Therapeutic efficacy by targeting correction of notch1-induced aberrants in uveal tumors

There is a need for more effective treatments for uveal melanoma. The recombinant oncolytic adenovirus H101 replicates specifically in p53-depleted tumor cells, and has been approved for use by the Chinese State Food and Drug Administration. However, this treatment is associated with subsequent remission. Transfection of uveal melanoma cells with a small interfering RNA against Notch1 (siNotch1) effectively suppressed Notch1 expression, resulting in significant cell growth inhibition when combined with H101 treatment. Combined treatment with siNotch1 and H101 (H101-Notch1-siRNA) greatly enhanced apoptosis and cell cycle arrest in vitro as compared to treatment with H101 or siNotch1 alone. For in vivo treatments, the combined treatment of siNotch1 and H101 showed remarkable tumor growth inhibition and prolonged mouse survival in the OCM1 xenograft model. We predict that Notch pathway deregulation could be a feature of uveal melanoma, and could be a therapeutic target, especially if p53 is concurrently targeted.     

嵌合型E1B 55-kDa蛋白缺陷型腺病毒载体治疗肿瘤的评价

ONYX-015和H101为可复制E1B 55-kDa蛋白缺陷的C族腺病毒,它们正作为抗癌药物进行临床研究. 然而它们在癌症基因治疗中的应用却受到了C族腺病毒天然特性的制约,部分原因是由于在恶性肿瘤中C族腺病毒受体(coxsackievirus-adenovirus receptor,CAR) 的表达量较低. 构建了一个以H101为骨架的含有编码35型腺病毒鞭毛区域的基因,替代5型腺病毒鞭毛基因的嵌合型腺病毒载体. 这一改动使得腺病毒载体可以通过一种在肿瘤中高表达的膜蛋白CD46感染肿瘤细胞. 应用RT-PCR方法检测不同肿瘤细胞株中CAR和CD46表达量的区别. 在CAR受体低表达的细胞株中(MDA-MB-435和MCF-7),H101-F35表现出比H101和ONYX-015更强的细胞杀伤效果;在CAR受体高表达的细胞株中(A549,NCI-H446,Hep3B,LNCaP,ZR-75-30和Bcap-37),H101-F35、 H101和ONYX-015的细胞杀伤效果则相似. 在荷MDA-MB-435肿瘤的裸鼠模型中,注射H101-F35的抑瘤效果比注射H101的抑瘤效果更明显. 这些结果表明嵌合型溶瘤腺病毒载体H101-F35在肿瘤基因治疗中将有很好的应用前景.     

溶瘤腺病毒靶向杀灭葡萄膜黑色素瘤的实验研究

葡萄膜黑色素瘤是成人最严重的原发性恶性肿瘤之一.传统的治疗方法,包括手术、放射治疗和化学治疗效果都不是很理想.溶瘤腺病毒H101,能够特异性地在p53突变的肿瘤细胞中复制并杀伤肿瘤细胞,同时对正常细胞影响较少,且已由中国国家食品药品监督管理总局批准上市.为了研究H101对葡萄膜黑色素瘤的治疗效果,通过体外感染葡萄膜黑色素瘤细胞,发现H101能够显著抑制葡萄膜黑色素瘤细胞的增殖并促进细胞凋亡,抑制细胞周期,而对正常的ARPE-19细胞没有影响.在体内实验中,建立了SP6.5细胞的荷瘤小鼠模型,在H101治疗后抑制了肿瘤的生长,延长了动物寿命.上述结果表明,溶瘤腺病毒H101治疗葡萄膜黑色素瘤是一种可行的方法.     

