A double tyrosine phosphorylation of P68 RNA helicase confers resistance to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent with the capability of inducing apoptosis specifically in tumor cells. However, cancer cells of many cancer types developed TRAIL resistance, limiting the applications of TRAIL in cancer therapies. We show here that p68 acquires a double tyrosine phosphorylation at Y593 and Y595 in TRAIL-resistant T98G glioblastoma cells. The double phosphorylations are induced by platelet-derived growth factor autocrine loop. The double phosphorylation mediates resistance to TRAIL-induced apoptosis. Our data suggest that the phosphorylated p68 protects the cells from programmed cell death by preventing procaspase-8 from proteolytic cleavage. The double-phosphorylated p68 may also confer apoptosis resistance by upregulation of X-chromosome-linked inhibitor apoptosis protein-associated factor 1. In addition, exogenous expression of p68 mutant that carries mutations at the phosphorylation sites (Y593/595F) dramatically sensitizes TRAIL-resistant cells to TRAIL-induced apoptosis, suggesting a potential therapeutic strategy to overcome TRAIL resistance.     

SKP2 confers resistance of pancreatic cancer cells towards TRAIL-induced apoptosis

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dismal prognosis and no effective conservative therapy exists. Although the F-box protein S-phase kinase associated protein 2 (SKP2) is highly expressed and regulates cell cycle progression in PDAC, alternative SKP2 functions in PDAC are unknown. Using RNA interference we now demonstrate that SKP2 confers resistance of a subset of PDAC cell lines towards the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not the topoisomerase II inhibitor etoposide. We observed accelerated cleavage of the BH3-only protein Bid and augmented downregulation of cFLIPL, XIAP and MCL1 upon treatment of SKP2-depleted MiaPaCa2 cells with TRAIL. Our data disclose a novel SKP2 function in PDAC cells and therefore define SKP2 as a molecular target.     

Mechanisms of resistance to TRAIL-induced apoptosis in cancer

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 TRAIL-induced 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-1beta-converting enzyme-inhibitory 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 mitochondria-dependent 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.     

Mechanisms of resistance to TRAIL-induced apoptosis in primary B cell chronic lymphocytic leukaemia

Primary B cells from B cell chronic lymphocytic leukaemia (B-CLL) were resistant to the novel selective cytotoxic agent, TNF-related apoptosis-inducing ligand (TRAIL). Low levels of the death-inducing TRAIL receptors, TRAIL-R1 and TRAIL-R2 but not the putative 'decoy' receptors, TRAIL-R3 and TRAIL-R4, were expressed on the surface of B-CLL cells. Resistance to TRAIL was upstream of caspase-8 activation, as little or no caspase-8 was processed in TRAIL-treated B-CLL cells. Low levels of a TRAIL death-inducing signalling complex (DISC) were formed in these cells, accompanied by the recruitment of endogenous FADD, caspase-8 and c-FLIP(L) but not c-FLIP(S). Both caspase-8 and c-FLIP(L) were cleaved to form two stable intermediates of approximately 43 kDa, which remained associated with the DISC. Caspase-8 was not further processed to its active heterotetramer. Thus the resistance of B-CLL cells to TRAIL may be due partly to low surface expression of the death receptors resulting in low levels of DISC formation and also to the high ratio of c-FLIP(L) to caspase-8 within the DISC, which would prevent further activation of caspase-8. Our results highlight the possibility of sensitising B-CLL cells to TRAIL by modulation of c-FLIP levels or by upregulation of surface expression of death receptors.     

Aspirin sensitizes cancer cells to TRAIL-induced apoptosis by reducing survivin levels.

