Engineering death receptor ligands for cancer therapy

CD95, TNFR1, TRAILR1 and TRAILR2 belong to a subgroup of TNF receptors which is characterized by a conserved cell death-inducing protein domain that connects these receptors to the apoptotic machinery of the cell. Activation of death receptors in malignant cells attracts increasing attention as a principle to fight cancer. Besides agonistic antibodies the major way to stimulate death receptors is the use of their naturally occurring “death ligands” CD95L, TNF and TRAIL. However, dependent from the concept followed to develop a death ligand-based therapy various limiting aspects have to be taken into consideration on the way to a “bedside” usable drug. Problems arise in particular from the cell associated transmembrane nature of the death ligands, the poor serum half life of the soluble fragments derived from the transmembrane ligands, the ubiquitous expression of the death receptors and the existence of additional non-death receptors of the death ligands. Here, we summarize strategies how these limitations can be overcome by genetic engineering.     

TRAIL as a target in anti-cancer therapy

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can initiate apoptosis through the activation of their death receptors. The ability of TRAIL to selectively induce apoptosis of transformed or tumor cells but not normal cells promotes the development of TRAIL-based cancer therapy. Accumulating preclinical studies demonstrate that the TRAIL ligand can effectively induce cancer cell apoptosis. Completed and ongoing Phases I and II clinical trials using TRAIL are showing clinically promising outcomes without significant toxicity. Importantly, TRAIL, DR4 and DR5 can all be induced by chemotherapeutics and/or radiation, which can sensitize cancer cells to TRAIL. Thus, understanding the regulation of the TRAIL apoptosis pathway can help develop more selective TRAIL-based agents for the treatment of human cancer.     

TRAIL death receptor-4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma

PURPOSE: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells, and a number of clinical trials have recently been initiated to test the safety and antitumoral potential of TRAIL in cancer patients. Four different receptors have been identified to interact with TRAIL: two are death-inducing receptors (TRAIL-R1 [DR4] and TRAIL-R2 [DR5]), whereas the other two (TRAIL-R3 [DcR1] and TRAIL-R4 [DcR2]) do not induce death upon ligation and are believed to counteract TRAIL-induced cytotoxicity. Because high levels of DcR2 expression have recently been correlated with carcinogenesis in the prostate and lung, this study investigated the importance of TRAIL and TRAIL receptor expression in breast cancer patients with invasive ductal carcinoma, taking various prognostic markers into consideration. METHODS AND MATERIALS: Immunohistochemical analyses were performed on 90 breast cancer patients with invasive ductal carcinoma using TRAIL and TRAIL receptor-specific antibodies. Age, menopausal status, tumor size, lymph node status, tumor grade, lymphovascular invasion, perineural invasion, extracapsular tumor extension, presence of an extensive intraductal component, multicentricity, estrogen and progesterone receptor status, and CerbB2 expression levels were analyzed with respect to TRAIL/TRAIL receptor expression patterns. RESULTS: The highest TRAIL receptor expressed in patients with invasive ductal carcinoma was DR4. Although progesterone receptor-positive patients exhibited lower DR5 expression, CerbB2-positive tissues displayed higher levels of both DR5 and TRAIL expressions. CONCLUSIONS: DR4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma.     

Targeting pro-apoptotic trail receptors sensitizes HeLa cervical cancer cells to irradiation-induced apoptosis

PURPOSE: To investigate the potential of irradiation in combination with drugs targeting the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor (DR)4 and DR5 and their mechanism of action in a cervical cancer cell line. METHODS AND MATERIALS: Recombinant human TRAIL (rhTRAIL) and the agonistic antibodies against DR4 and DR5 were added to irradiated HeLa cells. The effect was evaluated with apoptosis and cytotoxicity assays and at the protein level. Membrane receptor expression was measured with flow cytometry. Small-interfering RNA against p53, DR4, and DR5 was used to investigate their function on the combined effect. RESULTS: rhTRAIL and the agonistic DR4 and DR5 antibodies strongly enhanced 10-Gy-induced apoptosis. This extra effect was 22%, 23%, and 29% for rhTRAIL, DR4, and DR5, respectively. Irradiation increased p53 expression and increased the membrane expression of DR5 and DR4. p53 suppression, as well as small-interfering RNA against DR5, resulted in a significant downregulation of DR5 membrane expression but did not affect apoptosis induced by irradiation and rhTRAIL. After small-interfering RNA against DR4, rhTRAIL-induced apoptosis and the additive effect of irradiation on rhTRAIL-induced apoptosis were abrogated, implicating an important role for DR4 in apoptosis induced through irradiation in combination with rhTRAIL. CONCLUSION: Irradiation-induced apoptosis is strongly enhanced by targeting the pro-apoptotic TRAIL receptors DR4 or DR5. Irradiation results in a p53-dependent increase in DR5 membrane expression. The sensitizing effect of rhTRAIL on irradiation in the HeLa cell line is, however especially mediated through the DR4 receptor.     