Proteasome inhibitor MG-132 modifies coxsackie and adenovirus receptor expression in colon cancer cell line lovo

The efficacy of adenovirus vector-based cancer gene therapy is controversial. Its uptake by cells in many cases requires the major receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR). Low transduction is believed to be one of the main barriers as the expression of CAR on tumor cells is frequently reduced. Increasing CAR expression on tumor cells thus offers a promising opportunity for more effective adenovirus based treatment. Expression of CAR in 62 cases of colon tumor specimens were examined with immunohistochemistry. To modify the CAR expression, the effects of proteasome inhibitor MG132 on CAR expression of colon cancer cell lines were determined by flow cytometry, RT-PCR, and western blot. To evaluate adenovirus transfer, we further used rAd.EGFP, rAd.p53, and oncolytic adenovirus to infect target cells. The CAR expression was significantly decreased in colon carcinomas, both in primary tumors and lymphonode metastasis. Though the deregulation of CAR occurred in early disease and showed no relationship with TNM stage, when primary tumors are more than 5 cm in diameter, this deregulation becomes more frequent. More importantly, proteasome inhibitor MG-132 could enhance CAR expression in colon carcinoma cell line lovo, accompanied with enhanced adenovirus transfer, target gene expression, and oncolysis. These data provide a rational basis for evaluation of CAR expression in tumors and pretreatment with CAR conditioner prior to adenovirus vector-based gene therapy.     

p53选择性抗瘤腺病毒

多种肿瘤的抑癌基因p53发生了突变。一种腺病毒E1B缺失体ONYX-015能够在p53突变的肿瘤细胞内有效地复制而导致痛细胞的裂解,但不能在p53正常的细胞内复制。这种p53选择性抗瘤病毒代表了一类新的抗癌武器:溶癌病毒。     

Notch1 activation reduces proliferation in the multipotent hematopoietic progenitor cell line FDCP-mix through a p53-dependent pathway but Notch1 effects on myeloid and erythroid differentiation are independent of p53

Signaling mediated by activation of the transmembrane receptor Notch influences cell-fate decisions, differentiation, proliferation, and cell survival. Activated Notch reduces proliferation by altering cell-cycle kinetics and promotes differentiation in hematopoietic progenitor cells. Here, we investigated if the G(1) arrest and differentiation induced by activated mNotch1 are dependent on tumor suppressor p53, a critical mediator of cellular growth arrest. Multipotent wild-type p53-expressing (p53(wt)) and p53-deficient (p53(null)) hematopoietic progenitor cell lines (FDCP-mix) carrying an inducible mNotch1 system were used to investigate the effects of proliferation and differentiation upon mNotch1 signaling. While activated Notch reduced proliferation of p53(wt)-cells, no change was observed in p53(null)-cells. Activated Notch upregulated the p53 target p21(cip/waf) in p53(wt)-cells, but not in p53(null)-cells. Induction of the p21(cip/waf) gene by activated Notch was mediated by increased binding of p53 to p53-binding sites in the p21(cip/waf) promoter and was independent of the canonical RBP-J binding site. Re-expression of p53(wt) in p53(null) cells restored the inhibition of proliferation by activated Notch. Thus, activated Notch inhibits proliferation of multipotent hematopoietic progenitor cells via a p53-dependent pathway. In contrast, myeloid and erythroid differentiation was similarly induced in p53(wt) and p53(null) cells. These data suggest that Notch signaling triggers two distinct pathways, a p53-dependent one leading to a block in proliferation and a p53-independent one promoting differentiation.     

Notch1 Signaling Sensitizes Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis in Human Hepatocellular Carcinoma Cells by Inhibiting Akt/Hdm2-mediated p53 Degradation and Up-regulating p53-dependent DR5 Expression

