E1A Gene Research Articles

Combining histone deacetylase inhibitors with MDA-7/IL-24 enhances killing of renal carcinoma cells

In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca2+. Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda-7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.

Switching a Replication-Defective Adenoviral Vector into a Replication-Competent, Oncolytic Adenovirus

The adenovirus immediate early gene E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. For adenoviral (Ad) vector-mediated gene transfer and therapy approaches, where replication-defective (RD) gene transfer is required, E1A has thus been the primary target for deletions. For oncolytic gene therapy for cancer, where replication-competent (RC) Ad viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. A novel Ad vector that initially infected target tumor cells in an RD manner for transgene expression but that could be "switched" into an RC, oncolytic state when needed might represent an advance in vector technology. Here, we report that we designed such an Ad vector (proAdΔ24.GFP), where initial Ad replication is silenced by a green fluorescent protein (GFP) transgene that blocks cytomegalovirus (CMV)-mediated transcription of E1A. This vector functions as a bona fide E1A-deleted RD vector in infected tumor cells. However, because the silencing GFP transgene is flanked by FLP recombination target (FRT) sites, we show that it can be efficiently excised by Flp recombinase site-specific recombination, either when Flp is expressed constitutively in cells or when it is provided in trans by coinfection with a second RD herpes simplex virus (HSV) amplicon vector. This switches the RD Ad, proAdΔ24.GFP, into a fully RC, oncolytic Ad (rAdΔ24) that lyses tumor cells in culture and generates oncolytic progeny virions. In vivo, coinfection of established flank tumors with the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon leads to generation of RC, oncolytic rAdΔ24. In an orthotopic human glioma xenograft tumor model, coinjection of the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon also led to a significant increase in animal survival, compared to controls. Therefore, Flp-FRT site-specific recombination can be applied to switch RD Ad into fully oncolytic RC Ad for tumor therapy and is potentially applicable to a variety of gene therapy approaches.

E1a promotes c-Myc-dependent replicative stress

The E1a gene from adenovirus is known to be a potent inducer of chemo/radiosensitivity in a wide range of tumors. However, the molecular bases of its radiosensitizer properties are still poorly understood. In an attempt to study this effect, U87MG cells, derived from a radio-resistant tumor as glioblastoma, where infected with lentivirus carrying E1a gene developing an acute sensitivity to ionizing radiation. The induction of radiosensitivity correlated with a marked G2/M phase accumulation and a potent apoptotic response. Our findings demonstrate that c-Myc plays a pivotal role in E1a-associated radiosensitivity through the induction of a replicative stress situation, as our data support by genetic approaches, based in interference and overexpression in U87MG cells. In fact, we present evidence showing that Chk1 is a novel transcriptional target of E1a gene through the effect exerted by this adenoviral protein onto c-Myc. Moreover, c-Myc upregulation also explains the marked phosphorylation of H2AX associated to E1a expression in the absence of DNA damage. Indeed, all these observations were applicable to other experimental models, such as T98G, LN-405 and A172, rendering the same pattern in terms of radiosensitivity, cell cycle distribution, upregulation of Chk1, c-Myc, and phosphorylation pattern of H2AX. In summary, our data propose a novel mechanism to explain how E1a mediates radiosensitivity through the signaling axis E1a→c-Myc→ replicative stress situation. This novel mechanism of E1a-mediated radiosensitivity could be the key to open new possibilities in the current therapy of glioblastoma.

Anti-Tumor Activity of a miR-199-dependent Oncolytic Adenovirus

The down-regulation of miR-199 occurs in nearly all primary hepatocellular carcinomas (HCCs) and HCC cell lines in comparison with normal liver. We exploited this miR-199 differential expression to develop a conditionally replication-competent oncolytic adenovirus, Ad-199T, and achieve tumor-specific viral expression and replication. To this aim, we introduced four copies of miR-199 target sites within the 3’ UTR of E1A gene, essential for viral replication. As consequence, E1A expression from Ad-199T virus was tightly regulated both at RNA and protein levels in HCC derived cell lines, and replication controlled by the level of miR-199 expression. Various approaches were used to asses in vivo properties of Ad-199T. Ad-199T replication was inhibited in normal, miR-199 positive, liver parenchyma, thus resulting in reduced hepatotoxicity. Conversely, the intrahepatic delivery of Ad-199T in newborn mice led to virus replication and fast removal of implanted HepG2 liver cancer cells. The ability of Ad-199T to control tumor growth was also shown in a subcutaneous xenograft model in nude mice and in HCCs arising in immune-competent mice. In summary, we developed a novel oncolytic adenovirus, Ad-199T, which could demonstrate a therapeutic potential against liver cancer without causing significant hepatotoxicity.