PURPOSE: Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic antibodies targeting its receptors are promising cancer therapies because of their tumor selectivity, many tumors are resistant to TRAIL-based therapies. We examined whether the nonsteroidal anti-inflammatory drug aspirin sensitized cancer cells to TRAIL agonists in vitro and in vivo and investigated the underlying mechanism. EXPERIMENTAL DESIGN: The effects of aspirin on sensitivity to TRAIL agonists and expression of apoptosis regulators was determined in human breast cancer cell lines and xenograft tumors. The specific role of survivin depletion in the TRAIL-sensitizing effects of aspirin was determined by silencing survivin. RESULTS: Aspirin sensitized human breast cancer cells, but not untransformed human mammary epithelial cells, to TRAIL-induced caspase activation and apoptosis by a cyclooxygenase-2-independent mechanism. Aspirin also sensitized breast cancer cells to apoptosis induced by a human agonistic TRAIL receptor-2 monoclonal antibody (lexatumumab). Aspirin treatment led to G1 cell cycle arrest and a robust reduction in the levels of the antiapoptotic protein survivin by inducing its proteasomal degradation, but did not affect the levels of many other apoptosis regulators. Silencing survivin with small interfering RNAs sensitized breast cancer cells to TRAIL-induced apoptosis, underscoring the functional role of survivin depletion in the TRAIL-sensitizing actions of aspirin. Moreover, aspirin acted synergistically with TRAIL to promote apoptosis and reduce tumor burden in an orthotopic breast cancer xenograft model. CONCLUSIONS: Aspirin sensitizes transformed breast epithelial cells to TRAIL-based therapies in vitro and in vivo by a novel mechanism involving survivin depletion. These findings provide the first in vivo evidence for the therapeutic utility of this combination.     

Laforin confers cancer resistance to energy deprivation-induced apoptosis

A long-standing but poorly understood observation in experimental cancer therapy is the heterogeneity in cancer susceptibility to energy deprivation. Here, we show that the hexose kinase inhibitor 2-deoxyglucose (2-dG) preferentially kills cancer cells with defective laforin expression and significantly increases the survival of mice with aggressive lymphoma due to a genetic defect of the laforin-encoding Epm2a gene. Normal cells from Epm2a(-/-) mice also had greatly increased susceptibility to 2-dG. Thus, laforin is a novel regulator for cellular response to energy deprivation and its defects in cancer cells may be targeted for cancer therapy.     

Calcitonin induces apoptosis resistance in prostate cancer cell lines against cytotoxic drugs via the Akt/survivin pathway

The expression of calcitonin (CT) and CT-receptor (CTR) mRNA in primary prostate tumors increase with tumor progression. Since advanced prostate tumors display chemoresistance, we tested a hypothesis that CT increases apoptosis resistance of prostate cells against cytotoxic drugs. We examined the effect of CT on etoposide-induced apoptosis in PC-3M, LNCaP and NRP-152 cell lines. The cytoprotective actions of CT were then tested on paclitaxel-, dexamethasone- and selenite-induced apoptosis. We also examined cytotoxic actions of these drugs in CTR-silenced PC-3M cells. Since the role of Akt and inhibitors of apoptosis proteins (IAPs) in chemoresistance of advanced prostate cancers has been established, we tested the effect of CT on phospho-Akt and survivin levels in PC-3M cells. Finally, the cytoprotective effect of CT on PC-3M cells was tested in the presence of PI3K inhibitors such as LY 294002 and wortmannin. Acutely added CT significantly attenuated apoptosis of PC cell lines in response to etoposide, dexamethasone and selenite treatment, but could not reduce paclitaxel-induced apoptosis. CT potently stimulated phospho-Akt and survivin synthesis in PC-3M cells in a sustained manner, and LY 294002 attenuated CT-induced survivin synthesis as well as apoptosis resistance. These results suggest that CT induces chemoresistance to etoposide, dexamethasone and selenite but not to paclitaxel in prostate cells. Cytoprotective action of CT is mediated by CTR-induced activation of Akt-survivin pathway. Since CT/CTR expression in prostate cancers increases with tumor progression, the suppression of "CT System" may enhance the effectiveness of chemotherapy.     

