Contribution of Epigenetic Silencing of Tumor Necrosis Factor–Related Apoptosis Inducing Ligand Receptor 1 (DR4) to TRAIL Resistance and Ovarian Cancer (original) (raw)
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Gynecologic Oncology, 2004
TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, is a recently identified cytokine that preferentially kills transformed cells while sparing most normal cells. We investigated the ability of TRAIL alone and TRAIL in combination with cytotoxic drugs to induce apoptosis in six ovarian cancer cell lines. To get some insight into the resistance to TRAIL, the expression of TRAIL receptors and selected downstream signaling elements was determined. TRAIL induced significant apoptosis (up to 80%) in three out of six ovarian cancer cell lines (MZ-26, CaOV-3, ES-2). In A2780 and A2780ADR cells, resistance to TRAIL-induced apoptosis correlated with their lack of DR4-expression. MZ-15 cells, which expressed the processed form of FLIP(L), p43 (FADD-like IL-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP)), and FLIP(S), were resistant to TRAIL in spite of the presence of DR4. When TRAIL-resistant cell lines were co-incubated with routinely used cytotoxic agents, TRAIL exerted a synergistic effect leading to apoptosis rates unachievable by incubation with cytotoxic agents alone. The ability of TRAIL to induce apoptosis in ovarian cancer cells as well as to potentiate the activity of chemotherapeutic agents even in cell lines that are resistant to TRAIL-induced cytotoxicity is a powerful promise in the fight against this deadly disease.
Molecular Cancer …, 2009
Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is being evaluated clinically in treating various malignancies. Previous studies have shown that repeated application of high doses of rhTRAIL results in a subpopulation of parental cells that is unresponsive to the death ligand. However, it is not clear whether TRAIL-sensitive cancer cells could acquire resistance to TRAIL treatment. Here, we found that MDA-MB-231 breast cancer cells, which are highly sensitive to TRAIL-induced apoptosis, became resistant to TRAIL killing after a prolonged exposure to subtoxic doses of rhTRAIL. The resulting TRAIL-resistant cells were cross-resistant to antibodies against its death receptors (DR4 and DR5); however, they retained sensitivity to several clinically relevant chemotherapies. Surface expression of DR4 and DR5 was significantly reduced in the selected cells, resulting in failure in death-inducing signaling complex formation and caspase activation. In addition, real-time PCR analysis revealed an upregulation in multiple apoptosis-regulator genes, including c-FLIP, Stat5a, and Stat5b. Inhibition of Janus-activated kinase, an upstream activator of signal transducer and activator of transcription 5 (Stat5), or knockdown of Stat5 itself partially restored cellular sensitivity to TRAIL-induced apoptosis, suggesting that Stat5 signaling is also involved in the development of TRAIL resistance. Furthermore, we showed that acquired TRAIL resistance was effectively eliminated by combination with etoposide, doxorubicin, or paclitaxel. These results suggest that tumor cells could acquire resistance to TRAIL therapy especially when they are repeatedly exposed to low levels of the death ligand, highlighting the necessity of combination with therapies that target the resistance mechanisms. (Mol Cancer Res mixture of three siRNA sequences (see Materials and Methods) targeting different regions of the human c-FLIP transcript. After 24 h, cells were analyzed by immunoblotting for c-FLIP protein expression (top) or treated with rhTRAIL (20 ng/mL) at 37°C for an additional 4 h followed by FACS analysis for apoptosis (bottom). E. The effect of Stat5b knockdown was assessed similarly as in D (top, immunoblots for Stat5 using an anti-pan Stat5 antibody recognizing both Stat5a and Stat5b, Bcl-XL, and cyclin D1; bottom, apoptosis after incubation with TRAIL for 4 h at 20 ng/mL). F. Cells were pretreated with a cell-permeable JAK kinase inhibitor at the indicated concentrations for 1 h, followed by rhTRAIL (20 ng/mL) for an additional 4 h. The results are expressed as JAK inhibitioninduced apoptosis (apoptosis % induced by JAK inhibitor combining with rhTRAIL − apoptosis % by rhTRAIL alone; n = 3, mean ± SD).
Clinical Cancer Research, 2005
Epithelial ovarian cancer is the most common cause of mortality from gynecologic malignancies. Due to advanced stage at diagnosis, most patients need systemic treatment in addition to surgery. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with a promising toxicity profile and synergistic activity with chemotherapeutic agents. We used an arrayed panel of epithelial ovarian cancer tissue to assess the protein expression of TRAIL and the clinically most relevant members of its pathway death receptors 4 and 5 (DR4 and DR5) and the long form of FLICE inhibitory protein (FLIPL). We could show that a majority (66.2%) of the tumor tissues displayed either reduced DR4/DR5 expression (20.6%), increased FLIPL expression (39.7%), or both (5.9%) as determined by immunohistochemistry. Furthermore, higher TRAIL expression in the surrounding connective tissue but not in the tumor cells is significantly (P<0.05) linked with favorable overall survival in advanced-stage patients. Mechanisms to escape the immune surveillance mediated by TRAIL are developed by ovarian cancer cells in a high percentage. TRAIL expression in the ovarian cancer microenvironment has an effect on overall survival. These findings enhance our understanding of ovarian cancer pathology and might be helpful in guiding TRAIL-based therapy in future.