Homologous recombination-based adenovirus vector system for tumor cell-specific gene delivery

Cancer gene therapy requires tumor-specific delivery and expression of a transgene to maximize antitumor efficacy and minimize side effects. In this study, we developed a new tumor-targeting, homologous recombination-based adenovirus vector system, HRAVS. HRAVS is composed of two adenovirus vectors, Ad.CMV.IR containing reverse sequence (IR) and a CMV promoter and Ad.IR.EGFP comprising the report gene EGFP and IR. For improved viral DNA replication and transgene expression, the E1a gene was added to HRAVS to generate the enhanced HRAVS, EHRAVS, which consists of Ad.CMV.IR and Ad.IR.EGFP/E1a. The optimal vector composition ratio of Ad.CMV.IR to Ad.IR.EGFP or Ad.IR.EGFP/E1a was identified as 30:70 based on EGFP expression efficiency in tumor cells. The transgene expression of HRAVS and EHRAVS was efficiently and specifically activated in tumor cells only and not in normal cells. Moreover, compared with HRAVS, EHRAVS infection led to higher virus yields and transgene expression and higher toxicity to tumor cells, and these results could be related to the involvement of E1a genes. The results in present study suggest the need for in vivo antitumor study using these new dual-Ad vector systems based on the homologous recombination.

Bicalutamide-activated oncolytic adenovirus for the adjuvant therapy of high-risk prostate cancer

Conditionally replicating adenoviruses (CRAds) utilize tissue specific promoters to control the expression of the early genes, E1A and E1B, to preferentially replicate and lyse tumor cells (oncolysis). Previous CRAds used in prostate cancer gene therapy require androgens to activate prostate specific promoters and induce viral replication. Unfortunately, these CRAds have reduced activity in patients on androgen suppressive therapy. We describe a novel prostate specific CRAd generated by fusing the E1A gene to the androgen receptor (AR) cDNA with a point mutation in codon 685 (C685Y). The E1A-AR fusion neutralizes the previously described mutual inhibition of E1A & AR, and the C685Y point mutation alters specificity of steroid ligand binding to the AR, such that both androgens and non-steroidal anti-androgens can activate viral replication. We demonstrate that the mutated E1A-AR retained the ability to function in regulating AR responsive genes and E1A responsive viral genes. In combination therapy of virus, bicalutamide (anti-androgen) and radiation, a profound impact on cell death by viral oncolysis was seen both in vitro and tumor xenografts. To our knowledge, this is the first gene therapy engineered to be enhanced by anti-androgens, and a particularly attractive adjuvant strategy for intensity modulated radiation therapy (IMRT) of high-risk prostate cancers.

Experimental studies on treatment of pancreatic cancer with double-regulated duplicative adenovirus AdTPHre-hEndo carrying human endostatin gene

