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2.  Complete Eradication of Xenograft Hepatoma by Oncolytic Adenovirus ZD55 Harboring TRAIL-IETD-Smac Gene with Broad Antitumor Effect 
Human Gene Therapy  2012;23(9):992-1002.
Cancer-targeting dual-gene virotherapy (CTGVT-DG) is an important modification of CTGVT, in which two suitable genes are used to obtain an excellent antitumor effect. A key problem is to join the two genes to form one fused gene, and then to clone it into the oncolytic viral vector so that only one investigational new drug application, instead of two, is required for clinical use. Many linkers (e.g., internal ribosome entry site) are used to join two genes together, but they are not all equally efficacious. Here, we describe finding the best linker, that is, sequence encoding the four amino acids IETD, to join the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene and the second mitochondria-derived activator of caspase (Smac) gene to form TRAIL-IETD-Smac and inserting it into oncolytic viral vector ZD55 to construct ZD55-TRAIL-IETD-Smac, which matched ZD55-TRAIL plus ZD55-Smac in completely eliminating xenograft hepatoma. ZD55-TRAIL-IETD-Smac works by quantitative cleavage at IETD↓by inducing caspase-8; activation or inhibition of caspase-8 could up- or downregulate cleavage, respectively. The cleaved product, TRAIL-IETD, does not affect the function of TRAIL. Numerous experiments have shown that the combined use of ZD55-TRAIL plus ZD55-X could completely eradicate many xenograft tumors, and therefore the IETD is potentially a useful linker to construct many antitumor drugs, for example, ZD55-TRAIL-IETD-X, where X has a compensative or synergetic effect on TRAIL. We found that the antitumor effect of ZD55-IL-24-IETD-TRAIL also has an equivalent antitumor effect compared with the combined use of ZD55-IL-24 plus ZD55-TRAIL, because ZD55-IL-24 could also induce caspase-8. This means that IETD, as a two-gene linker, may have broad use.
Wang and colleagues identify the amino acid sequence IETD as the most effective linker to mediate cancer-targeting dual gene virotherapy. IETD-linked tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and second mitochondria-derived activator of caspase (Smac) were cloned into the ZD55 oncolytic viral vector. ZD55-TRAIL-IETD-Smac completely eliminated xenografted hepatomas in nude mice.
PMCID: PMC3440030  PMID: 22530834
3.  New Advance in Caspase-Independent Programmed Cell Death and its Potential in Cancer Therapy 
Caspase activation has been frequently viewed as synonymous with programmed cell death (PCcD); however, accumulating evidence showed that there existing caspase-independent PCcD pathways displaying morphologies that are not fully consistent with classical apoptosis. In this article, we will focus on the most recent progresses of different models of PCcD independent of caspases activity. Since some tumor cells can unexpectedly survive the activation of caspases, and tumor suppressor proteins that activate caspase-independent PCcD are commonly mutated in human cancer, the alternative cell death pathways are gaining increasing interest among cancer researchers. Though the mechanism of this cell death pathway is poorly understood, it is clear that a full understanding of the regulation of caspase-independent PCcD could provide new means of improving current diagnosis and promoting conceptual advances for the design of new therapeutic strategies for cancer therapy.
PMCID: PMC3614612  PMID: 23674984
apoptosis; autophagy; cancer therapy; caspase-independent programmed cell death
4.  Acetylcholinesterase overexpression mediated by oncolytic adenovirus exhibited potent anti-tumor effect 
BMC Cancer  2014;14(1):668.
Acetylcholinesterase (AChE) mainly functions as an efficient terminator for acetylcholine signaling transmission. Here, we reported the effect of AChE on gastric cancer therapy.
The expression of AChE in gastric cancerous tissues and adjacent non-cancerous tissues was examined by immunohistochemistry. Gastric cancer cells were treated with AChE delivered by replication-deficient adenoviral vector (Ad.AChE) or oncolytic adenoviral vector (ZD55-AChE), respectively, followed by measurement of cell viability and apoptosis by MTT assay and apoptosis detection assays. In vivo, the tumor growth of gastric cancer xenografts in mice treated with Ad.AChE or ZD55-AChE (1 × 109 PFU) were measured. In addition, the cell viability of gastric cancer stem cells treated with Ad.AChE or ZD55-AChE were evaluated by MTT assay.