miR-3132 upregulates surface TRAIL to induce apoptotic cell death in cancer cells

TRAIL-based therapies are of significant clinical interest because of its unique ability to induce apoptosis in cancer cells while sparing normal and untransformed cells. This selective antitumor potential of the TRAIL pathway has been harnessed by development of therapeutics including recombinant (rh)TRAIL and TRAIL-receptor agonist antibodies such as mapatumumab and lexatumumab. While these TRAIL-based therapies have proven successful in preclinical studies and safe in early phase clinical trials, the limited serum half-life has been a hurdle for further clinical development. Here we characterize miR-3132, a novel and first-in class TRAIL-inducing miRNA with potent anti-proliferative and pro-apoptotic effects in cancer cell lines. Initial mechanistic studies indicate that miR-3132 engages the interferon signaling pathway to induce TRAIL and subsequent TRAIL-dependent apoptosis in cancer cell lines. Our data further suggests that the binding of miR-3132 to toll-like receptors could be the upstream pathway for the interferon response. The current study the first report to demonstrate miR-3132’s in vitro efficacy and preliminary mechanism of action in cancer cell lines.     

Engineered vascular-targeting antibody-interferon-gamma fusion protein for cancer therapy

A number of cytokines are either approved drugs or are in advanced clinical trials, yet these biopharmaceuticals do not typically localize efficiently in solid tumors and manifest their therapeutic potential at the expense of severe side effects. The targeted delivery of cytokines to solid tumors is a promising avenue for increasing the therapeutic index of these biopharmaceuticals. We engineered a fusion protein between scFv(L19), a human antibody fragment specific to the EDB domain of fibronectin, and a cysteine-free mutant of murine interferon-gamma. The resulting fusion protein was capable of targeting new blood vessels in solid tumors, and the targeting efficiency was strikingly increased in tumor-bearing knockout mice lacking the interferon-gamma receptor. ScFv(L19)-interferon-gamma displayed a strong antitumor effect in both subcutaneous and metastatic murine F9 teratocarcinomas, but was not efficacious as single agent when used to treat C51 and CT26 tumors. The potency of this fusion protein could be substantially enhanced by combination with doxorubicin and other immunocytokines. These findings are of clinical relevance, as the EDB domain is a marker of angiogenesis, with identical sequence in mouse and man, which is abundantly expressed in a variety of aggressive solid tumors but is undetectable in most normal tissues.     

重组腺病毒TRAIL基因联合抗EGFR靶向药物对肺癌细胞增殖的影响

目的:观察重组腺病毒TRAIL基因制剂联合抗EGFR靶向药物对H460肺癌细胞株及裸鼠移植瘤增殖的影响。方法:重组腺病毒TRAIL基因治疗制剂分别与EGFR信号通路靶向治疗药物Iressa、Tarceva、以及C225联合应用于H460肺腺癌细胞株,采用MTT法以及流式细胞仪检测不同用药方案的抗肿瘤作用;在裸鼠H460肺癌模型中验证重组腺病毒TRAIL制剂与C225的协同抗肿瘤作用。结果:在体外实验中发现Iressa和Tarceva可以增加重组腺病毒TRAIL基因制剂对H460肺癌细胞的抗肿瘤作用(P<0.05);重组腺病毒TRAIL基因制剂在体外实验中与C225并不存在协同效应(P>0.05),但在裸鼠H460肺癌模型中重组腺病毒TRAIL基因制剂与C225有明显的协同抗肿瘤作用(P<0.05)。结论:本研究初步探讨了基因治疗与靶向治疗的联合抗肿瘤作用,实验发现EGFR信号通路上的靶向治疗药物包括小分子酪氨酸激酶抑制剂以及EGFR单克隆抗体均可以增加重组腺病毒TRAIL基因制剂抗肺癌作用。     

Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells

Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future.     