Notch signaling plays a critical role in regulating cell proliferation, differentiation, and apoptosis. Our previous study showed that overexpression of Notch1 could inhibit human hepatocellular carcinoma (HCC) cell growth by arresting the cell cycle and inducing apoptosis. HCC cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), so new therapeutic approaches have been explored to sensitize HCC cells to TRAIL-induced apoptosis. We are wondering whether and how Notch1 signaling can enhance the sensitivity of HCC cells to TRAIL-induced apoptosis. In this study, we found that overexpression of ICN, the constitutive activated form of Notch1, up-regulated p53 protein expression in HCC cells by inhibiting proteasome degradation. p53 up-regulation was further observed in human primary hepatocellular carcinoma cells after activation of Notch signaling. Inhibition of the Akt/Hdm2 pathway by Notch1 signaling was responsible for the suppression of p53 proteasomal degradation, thus contributing to the Notch1 signaling-mediated up-regulation of p53 expression. Accordingly, Notch1 signaling could make HCC cells more sensitive to TRAIL-induced apoptosis, whereas Notch1 signaling lost the synergistic promotion of TRAIL-induced apoptosis in p53-silenced HepG2 HCC cells and p53-defective Hep3B HCC cells. The data suggest that enhancement of TRAIL-induced apoptosis by Notch1 signaling is dependent upon p53 up-regulation. Furthermore, Notch1 signaling could enhance DR5 expression in a p53-dependent manner. Taken together, Notch1 signaling sensitizes TRAIL-induced apoptosis in HCC cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression. Thus, our results suggest that activation of Notch1 signaling may be a promising approach to improve the therapeutic efficacy of TRAIL-resistant HCC.Notch signaling determines cell fate and affects cell proliferation, differentiation, and apoptosis during cell development (1). As a highly conserved family, Notch coordinates a signaling cascade present in all animal species studied to date (2). Mammals have four Notch receptors that bind five different ligands, among which Notch1 signaling functions in many physiological and pathophysiological processes of numerous cell types, and its dysfunction results in a variety of developmental defects, including embryonic lethality and adult disorders. For example, the Notch1/Jagged1 signaling pathway is activated during liver regeneration and is potentially contributing to signals affecting hepatocyte growth (3, 4). Inducible inactivation of Notch1 has been shown to cause nodular regenerative hyperplasia in mouse liver (5). These studies suggest that Notch1 signaling may be involved in the liver functions and the pathogenesis of liver diseases. Our previous study demonstrated that Notch1 signaling could suppress the growth of human hepatocellular carcinoma (HCC)⁴ cells by arresting the cell cycle and inducing apoptosis (6). However, the underlying molecular mechanisms remain to be fully understood.p53, an important tumor suppressor gene, is involved in cell cycle arrest and cellular apoptosis. Its activity is mostly regulated by complex networks of post-translational modifications, including phosphorylation, ubiquitination, and proteasome degradation. One protein that is essential for determining p53 stability is Mdm2 (mouse double minute protein 2) (7). Mdm2, a nuclear phosphoprotein and an E3 ubiquitin ligase, binds to p53 and ubiquitinates p53, leading to proteosome degradation of p53 (8). Another important mechanism of p53 stability is related to its phosphorylation status, which is Mdm2-dependent or Mdm2-independent (9). As to the regulation of p53 by Notch1, there are controversial reports that Notch1 activation increased p53 expression in neural progenitor cells (10); however, suppression of p53 by Notch signaling was also well established in lymphomagenesis (11). We also reported that Notch1 signaling significantly up-regulated p53 expression in SMMC7721 HCC cells (6); however, the molecular mechanisms remained unclear and needed to be further characterized.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of a superfamily of cell death-inducing ligands, induces apoptosis in a broad range of transformed cells and tumor cells but has little or no effect on normal cells (12). Therefore, TRAIL has been regarded as a potential drug for cancer therapy (12, 13). However, several kinds of cancer, including HCC, are not sensitive to soluble TRAIL treatment (14). HCC accounts for 80–90% of liver cancers and is one of the most prevalent carcinomas throughout the world, especially in Africa and Asia. Thus, it is worthwhile to find a new strategy to overcome the resistance of HCC cells to TRAIL-induced apoptosis.Considering that Notch1 signaling up-regulates p53 and induces apoptosis of HCC cells and that there are no reports to date that address the relationship between Notch1 signaling and TRAIL-induced apoptosis, in this study, we investigated whether and how Notch1 signaling could sensitize HCC cells to TRAIL-induced apoptosis. We demonstrate that Notch1 signaling up-regulates p53 expression by inhibiting proteasome degradation via, at least in part, suppressing the phosphatidylinositol 3-kinase/Akt/Hdm2 pathway. In addition, we here report that Notch1 signaling enhances DR5 (death receptor 5) expression in a p53-dependent manner, and DR5 contributes, at least in part, to the enhancement of TRAIL-induced apoptosis by Notch1 signaling. Accordingly, Notch1 signaling sensitizes HCC cells to TRAIL-induced apoptosis.     