A positive correlation was found between higher level of AChE expression in gastric cancer patient samples and longer survival time of the patients. Ad.AChE and ZD55-AChE inhibited gastric cancer cell growth, and low dose of ZD55-AChE induced mitochondrial pathway of apoptosis in cells. ZD55-AChE repressed tumor growth in vivo, and the anti-tumor efficacy is greater than Ad.AChE. Moreover, ZD55-AChE suppressed the growth of gastric cancer stem cells.
ZD55-AChE represented potential therapeutic effect for human gastric cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-668) contains supplementary material, which is available to authorized users.
PMCID: PMC4169801  PMID: 25220382
AChE; Oncolytic adenovirus; Gastric cancer; Apoptosis
5.  Spectrum of LKB1, EGFR, and KRAS Mutations in Chinese Lung Adenocarcinomas 
Somatic LKB1 mutations are found in lung adenocarcinomas at different frequencies in Caucasian and East Asian (Japanese and Korean) populations. This study was designed to characterize the frequency of LKB1 mutations, their relationship to EGFR and KRAS mutations, and their associated clinicopathologic characteristics in Chinese patients.
Two hundred thirty-nine lung adenocarcinomas consecutively collected from October 2007 to July 2009 were dissected into 3 to 4 small (3 mm) pieces for histopathological analyses of tumor content. Genomic DNA and/or cDNA from 86 samples with more than 70% tumor content were used for sequencing of LKB1 (exons 1–9), EGFR (exons 18–21), and KRAS (exon 2). LKB1 germline mutation status was determined by sequencing of genomic DNA from matched histologically distant lung tissues that are histologically normal.
6.9% of lung adenocarcinomas harbored LKB1 somatic mutations. A total of 10.5% of patients had an LKB1 germline polymorphism, F354L. Interestingly, in two of these patients, tumors displayed loss of heterozygosity at this allele. EGFR kinase domain and KRAS mutations were found in 66.3% and 2.3% of Chinese lung adenocarcinomas, respectively. Concurrent LKB1 and EGFR somatic mutations were observed in one patient. Both KRAS-mutant tumors harbored LKB1 mutations.
These data provide important clinical and molecular characteristics of lung adenocarcinomas from Chinese patients.
PMCID: PMC4009449  PMID: 20559149
Chinese lung adenocarcinoma; LKB1; EGFR; KRAS; Mutation
6.  Two co-existing germline mutations P53 V157D and PMS2 R20Q promote tumorigenesis in a familial cancer syndrome 
Cancer letters  2013;342(1):36-42.
Germline mutations are responsible for familial cancer syndromes which account for approximately 5–10% of all types of cancers. These mutations mainly occur at tumor suppressor genes or genome stability genes, such as DNA repair genes. Here we have identified a cancer predisposition family, in which eight members were inflicted with a wide spectrum of cancer including one diagnosed with lung cancer at 22 years old. Sequencing analysis of tumor samples as well as histologically normal specimens identified two germline mutations co-existing in the familial cancer syndrome, the mutation of tumor suppressor gene P53 V157D and mismatch repair gene PMS2 R20Q. We further demonstrate that P53 V157D and/or PMS2 R20Q mutant promotes lung cancer cell proliferation. These two mutants are capable of promoting colony formation in soft agar as well as tumor formation in transgenic drosophila system. Collectively, these data have uncovered the important role of co-existing germline P53 and PMS2 mutations in the familial cancer syndrome development.
PMCID: PMC3981830  PMID: 23981578
P53 V157D; PMS2 R20Q; Germline mutation; Familial cancer syndrome; Co-existing
7.  Generation of haploid embryonic stem cells from Macaca fascicularis monkey parthenotes 
Cell Research  2013;23(10):1187-1200.