EGFR inhibitors sensitize non-small cell lung cancer cells to TRAIL-induced apoptosis

Apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be regulated by the epidermal growth factor (EGF) signaling pathway. In this study, recombinant adenoviral vectors that encode TRAIL gene from the hTERT/RGD promoter (AdTRAIL) was combined with drugs including gefitinib, elotinib, and cetuximab that inhibit EGFR and the EGF signaling pathway in non-small cell lung cancer (NSCLC) cell lines to investigate their antitumor activity. In vitro, compared to single reagent, AdTRAIL combined with EGFR inhibitors reduced proliferation and enhanced apoptosis in H460, A549, and SW1573 cell lines. Western blot results suggested that these effects were relative to up-regulation of pro-apoptosis protein BAX and down-regulation of p-AKT. In vivo, AdTRAIL combined with cetuximab resulted in a significant growth reduction in H460 xenografts without damage to the main organs of nude mice. Histological examination and TUNEL analyses of xenografts showed that cetuximab enhanced cell apoptosis induced by AdTRAIL. These results indicate that EGFR inhibitors enhanced AdTRAIL anti-tumor activity in NSCLC cell lines and that inhibiting the AKT pathway played an important role in this enhancement.     

OPG is regulated by beta-catenin and mediates resistance to TRAIL-induced apoptosis in colon cancer

PURPOSE: Resistance to apoptosis is a hallmark of cancer and correlates with aggressiveness of tumors and poor prognosis. The Wnt/beta-catenin pathway plays a pivotal role in the genesis of colorectal cancer by mechanisms not fully elucidated yet. Previous studies have linked regulation of osteoprotegerin (OPG) in bone to Wnt/beta-catenin signaling. As OPG also serves as a decoy receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we hypothesized that OPG might play a role in mediating resistance to apoptosis in colorectal cancer cells. EXPERIMENTAL DESIGN: Expression analysis and functional studies in human colorectal cancer cell lines and determination of expression in primary tumors and sera from patients with colorectal cancer. RESULTS: We found production of OPG in colorectal cancer cells to be regulated by beta-catenin/Tcf-4. Addition of exogenous OPG to colorectal cancer cells caused resistance to TRAIL. Similarly, accumulation of OPG in medium of cultivated cells caused resistance to TRAIL, and this could be reverted by removal of OPG. Furthermore, OPG levels were significantly increased in serum of patients with advanced disease. CONCLUSIONS: We conclude that the Wnt/beta-catenin pathway contributes to carcinogenesis and cancer cell survival by driving expression of OPG. Expression of the survival factor OPG might provide colorectal cancer cells with an essential growth advantage and contribute to cell invasion and metastasis. Inhibition of OPG expression might offer a new therapeutic approach for the treatment of patients with colorectal tumors overexpressing OPG and make these tumors sensitive to TRAIL-induced apoptosis.     

Inhibiting survivin expression enhances TRAIL-induced tumoricidal activity in human hepatocellular carcinoma via cell cycle arrest

Human Hepatocellular carcinoma (HCC) cell types exhibit a major resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death, and the key determinants of mechanisms accounting for TRAIL susceptibility, still remain controversial. Our previous studies showed that overexpression of survivin reduced sensitivity of HCC cells to TRAIL. The aim of this study is to investigate how tumor cells escape TRAIL-mediated surveillance through survivin expression and how to reverse the resistance of TRAIL-inducing apoptosis. Seven tumor cell lines were treated with or without TRAIL protein and antisense oligodeoxynucleotides (ODNs) against survivin in culture. HepG(2) and SMMC7721 cells were treated with mimosine, thymidine or nocodazole to synchronize their cell cycle phases and then used to test their sensitivity to TRAIL. In vivo effects of TRAIL plasmid alone or in combination with survivin antisense ODNs on tumor growth were evaluated in a nude mouse hepatoma model of HepG(2) cell grafts. Varied levels of survivin mRNA in various cell lines were evaluated and negatively correlated to TRAIL-induced apoptosis. Hepatoma HepG(2) and SMMC7721 cells in G (1) or S phase are more sensitive to TRAIL than those in G(2) phase. Treatment with survivin antisense ODNscaused S phase arrest and significantly enhanced TRAIL-induced apoptosis. TRAIL protein caused G(2)/M arrest and resulted in an increase of survivin in HepG(2) cells. Combined TRAIL plasmid and survivin antisense ODNs significantly supressed the growth of tumor xenografts as compared to TRAIL plamid or antisense ODNs alone during four weeks of observation. The findings indicate that survivin may play a role in tumor cell resistance to TRAIL-induced apoptosis, at least in part, through cell cycle regulation. Manipulation of survivin expression levels may sensitizes tumor cells to TRAIL-induced apoptosis.     