溶瘤腺病毒在肿瘤靶向治疗中的研究进展

溶瘤腺病毒是一组通过基因工程构建的腺病毒、能够选择性在肿瘤细胞中完成感染-复制周期,从而特异性地杀伤、溶解肿瘤而不伤及其他正常细胞、组织,其作用机制包括:通过基因的缺失突变、插入特异性启动子、以及通过病毒结构蛋白的修饰等方面,实现肿瘤靶向治疗作用。本文就相关研究及进展进行综述。     

溶瘤腺病毒在肿瘤靶向治疗中的研究进展

溶瘤腺病毒是一组通过基因工程构建的腺病毒、能够选择性在肿瘤细胞中完成感染-复制周期,从而特异性地杀伤、溶解肿瘤而不伤及其他正常细胞、组织,其作用机制包括：通过基因的缺失突变、插入特异性启动子、以及通过病毒结构蛋白的修饰等方面,实现肿瘤靶向治疗作用。本文就相关研究及进展进行综述。     

Contribution of CD95 ligand-induced neutrophil infiltration to the bystander effect in p53 gene therapy for human cancer

Clinical trials of adenoviral p53 gene therapy provide the evidence that the bystander effect induced by the wild-type p53 gene transfer on adjacent tumor cells contributes to tumor progression; its mechanism, however, remains uncharacterized. We report in this work that injection of adenovirus expressing the human wild-type p53 gene (Ad5CMVp53) into established human colorectal tumors in nu/nu mice resulted in CD95 ligand (CD95L) overexpression, followed by a massive neutrophil infiltration. Culture supernatants of human colorectal cancer cells infected with Ad5CMVp53 exhibited a potent chemotactic activity against murine polymorphonuclear neutrophils, which could be abolished by the anti-CD95L mAb (NOK-1). In vivo cell depletion experiments indicated that neutrophils were in part responsible for the antitumor effect of the Ad5CMVp53 infection. Our data directly suggest that overexpression of CD95L by the wild-type p53 gene transfer induces neutrophil infiltration into human colorectal tumors, which may play a critical role in the bystander effect of p53 gene therapy.     

Cancer selective adenoviruses

Ten years ago Frank McCormick proposed dl1520 as an oncolytic adenovirus. Although great as an inspiration for better oncolytic viruses it was far from a good product. As Onyx-015, it underwent a wish-fulfilling clinical development program seizing the opportunity left by its p53-targeted non-replicative counterpart Ad-p53. Now, facing a skeptical environment, more selective and potent oncolytic adenoviruses await their clinical opportunity. However, advance in key issues remains elusive, such as, selectivity or retargeting at the level of cell receptors to improve pharmacokinetics. Preclinical models and a few clinical data on biodistribution show that only a minimal proportion of the injected dose reaches the tumors after systemic administration. Once in the tumor, the virus must overcome barriers to efficient spread imposed by stroma and immune responses. Arming the oncolytic virus with transgenes is a natural combination of virotherapy and gene therapy strategies. Transgenes that increase virus production or cellular spread may help to overcome these barriers. Cytotoxic transgenes can help to eliminate tumor cells but need to be compatible with efficient virus replication. These challenges require a careful approach to clinical development and a great deal of collaboration to launch clinical tests with a virus backbone that contains intellectual property from multiple sources.     

Epigenetic silencing of Notch signaling in gastrointestinal cancers

The Notch signaling pathway drives proliferation, differentiation, apoptosis, cell fate choices and maintenance of stem cells during embryogenesis and in self-renewing tissues of the adult. In addition, aberrant Notch signaling has been implicated in several tumors, where Notch can function both as an oncogene or a tumor-suppressor gene, depending on the context. This Extra View aims to review what is currently known about Notch signaling, in particular in gastrointestinal tumors, providing a summary of our data on Notch1 signaling in gastric cancer with results obtained in colorectal cancer (CRC). We have already reported that the epigenetic regulation of the Notch ligand DLL1 controls Notch1 signaling activation in gastric cancer, and that Notch1 inhibition is associated with the diffuse type of gastric cancer. Here, we describe additional data showing that in CRC cell lines, unlike gastric cancer, DLL1 expression is not regulated by promoter methylation. Moreover, in CRC, Notch1 receptor is not affected by any mutation. These data suggest a different regulation of Notch1 signaling between gastric cancer and CRC.     