Recent success in the derivation of haploid embryonic stem cells (haESCs) from mouse via parthenogenesis and androgenesis has enabled genetic screening in mammalian cells and generation of gene-modified animals. However, whether haESCs can be derived from primates remains unknown. Here, we report the derivation of haESCs from parthenogenetic blastocysts of Macaca fascicularis monkeys. These cells, termed as PG-haESCs, are pluripotent and can differentiate to cells of three embryonic germ layers in vitro or in vivo. Interestingly, the haploidy of one monkey PG-haESC line (MPH1) is more stable compared with that of the other one (MPH2), as shown by the existence of haploid cells for more than 140 days without fluorescence-activated cell sorting (FACS) enrichment of haploid cells. Importantly, transgenic monkey PG-haESC lines can be generated by lentivirus- and piggyBac transposon-mediated gene transfer. Moreover, genetic screening is feasible in monkey PG-haESCs. Our results demonstrate that PG-haESCs can be generated from monkeys, providing an ideal tool for genetic analyses in primates.
PMCID: PMC3790242  PMID: 23856644
embryonic stem cell; haploid cells; monkey
8.  Par-1 Regulates Tissue Growth by Influencing Hippo Phosphorylation Status and Hippo-Salvador Association 
PLoS Biology  2013;11(8):e1001620.
Par-1 regulates the Hippo signaling pathway in Drosophila melanogaster by modifying the phosphorylation status of Hippo and also by inhibiting the interaction of Hippo and Salvador.
The evolutionarily conserved Hippo (Hpo) signaling pathway plays a pivotal role in organ size control by balancing cell proliferation and cell death. Here, we reported the identification of Par-1 as a regulator of the Hpo signaling pathway using a gain-of-function EP screen in Drosophila melanogaster. Overexpression of Par-1 elevated Yorkie activity, resulting in increased Hpo target gene expression and tissue overgrowth, while loss of Par-1 diminished Hpo target gene expression and reduced organ size. We demonstrated that par-1 functioned downstream of fat and expanded and upstream of hpo and salvador (sav). In addition, we also found that Par-1 physically interacted with Hpo and Sav and regulated the phosphorylation of Hpo at Ser30 to restrict its activity. Par-1 also inhibited the association of Hpo and Sav, resulting in Sav dephosphorylation and destabilization. Furthermore, we provided evidence that Par-1-induced Hpo regulation is conserved in mammalian cells. Taken together, our findings identified Par-1 as a novel component of the Hpo signaling network.
Author Summary
An organism's organ size is determined by cell number, the size of each cell, and the distance between cells. All of these factors are controlled by the coordination of different cell signaling pathways and other mechanisms. The Hippo signaling pathway controls organ size by restricting the number of cells that make up the organ. Malfunction of this pathway leads to abnormal overgrowth, and is involved in a large number of human diseases and cancers. We identify here a component of the Hippo pathway, Par-1, which controls tissue growth by negatively regulating the Hippo pathway. We show that overexpression or depletion of Par-1 influences tissue growth in fruit flies via Hippo signaling. Then, by genetic and biochemical experiments, we show that Par-1 interacts with Hippo, regulating the Hippo Ser30 phosphorylation status to alter Hippo activity. In addition, we found that Par-1 regulates Hippo signaling via inhibition of the Hippo-Salvador association in a kinase-dependent fashion. We predict that Par-1 is a potential oncogene and that its regulatory role in Hippo signaling could be conserved.
PMCID: PMC3735459  PMID: 23940457
10.  Drosophila Ste-20 Family Protein Kinase, Hippo, Modulates Fat Cell Proliferation 
PLoS ONE  2013;8(4):e61740.
Evolutionarily conserved Hippo (Hpo) pathway plays a pivotal role in the control of organ size. Although the Hpo pathway regulates proliferation of a variety of epidermal cells, its function in non-ectoderm-derived cells is largely unknown.