Mechanisms of resistance to TRAIL-induced apoptosis in cancer
Cancer Gene Therapy, 2005
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anticancer agent. However, considerable numbers of cancer cells, especially some highly malignant tumors, are resistant to apoptosis induction by TRAIL, and some cancer cells that were originally sensitive to TRAIL-induced apoptosis can become resistant after repeated exposure (acquired resistance). Understanding the mechanisms underlying such resistance and developing strategies to overcome it are important for the successful use of TRAIL for cancer therapy. Resistance to TRAIL can occur at different points in the signaling pathways of TRAILinduced apoptosis. Dysfunctions of the death receptors DR4 and DR5 due to mutations can lead to resistance. The adaptor protein Fas-associated death domain (FADD) and caspase-8 are essential for assembly of the death-inducing signaling complex, and defects in either of these molecules can lead to TRAIL resistance. Overexpression of cellular FADD-like interleukin-1b-converting enzymeinhibitory protein (cFLIP) correlates with TRAIL resistance in several types of cancer. Overexpression of Bcl-2 or Bcl-X L , loss of Bax or Bak function, high expression of inhibitor of apoptosis proteins, and reduced release of second mitochondria-derived activator of caspases (Smac/Diablo) from the mitochondria to the cytosol have all been reported to result in TRAIL resistance in mitochondriadependent type II cancer cells. Finally, activation of different subunits of mitogen-activated protein kinases or nuclear factor-kappa B can lead to development of either TRAIL resistance or apoptosis in certain types of cancer cells.
Trail activity in human ovarian cancer cells
European Journal of Cancer, 2002
The ability of the TRAIL ligand to induce cell killing in three ovarian cancer cell lines was investigated using a glutathione-Stransferase (GST)-TRAIL fusion protein. One of the three lines was sensitive to TRAIL, which induced cell killing in a range of concentrations similar to those necessary to kill the TRAIL-sensitive leukaemic cell line Jurkat. The relative mRNA expression of the four TRAIL receptors did not explain the different sensitivities of the three ovarian cancer cell lines to TRAIL treatment. The TRAIL-sensitive IGROV-1 cell line expressed slightly lower levels of the anti-apoptotic protein FLIP than the two TRAIL-insensitive cell lines (A2780 and SKOV-3). Nevertheless, although TRAIL did not significantly reduce cell growth in the A2780 and SKOV-3 cells it did enhance the activity of paclitaxel and cisplatin (DDP), the two most widely used drugs for the treatment of ovarian cancer, increasing their ability to induce apoptosis. The use of TRAIL in combination with classical anticancer agents might thus boost the apoptotic response, improving the activity of DDP and paclitaxel in ovarian cancer. # (M. Broggini).
Journal of Biological Chemistry, 2004
Many tumor cell types are sensitive to TNF-related apoptosis inducing ligand (TRAIL)induced apoptosis. Incubation of TRAIL-sensitive cells with TRAIL invariably leads to resistant survivors even when high doses of TRAIL are used. Because the emergence of resistance to apoptosis is a major concern in successful treatment of cancer, and TRAILsurvivors may contribute to therapeutic failure, we investigated potential resistance mechanisms. We selected TRAIL-resistant SW480 human colon adenocarcinoma cells by repeatedly treating them with high and/or low doses of TRAIL. The resulting TRAIL resistant clones were not cross-resistant to Fas or paclitaxel. Expression of modulators of apoptosis was not changed in the resistant cells, including TRAIL receptors, cFLIP, Bax, bid, or IAP's. Surprisingly, we found that DISC formation was deficient in multiple selected TRAIL-resistant clones. DR4 was not recruited to the DISC upon TRAIL treatment, and Caspase 8 was not activated at the DISC. Although total cellular DR4 mRNA and protein were virtually identical in TRAIL-sensitive parental and TRAILresistant clones, DR4 protein expression on the cell surface was essentially undetectable in the TRAIL-resistant clones. Moreover, exogenous DR4 and KILLER/DR5 were not properly transported to the cell surface in the TRAIL-resistant cells. Interestingly, TRAIL-resistant cells were re-sensitized to TRAIL by tunicamycin pre-treatment, which increased cell surface expression of DR4 and KILLER/DR5. Our data suggest that tumor cells may become resistant to TRAIL through regulation of the death receptor cell surface transport and that resistance to TRAIL may be overcome by the glycosylation inhibitor/ER-stress inducing agent tunicamycin.
Oncology Reports, 2009
The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a specific and potent inducer of apoptosis in cancer cells, but the resistance of many tumour cells to TRAIL still represents a major hurdle for the clinical treatment of tumours with TRAIL. As apoptosis is regulated by the balance of activities of several anti-apoptotic factors and pro-apoptotic factors, we analysed the relative contribution of the two sides and found that down-regulation of Bcl-x L and in particular XIAP, but not c-Flip, sensitised the TRAIL resistant pancreatic cancer cell line Panc-1. A combination of both XIAP and Bcl-x L knockdowns showed no substantial added benefit indicating that both act in the same pathway. Notably, the degree of sensitisation by silencing of anti-apoptotic genes was further elevated by concomitantly increasing the pro-apoptotic potential in Panc-1 cells through over-expression of TRAIL-R1 or IFN-cmediated increases in caspase-8 levels. Similar sensitisation effects were obtained for another TRAILresistant pancreatic tumour cell line, AsPC-1. Our findings demonstrate that modulation of the balance between anti-and pro-apoptotic pathways from both sides by inhibition of apoptosis-antagonists and stimulation of pro-apoptotic factors provides the best way to enhance the anti-tumourigenic effect of TRAIL.