Inhibition of proteasome activity sensitizes human granulosa tumor cells to TRAIL-induced cell death

Human granulosa tumor cell (GCT) lines (KGN and COV434) were utilized to establish the combinatorial effects of TRAIL treatment and a proteasome inhibitor on cell viability, in vitro. TRAIL induced a slight, but consistent, decrease in viability for both cell lines, and pharmacologic inhibition of proteasome activity, using Z-LLF-CHO (Z-LLF), synergistically enhanced TRAIL-induced loss of viability. This enhanced sensitization was associated with the up-regulation of a TRAIL receptor, DR5, and pro-apoptotic Bax. Targeted reduction of p53 expression revealed that the ability of Z-LLF to enhance DR5 and Bax expression occurs independent of p53 activity. These studies underscore the potential to develop targeted treatments for GCTs using established cell lines.     

甲状腺癌组织中凋亡相关蛋白Fas,FasL,Bcl-2和Bax的表达及意义

目的 研究凋亡相关蛋白Fas,FasL,Bcl-2和Bax在甲状腺癌组织中的表达及意义.方法应用免疫组织化学,采用TUMEL法,检测34例甲状腺癌及10例结节性甲状腺肿、9例甲状腺腺瘤组织病理标本中的细胞凋亡指数(Apoptosis Index, AI);并用通用型二步法对凋亡相关蛋白Fas,FasL,Bcl-2和Bax的表达进行检测.结果 (1)对照组细胞平均AI(%)为12.39±2.21,甲状腺癌细胞平均AI(%)为20.17±2.68,即肿瘤组的细胞凋亡指数高于对照组,二者有显著性差异(t＝4.138＞t(0.05),P＜0.05).(2)甲状腺癌组织中Fas,FasL,Bcl-2和Bax蛋白表达显著高于对照组.(3)Fas,FasL,Bcl-2和Bax蛋白表达的阳性率与其病理类型、临床分期无明显关系(P＞0.05).结论甲状腺癌组织中有较高水平细胞凋亡,Fas,FasL,Bcl-2和Bax的表达均明显增强,并且通过参与调节细胞凋亡而与甲状腺癌的发生有关,但与病理类型、临床分期无明显关系.     

DR4 specific TRAIL variants are more efficacious than wild-type TRAIL in pancreatic cancer

Current treatment modalities for pancreatic carcinoma afford only modest survival benefits. TRAIL, as a potent and specific inducer of apoptosis in cancer cells, would be a promising new treatment option. However, since not all pancreatic cancer cells respond to TRAIL, further improvements and optimizations are still needed. One strategy to improve the effectiveness of TRAIL-based therapies is to specifically target one of the 2 cell death inducing TRAIL-receptors, TRAIL-R1 or TRAIL-R2 to overcome resistance. To this end, we designed constructs expressing soluble TRAIL (sTRAIL) variants that were rendered specific for either TRAIL-R1 or TRAIL-R2 by amino acid changes in the TRAIL ectodomain. When we expressed these constructs, including wild-type sTRAIL (sTRAIL^wt), TRAIL-R1 (sTRAIL^DR4) and TRAIL-R2 (sTRAIL^DR5) specific variants, in 293 producer cells we found all to be readily expressed and secreted into the supernatant. These supernatants were subsequently transferred onto target cancer cells and apoptosis measured. We found that the TRAIL-R1 specific variant had higher apoptosis-inducing activity in human pancreatic carcinoma Colo357 cells as well as PancTu1 cells that were additionally sensitized by targeting of XIAP. Finally, we tested TRAIL-R1 specific recombinant TRAIL protein (rTRAIL^DR4) on Colo357 xenografts in nude mice and found them to be more efficacious than rTRAIL^wt. Our results demonstrate the benefits of synthetic biological approaches and show that TRAIL-R1 specific variants can potentially enhance the therapeutic efficacy of TRAIL-based therapies in pancreatic cancer, suggesting that they can possibly become part of individualized and tumor specific combination treatments in the future.     

Targeting apoptosis proteins in hematological malignancies

The apoptotic machinery plays a key role in hematopoietic cell homeostasis. Terminally differentiated cells are eliminated, at least in part, by apoptosis, whereas part of the apoptotic machinery, including one or several caspases, is required to go through very specific steps of the differentiation pathways. A number of hematological diseases involve a deregulation of this machinery, which in most cases is a decrease in cell sensitivity to pro-apoptotic signals through over-expression of anti-apoptotic molecules. In some situations however, e.g. in the erythroid lineage of low grade myelodysplastic syndromes, cell sensitivity to apoptosis is increased in a death receptor-dependent manner and cell death pathways are inhibited only when these diseases progress into high grade and acute leukemia. Therapeutic strategies targeting the apoptotic machinery specifically block cell death inhibitors that are over-expressed in transformed cells, mainly Bcl-2-related proteins and Inhibitor of Apoptosis Proteins (IAPs). Another strategy is the activation of the extrinsic pathway to apoptosis, mainly through the death receptor agonist Tumor necrosis factor-Related Apoptosis Inducing Ligand (TRAIL) or agonistic antibodies targeting TRAIL receptors. The use of inhibitors of death receptors could make sense when these receptors are involved in excessive cell death or activation of survival pathways. Most of the drugs targeting apoptotic pathways introduced in clinics have demonstrated their tolerability. Their efficacy, either alone or in combination with other drugs such as demethylating agents and histone deacetylase inhibitors, is currently tested in both myeloid and lymphoid hematological diseases.     