Methodology/Principal Findings
Through methods including fat quantification assays, starvation assays, in vivo labeling assays, we show that overexpression of Hpo in Drosophila melanogaster fat body restricts Drosophila body growth and reduces fat storage through regulation of adipocyte proliferation rather than through influencing the size of fat cells and lipid metabolism, whereas compromising Hpo activity results in weight gain and greater fat storage. Furthermore, we provide evidence that Yorkie (Yki, a transcriptional coactivator that functions in the Hpo pathway) antagonizes Hpo to modulate fat storage in Drosophila.
Our findings specify a role of Hpo in controlling mesoderm-derived cell proliferation. The observed anti-obesity effects of Hpo may indicate great potential for its utilization in anti-obesity therapeutics.
PMCID: PMC3630116  PMID: 23637896
11.  Tumor suppressor in lung cancer-1 (TSLC1) mediated by dual-regulated oncolytic adenovirus exerts specific antitumor actions in a mouse model 
Acta Pharmacologica Sinica  2013;34(4):531-540.
The tumor suppressor in lung cancer-1 (TSLC1) is a candidate tumor suppressor of lung cancer, and frequently inactivated in primary non-small cell lung cancer (NSCLC). In this study, we investigated the effects of TSLC1 mediated by a dual-regulated oncolytic adenovirus on lung cancer, and the mechanisms underlying the antitumor actions.
The recombinant virus Ad·sp-E1A(Δ24)-TSLC1 was constructed by inserting the TSLC1 gene into the dual-regulated Ad·sp-E1A(Δ24) vector, which contained the survivin promoter and a 24 bp deletion within E1A. The antitumor effects of Ad·sp-E1A(Δ24)-TSLC1 were evaluated in NCI-H460, A549, and H1299 lung cancer cell lines and the normal fibroblast cell line MRC-5, as well as in A549 xenograft model in nude mice. Cell viability was assessed using MTT assay. The expression of TSLC1 and activation of the caspase signaling pathway were detected by Western blot analyses. The tumor tissues from the xenograft models were examined using H&E staining, IHC, TUNEL, and TEM analyses.
Infection of A549 lung cancer cells with Ad·sp-E1A(Δ24)-TSLC1 induced high level expression of TSLC1. Furthermore, the Ad·sp-E1A(Δ24)-TSLC1 virus dose-dependently suppressed the viability of NCI-H460, A549, and H1299 lung cancer cells, and did not affect MRC-5 normal fibroblast cells. Infection of NCI-H460, A549, and H1299 lung cancer cells with Ad·sp-E1A(Δ24)-TSLC1 induced apoptosis, and increased activation of caspase-8, caspase-3 and PARP. In A549 xenograft model in nude mice, intratumoral injection of Ad·sp-E1A(Δ24)-TSLC1 significantly suppressed the tumor volume, and increased the survival rate (from less than 15% to 87.5% at d 60). Histological studies showed that injection of Ad·sp-E1A(Δ24)-TSLC1 caused tumor cell apoptosis and virus particle propagation in tumor tissues.
The oncolytic adenovirus Ad·sp-E1A(Δ24)-TSLC1 exhibits specific antitumor effects, and is a promising agent for the treatment of lung cancer.
PMCID: PMC4002789  PMID: 23503473
lung cancer; tumor suppressor in lung cancer-1; oncolytic adenovirus; survivin; apoptosis; caspase signaling pathway; tumor xenograft model
12.  Correction: Temporal Dissection of K-rasG12D Mutant In Vitro and In Vivo Using a Regulatable K-rasG12D Mouse Allele 
PLoS ONE  2012;7(7):10.1371/annotation/0671c124-a263-49e9-9c23-421fd125db2c.
PMCID: PMC3394811
13.  Temporal Dissection of K-rasG12D Mutant In Vitro and In Vivo Using a Regulatable K-rasG12D Mouse Allele 
PLoS ONE  2012;7(5):e37308.
Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-rasG12D (ER-K-rasG12D) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 µM works optimally for activation of ER-K-rasG12D independent of the gender status. Furthermore, tamoxifen-inducible activation of K-rasG12D promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-rasG12D in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis.