Epigenetic silencing of Notch signaling in gastrointestinal cancers

The Notch signaling pathway drives proliferation, differentiation, apoptosis, cell fate choices and maintenance of stem cells during embryogenesis and in self-renewing tissues of the adult. In addition, aberrant Notch signaling has been implicated in several tumors, where Notch can function both as an oncogene or a tumor-suppressor gene, depending on the context.

This Extra View aims to review what is currently known about Notch signaling, in particular in gastrointestinal tumors, providing a summary of our data on Notch1 signaling in gastric cancer with results obtained in colorectal cancer (CRC).

We have already reported that the epigenetic regulation of the Notch ligand DLL1 controls Notch1 signaling activation in gastric cancer, and that Notch1 inhibition is associated with the diffuse type of gastric cancer. Here, we describe additional data showing that in CRC cell lines, unlike gastric cancer, DLL1 expression is not regulated by promoter methylation. Moreover, in CRC, Notch1 receptor is not affected by any mutation. These data suggest a different regulation of Notch1 signaling between gastric cancer and CRC.     

In vivo inhibition of endogenous brain tumors through systemic interference of Hedgehog signaling in mice

The full spectrum of developmental potential includes normal as well as abnormal and disease states. We therefore subscribe to the idea that tumors derive from the operation of paradevelopmental programs that yield consistent and recognizable morphologies. Work in frogs and mice shows that Hedgehog (Hh)-Gli signaling controls stem cell lineages and that its deregulation leads to tumor formation. Moreover, human tumor cells require sustained Hh-Gli signaling for proliferation as cyclopamine, an alkaloid of the lily Veratrum californicum that blocks the Hh pathway, inhibits the growth of different tumor cells in vitro as well as in subcutaneous xenografts. However, the evidence that systemic treatment is an effective anti-cancer therapy is missing. Here we have used Ptc1(+/-); p53(-/-) mice which develop medulloblastoma to test the ability of cyclopamine to inhibit endogenous tumor growth in vivo after tumor initiation through intraperitoneal delivery, which avoids the brain damage associated with direct injection. We find that systemic cyclopamine administration improves the health of Ptc1(+/-);p53(-/-) animals. Analyses of the cerebella of cyclopamine-treated animals show a severe reduction in tumor size and a large decrease in the number of Ptc1-expressing cells, as a readout of cells with an active Hu-Gli pathway, as well as an impairment of their proliferative capacity, always in comparison with vehicle treated mice. Our data demonstrate that systemic treatment with cyclopamine inhibits tumor growth in the brain supporting its therapeutical value for human HH-dependent tumors. They also demonstrate that even the complete loss of the well-known tumor suppressor p53 does not render the tumor independent of Hh pathway function.     

Multifaceted p21 in carcinogenesis,stemness of tumor and tumor therapy

Cancer cells possess metabolic properties that are different from those of benign cells. p21, encoded by CDKN1A gene, also named p21^Cip1/WAF1, was first identified as a cyclin-dependent kinase regulator that suppresses cell cycle G1/S phase and retinoblastoma protein phosphorylation. CDKN1A (p21) acts as the downstream target gene of TP53 (p53), and its expression is induced by wild-type p53 and it is not associated with mutant p53. p21 has been characterized as a vital regulator that involves multiple cell functions, including G1/S cell cycle progression, cell growth, DNA damage, and cell stemness. In 1994, p21 was found as a tumor suppressor in brain, lung and colon cancer by targeting p53 and was associated with tumorigenesis and metastasis. Notably, p21 plays a significant role in tumor development through p53-dependent and p53-independent pathways. In addition, expression of p21 is closely related to the resting state or terminal differentiation of cells. p21 is also associated with cancer stem cells and acts as a biomarker for such cells. In cancer therapy, given the importance of p21 in regulating the G1/S and G2 check points, it is not surprising that p21 is implicated in response to many cancer treatments and p21 promotes the effect of oncolytic virotherapy.     

昆虫Notch信号通路研究进展

Notch 信号通路由 Notch 受体、Notch 配体(DSL 蛋白)、CSL[C promoter binding factor-1(CBF1),Suppressor of hairless(Su(H)),Lag-1]转录因子、其他效应子和Notch调节分子构成,在动物组织的发育和器官的细胞命运决定中起着基础性的...