Diosgenin induces death receptor-5 through activation of p38 pathway and promotes TRAIL-induced apoptosis in colon cancer cells

Previously, we demonstrated that diosgenin induced apoptosis in colorectal cancer cell lines HCT-116 and HT-29. HT-29 cells have been reported to be one of the most resistant colorectal cancer cell lines to TRAIL-induced apoptosis. In this study, we investigated the effect of diosgenin on TRAIL-induced apoptosis in HT-29 cells. We showed that diosgenin sensitizes HT-29 cells to TRAIL-induced apoptosis. Mechanisms underlying this sensitization mainly involved diosgenin-induced p38 MAPK pathway activation and subsequent DR5 overexpression. Furthermore, we showed that diosgenin alone, TRAIL alone or combination treatment increased COX-2 expression and that the use of a COX-2 inhibitor further increased apoptosis induction.     

顺铂通过促进死亡受体4在脂筏内聚集增强肿瘤坏死因子相关凋亡诱导配体对胃癌MGC803细胞凋亡的作用

目的 探讨顺铂影响胃癌细胞对肿瘤坏死因子相关凋亡诱导配体(TRAIL)敏感性的机制.方法 以顺铂和TRAIL单独及联合作用MGC803细胞,采用四甲基偶氮唑蓝(MTT)法测定各组细胞的增殖能力,流式细胞仪检测细胞凋亡,Western blot检测caspase-8和caspase-3的蛋白表达,免疫荧光显微技术观察脂筏和死亡受体4(DR4)的分布.以50 mg/L的制霉菌素预处理MGC803细胞1 h,之后加入顺铂和TRAIL,观察细胞凋亡的变化.结果 100 μg/L TRAIL作用MGC803细胞24 h,增殖抑制率为(8.51±3.45)%,细胞凋亡率为(3.26±0.89)%.8.49 mg/L顺铂作用MGC803细胞24 h,增殖抑制率为(52.58±4.57)%,细胞凋亡率为(23.10±3.41)%.100 μg/L TRAIL和8.49 mg/L顺铂联合作用时,细胞增殖抑制率提高至(76.43±5.35)%,细胞凋亡率提高到(42.56±4.11)%,均高于相同浓度顺铂和TRAIL单独作用组(P＜0.05).同时检测到caspase-8和caspase-3的裂解.TRAIL未引起明显的脂筏和DR4聚集,而顺铂明显促进了DR4在聚集脂筏内的定位.50 mg/L制霉菌素处理MGC803细胞24 h,细胞凋亡率为(3.66±0.52)%.经制霉菌素预处理后,顺铂作用下MGC803细胞的凋亡率为(22.76±2.97)%,与未经制霉菌素预处理组[(25.74±3.28)%]差异无统计学意义(P=0.248);而顺铂和TRAIL联合作用下细胞凋亡率为(31.52±3.99)%,与未经制霉菌素预处理组[(43.16±4.26)%]差异有统计学意义(P＜0.001).结论 顺铂通过促进死亡受体在脂筏聚集增强了TRAIL诱导的胃癌MGC803细胞凋亡.