A Smac-mimetic sensitizes prostate cancer cells to TRAIL-induced apoptosis via modulating both IAPs and NF-kappaB

Background  

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for human cancer therapy, prostate cancer still remains resistant to TRAIL. Both X-linked inhibitor of apoptosis (XIAP) and nuclear factor-kappaB function as key negative regulators of TRAIL signaling. In this study, we evaluated the effect of SH122, a small molecule mimetic of the second mitochondria-derived activator of caspases (Smac), on TRAIL-induced apoptosis in prostate cancer cells.     

Targeting Aurora B kinase prevents and overcomes resistance to EGFR inhibitors in lung cancer by enhancing BIM- and PUMA-mediated apoptosis

1. Download : Download high-res image (249KB)
2. Download : Download full-size image

     

Inhibition of Aurora A promotes chemosensitivity via inducing cell cycle arrest and apoptosis in cervical cancer cells

Aurora kinase A (AurA) regulates genomic instability and tumorigenesis in multiple cancer types. Although some studies have reported that Aur A may predict cervical cancer outcomes, its precise function and molecular mechanism in cervical cancer pathogenesis remain unclear. In this study, by overexpression or silencing of Aur A in cervical cancer cell lines, we found that overexpression of Aur A promoted cell proliferation through G1/S cell cycle transition and anti-apoptosis, xenograft tumor growth and chemoresistance to Taxol. We further found that inhibition of Aur A with its specific inhibitor VX-680 enhanced the antitumor effect of Taxol via inducing apoptosis. Moreover, the clinical analysis from tissue samples demonstrated that Aur A was overexpressed, and the expression of Aur A and pERK1/2 was negatively correlated in cervical cancer tissues. The above results may provide some potential insights in treatment of cervical cancer in clinic.     

Perifosine synergistically enhances TRAIL-induced myeloma cell apoptosis via up-regulation of death receptors.

PURPOSE: The aim of this study is to investigate the efficacy of a novel Akt inhibitor, perifosine, in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human myeloma cells and primary patient samples. EXPERIMENTAL DESIGN: The activity of perifosine in combination with TRAIL was evaluated with experiments testing the effect of perifosine on DR4/DR5 expression by the use of chimeric blocking antibodies, as well as siRNA. RESULTS: DR4 and DR5 expression was induced by exposure to single-agent perifosine. After exposure of human myeloma cell lines or primary patient samples to increasing doses of perifosine with exogenous TRAIL, we identified synergistically enhanced apoptosis when compared with the perifosine alone, which was achieved with levels well below clinically achievable concentrations for both agents. Transfection with siRNA against DR4, and DR5 reduced the level of apoptosis induced by the combination but did not result in total abrogation of the combination effect. Overexpression of activated Akt, the proposed target for perifosine, did not inhibit the combination effect. Anti-DR4 and DR5 chimeric proteins blocked the cytotoxicity induced by the combination, and the use of c-FLICE-like interleukin protein (FLIP) siRNA enhanced the efficacy at the combination, further supporting the importance of the DR4/DR5 axis in the effect of perifosine. CONCLUSION: Our observation seems to be independent of the effects of perifosine on Akt signaling, and may represent an additional mechanism of action for this agent, and supports future clinical trials combining these two agents.