment that increased DTX1 expression also sensitized gastric cancer to TRAIL treatment. Our results suggest that induction of DTX1 could be a new approach to enhancing the benefits of TRAIL-mediated cancer therapy. We also found that DTX1 enhanced c-FLIP degradation and Fas-induced and TRAIL-induced apoptosis in T cells, indicating that DTX1 constitutes one of the physiological mechanisms regulating c-FLIP stability. Results DTX1 expression is negatively correlated with gastric cancer progression We found that expression of the ubiquitin E3 ligase (and is reduced in most of the gastric cancer cell lines examined (Fig.?1d). Expression of is variable in different DUSP5 gastric cancer cell lines, whereas the expression of is increased in the same group of gastric cancer cell lines (Fig.?1e, f), suggesting that the ITCH-mediated and CBL-mediated c-FLIP degradation processes are not operational in gastric cancer. Gene expression-based prognosis risk score analyses in gastric cancer have also shown that gastric cancer tissues from relapse-free survival (RFS) patients46 expressed higher levels of mRNA (Fig.?1g, h). This is in contrast to no correlation being found between the expression of or and the RFS of gastric cancer patients (Fig.?1i, j). Therefore, is downregulated in gastric cancer tissues and expression is negatively correlated with gastric cancer progression. Open in a separate window Fig. 1 expression is lower while and expressions are normal or elevated in gastric intestinal type adenocarcinoma. Expression of (a), (b), and (c) mRNA in human gastric cancer tissue versus normal43C45. All three databases contain patients.