Abstract：

Objective Gastric cancer cells are insensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To sensitize gastric cancer cells to TRAIL, we treated gastric cancer MGC803 cells with TRAIL and cisplatin. Methods Cell proliferation was measured using MTT assay. Cell apoptosis was determined by flow cytometry. Expression of proteins was analyzed by Western blot. The distribution of lipid rafts and death receptors was analyzed by immunofluorescence microscopy. MGC803 cells were pretreated with 50 mg/L nystatin for 1 h, and followed by the treatment of cisplatin and TRAIL. Results 100 μg/L TRAIL resulted in (8.51±3.45)% inhibition of cell proliferation and caused (3.26±0.89)% cell apoptosis in MGC803 cells. Compared with the treatment with cisplatin alone, treatment with TRAIL (100 μg/L) and cisplatin (8.49 mg/L, IC50 dose of 24 h) led to a dramatic increase in both inhibition of cell proliferation [(52.58±4.57)% vs. (76.43±5.35)%, P＜0.05]and cell apoptosis [(23. 10±3. 41)% vs. (42.56±4.11)%, P＜0.05]. Moreover, cleavage of caspase-8 and caspase-3 was detected. TRAIL (100 μg/L) did not induce obvious lipid rafts aggregation and death receptor 4 (DR4) clustering, while cisplatin (8.49 mg/L) significantly promoted the localization of DR4 in aggregated lipid rafts. Pretreatment with 50 mg/L nystatin, a cholesterol-sequestering agent, triggered (3.66±0.52)% cell apoptosis after 24 h. Pretreatment with nystatin for 1 h before the addition of 8.49 mg/L cisplatin for 24 h caused a decreased tendency to cell apoptosis [(25.74±3.28)% vs. (22.76±2.97)%]. While, pretreatment with nystatin before the addition of cisplatin and TRAIL, the proportion of apoptotic cells decreased from (43. 16±4.26)% to (31.52 ± 3.99)% (P＜0.05). Conclusion Cisplatin enhances TRAIL-induced apoptosis in gastric cancer MGC803 cells through clustering death receptors into lipid rafts.     

TRAIL及其受体在膀胱癌中的表达及相关性研究

目的:观察肿瘤坏死因子相关诱导凋亡配体(TRAIL)及其受体DR4、DcR1在膀胱癌组织及正常膀胱黏膜中的表达,并探讨其与临床病理特征间的相关性.方法:采用免疫组织化学PV9001二步法检测64例膀胱癌石蜡标本及14例正常膀胱组织中的TRAIL、DR4、DcR1的表达,结合患者病理资料,分析其与患者临床病理特征之间的关系.结果:TRAIL、DR4在膀胱癌组织中的表达较正常组织中的表达低,DcR1表达与之相反(P<0.05).进一步统计分析得出TRAIL、DR4在高分化膀胱癌中表达较中低分化癌高,DcR1与之相反(P<0.05).TRAIL、DR4、DcR1的表达与性别、年龄、临床分期及是否淋巴转移等病理特征无关(P>0.05).结论:TRAIL、DR4、DcR1在膀胱癌组织及正常组织中的表达具有统计学差异,并且与病理分级具有一定的相关性,与其他临床病理特征无关.     

Livin/ML-IAP as a new target for cancer treatment

Livin is a member of the inhibitors of apoptosis protein (IAP) gene family, which encodes negative regulatory proteins that prevent cell apoptosis. Livin is selectively expressed in the most common human neoplasms and appears to be involved in tumor cell resistance to chemotherapeutic agents. Several studies in vitro and in vivo have demonstrated that down-regulation of Livin expression increases the apoptotic rate, reduces tumor growth potential and sensitized tumor cells to chemotherapeutic drugs. This review will focus on the role of this protein during cancer development and progression and will demonstrate possible targets for cancer therapy.     

TRAIL, FasL and a blocking anti-DR5 antibody augment paclitaxel-induced apoptosis in human non-small-cell lung cancer.

Lung carcinoma is one of the most frequent causes of malignancy-related mortality in the world. Paclitaxel (PA) is an antineoplastic agent used in the treatment of non-small-cell lung cancer (NSCLC) and possesses a single-agent response rate approaching 25%. PA kills tumor cells by inducing both cellular necrosis and apoptosis. Fas and Trail receptors (DR4 and DR5) are TNF family members and act as death signal transduction proteins in the apoptosis cascade. Despite the importance of PA in lung cancer treatment, the function of Fas, DR4 and DR5 in PA-induced apoptosis, as well as the effect of their respective ligands FasL and TRAIL alone or in combination with PA, remains poorly understood. We show here that 10 microM PA induces a significant 10- to 57-fold increase in primary lung cancer cell apoptosis and is associated with 20-215% increases in caspase-3 activity in various NSCLC cell types. All the lung cancer cells express Fas, FasL, DR4 and DR5; however PA did not significantly modify their levels. We provide here the first time evidence that TRAIL is a potent inducer of apoptosis in multiple NSCLC cell lines. Noticeably, CH11, the Fas receptor cross-linking and the antagonistic anti-DR5 antibody enhance considerably the spontaneous apoptotic rate in 3 out of 5 cell types. The combination treatments, FasL+PA, TRAIL+PA or PA+anti-DR5 antibody, greatly enhance PA-apoptotic effect in most cell lines. These data suggest that the use of new combination treatment with PA and ligands targeting Fas or TRAIL receptors would be particularly efficacious.