PMCID: PMC3350485  PMID: 22606359
14.  Prostate Cancer-Specific and Potent Antitumor Effect of a DD3-Controlled Oncolytic Virus Harboring the PTEN Gene 
PLoS ONE  2012;7(4):e35153.
Prostate cancer is a major health problem for men in Western societies. Here we report a Prostate Cancer-Specific Targeting Gene-Viro-Therapy (CTGVT-PCa), in which PTEN was inserted into a DD3-controlled oncolytic viral vector (OV) to form Ad.DD3.E1A.E1B(Δ55)-(PTEN) or, briefly, Ad.DD3.D55-PTEN. The woodchuck post-transcriptional element (WPRE) was also introduced at the downstream of the E1A coding sequence, resulting in much higher expression of the E1A gene. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical bio-diagnostic marker. PTEN is frequently inactivated in primary prostate cancers, which is crucial for prostate cancer progression. Therefore, the Ad.DD3.D55-PTEN has prostate cancer specific and potent antitumor effect. The tumor growth rate was almost completely inhibited with the final tumor volume after Ad.DD3.D55-PTEN treatment less than the initial volume at the beginning of Ad.DD3.D55-PTEN treatment, which shows the powerful antitumor effect of Ad.DD3.D55-PTEN on prostate cancer tumor growth. The CTGVT-PCa construct reported here killed all of the prostate cancer cell lines tested, such as DU145, 22RV1 and CL1, but had a reduced or no killing effect on all the non-prostate cancer cell lines tested. The mechanism of action of Ad.DD3.D55-PTEN was due to the induction of apoptosis, as detected by TUNEL assays and flow cytometry. The apoptosis was mediated by mitochondria-dependent and -independent pathways, as determined by caspase assays and mitochondrial membrane potential.
PMCID: PMC3324420  PMID: 22509396
15.  Spectrum of Oncogenic Driver Mutations in Lung Adenocarcinomas from East Asian Never Smokers 
PLoS ONE  2011;6(11):e28204.
We previously showed that 90% (47 of 52; 95% CI, 0.79 to 0.96) of lung adenocarcinomas from East Asian never-smokers harbored well-known oncogenic mutations in just four genes: EGFR, HER2, ALK, and KRAS. Here, we sought to extend these findings to more samples and identify driver alterations in tumors negative for these mutations.
Experimental Design
We have collected and analyzed 202 resected lung adenocarcinomas from never smokers seen at Fudan University Shanghai Cancer Center. Since mutations were mutually exclusive in the first 52 examined, we determined the status of EGFR, KRAS, HER2, ALK, and BRAF in stepwise fashion as previously described. Samples negative for mutations in these 5 genes were subsequently examined for known ROS1 fusions by RT-PCR and direct sequencing.
152 tumors (75.3%) harbored EGFR mutations, 12 (6%) had HER2 mutations, 10 (5%) had ALK fusions all involving EML4 as the 5′ partner, 4 (2%) had KRAS mutations, and 2 (1%) harbored ROS1 fusions. No BRAF mutation were detected.
The vast majority (176 of 202; 87.1%, 95% CI: 0.82 to 0.91) of lung adenocarcinomas from never smokers harbor mutant kinases sensitive to available TKIs. Interestingly, patients with EGFR mutant patients tend to be older than those without EGFR mutations (58.3 Vs 54.3, P = 0.016) and patient without any known oncogenic driver tend to be diagnosed at a younger age (52.3 Vs 57.9, P = 0.013). Collectively, these data indicate that the majority of never smokers with lung adenocarcinoma could benefit from treatment with a specific tyrosine kinase inhibitor.
PMCID: PMC3227646  PMID: 22140546
16.  HCCS1-armed, quadruple-regulated oncolytic adenovirus specific for liver cancer as a cancer targeting gene-viro-therapy strategy 
Molecular Cancer  2011;10:133.
In previously published studies, oncolytic adenovirus-mediated gene therapy has produced good results in targeting cancer cells. However, safety and efficacy, the two most important aspects in cancer therapy, remain serious challenges. The specific expression or deletion of replication related genes in an adenovirus has been frequently utilized to regulate the cancer cell specificity of a virus. Accordingly, in this study, we deleted 24 bp in E1A (bp924-bp947) and the entirety of E1B, including those genes encoding E1B 55kDa and E1B19kDa. We used the survivin promoter (SP) to control E1A in order to construct a new adenovirus vector named Ad.SP.E1A(Δ24).ΔE1B (briefly Ad.SPDD). HCCS1 (hepatocellular carcinoma suppressor 1) is a novel tumor suppressor gene that is able to specifically induce apoptosis in cancer cells. The expression cassette AFP-HCCS1-WPRE-SV40 was inserted into Ad.SPDD to form Ad.SPDD-HCCS1, enabling us to improve the safety and efficacy of oncolytic-mediated gene therapy for liver cancer.
Ad.SPDD showed a decreased viral yield and less toxicity in normal cells but enhanced toxicity in liver cancer cells, compared with the cancer-specific adenovirus ZD55 (E1B55K deletion). Ad.SPDD-HCCS1 exhibited a potent anti-liver-cancer ability and decreased toxicity in vitro. Ad.SPDD-HCCS1 also showed a measurable capacity to inhibit Huh-7 xenograft tumor growth on nude mice. The underlying mechanism of Ad.SPDD-HCCS1-induced liver cancer cell death was found to be via the mitochondrial apoptosis pathway.
These results demonstrate that Ad.SPDD-HCCS1 was able to elicit reduced toxicity and enhanced efficacy both in vitro and in vivo compared to a previously constructed oncolytic adenovirus. Ad.SPDD-HCCS1 could be a promising candidate for liver cancer therapy.
PMCID: PMC3222618  PMID: 22040050
liver cancer; quadruple regulated adenovirus; HCCS1; mitochondrial apoptosis pathway
17.  Induction of cell cycle arrest at G1 and S phases and cAMP-dependent differentiation in C6 glioma by low concentration of cycloheximide 
BMC Cancer  2010;10:684.
Differentiation therapy has been shown effective in treatment of several types of cancer cells and may prove to be effective in treatment of glioblastoma multiforme, the most common and most aggressive primary brain tumor. Although extensively used as a reagent to inhibit protein synthesis in mammalian cells, whether cycloheximide treatment leads to glioma cell differentiation has not been reported.
C6 glioma cell was treated with or without cycloheximide at low concentrations (0.5-1 μg/ml) for 1, 2 and 3 days. Cell proliferation rate was assessed by direct cell counting and colony formation assays. Apoptosis was assessed by Hoechst 33258 staining and FACS analysis. Changes in several cell cycle regulators such as Cyclins D1 and E, PCNA and Ki67, and several apoptosis-related regulators such as p53, p-JNK, p-AKT, and PARP were determined by Western blot analysis. C6 glioma differentiation was determined by morphological characterization, immunostaining and Western blot analysis on upregulation of GFAP and o p-STAT3 expression, and upregulation of intracellular cAMP.
Treatment of C6 cell with low concentration of cycloheximide inhibited cell proliferation and depleted cells at both G2 and M phases, suggesting blockade at G1 and S phases. While no cell death was observed, cells underwent profound morphological transformation that indicated cell differentiation. Western blotting and immunostaining analyses further indicated that changes in expression of several cell cycle regulators and the differentiation marker GFAP were accompanied with cycloheximide-induced cell cycle arrest and cell differentiation. Increase in intracellular cAMP, a known promoter for C6 cell differentiation, was found to be elevated and required for cycloheximide-promoted C6 cell differentiation.
Our results suggest that partial inhibition of protein synthesis in C6 glioma by low concentration of cycloheximide induces cell cycle arrest at G1 and M phases and cAMP-dependent cell differentiation.
PMCID: PMC3009684  PMID: 21159181

Results 1-17 (17)