BackgroundGene-virus targeted therapy is a promising new method of treating pancreatic cancer. To increase the efficacy and decrease the side-effect, we constructed a conditionally replicative adenovirus (CRAd) expressing human endostatin, with a human Telomoerase Reverse Transcriptase (hTERT) promoter for the regulation of the early stage of adenovirus expression of gene E1a and a Hypoxia Response Element (HRE) promoter to regulate the gene E1b. MethodsA gene recombination technique was adopted to construct and generate the adenovirus AdTPHre-hEndo. Pancreatic cancer cells were studied both in vitro and in vivo. Western blotting was adopted to observe the expressions of protein E1A and E1B; duplication assay was applied to observe the selective duplication capability of recombinant cells. MTT assay was applied to measure the lethal effects of virus on pancreatic cancer cells, and ELISA was adopted to detect the human endostatin gene expression. A pancreatic cancer transplantation tumor model of nude mice was constructed to observe the antitumor effects of the virus. ResultsDouble-regulated duplicative adenovirus AdTPHre-hEndo genes were successfully constructed. Duplication and lethal assays proved that AdTPHre-hEndo could replicate specifically in pancreatic cancer cells and kill them. The endostatin expression in a cultured supernatant from tumor cells was significantly higher than that obtained from non-duplicative adenovirus vectors carrying that gene. The animal experiment demonstrated that AdTPHre-hEndo has a high capability to limit pancreatic cancer growth. ConclusionsAdTPHre-hEndo has a special ability to duplicate and kill pancreatic cancer cells in in vitro and in vivo experiments, thus providing a new gene-virus-based treatment system for pancreatic cancer.

Target expression of Staphylococcus enterotoxin A from an oncolytic adenovirus suppresses mouse bladder tumor growth and recruits CD3+ T cell

We recently engineered an oncolytic adenovirus (PPE3-SEA) that expresses the superantigen, Staphylococcus enterotoxin A (SEA), and that has enhanced tumor specificity because the telomerase reverse transcriptase and hypoxia-inducible factor promoters regulate expression of E1A and E1B genes, respectively. Here, we evaluated the PPE3-SEA adenovirus anti-tumor activity against MB49 mouse bladder cancer cell proliferation in vitro and in vivo. PPE3-SEA infection of murine MB49 cells in vitro induced cytopathic effects, and significant expression of SEA mRNA and protein, as measured by RT-PCR and western blot, respectively. Subcutaneous MB29 bladder tumors were established in syngeneic C57BL/6 mice. After 10 days, tumors were injected with either oncolytic virus or PBS. Tumor dimensions were measured on days 1, 3, 5, 7, 9, and 11 post-treatment and tumor volumes were calculated. One of eight PPE3-SEA-treated mice had no tumor by day 9. PPE3-SEA treated group had significantly lower mean tumor volume beginning on day 5 post-treatment (p < 0.01), more fibrous tissue in the tumor, and increased presence of infiltrating CD3+ T cells than those of the control group. Gross appearance and histologic sections from the livers and kidneys of the PPE3-SEA-treated group were similar to those of the control group. In conclusion, oncolytic adenoviruses can provide a novel delivery vehicle for SEA to tumor sites, and PPE3-SEA warrants further study as a potential anti-tumor agent for bladder cancer.

482. Suppression of the Leaky Expression of Adenovirus Genes Following Transduction with a Replication-Incompetent Adenovirus Vector by Incorporating microRNA-Targeted Sequences into the 3'-Untranslated Region of the E2A, E4, or pIX Gene

482. Suppression of the Leaky Expression of Adenovirus Genes Following Transduction with a Replication-Incompetent Adenovirus Vector by Incorporating microRNA-Targeted Sequences into the 3'-Untranslated Region of the E2A, E4, or pIX Gene

Abstract 3316: Oncolytic adenovirus-armed p53 induces apoptosis significantly through upregulating miR-93 and 106b in human osteosarcoma cells..

Abstract Bone and soft tissue sarcomasare the third most common cancer in children and account for 15.4% of all childhood malignancies. About one fourth of the patients show a poor response to conventional chemo-radiotherapy, resulting in subsequent recurrence and leading to a poor prognosis. Therefore, the development of a novel therapeutic strategy is required. We previously developed an oncolytic adenovirus, OBP-301 (Telomelysin), in which the hTERT gene promoter drives the expression of the E1A and E1B genes linked to an internal ribosome entry site (IRES). Recently, we revealed that OBP-301 shows the cytopathic activity in human bone and soft tissue sarcoma cells. However, some human osteosarcoma cells (MNNG/HOS, SaOS-2) were less sensitive to cytopathic activity of OBP-301. Therefore, we developed a telomerase-specific p53-armed oncolytic adenovirus (OBP-702). In this study, we examined the antitumor effect of OBP-702 in OBP-301-resistant osteosarcoma cells, and revealed the mechanism. We compared the antitumor effects of OBP-702 and OBP-301 using XTT assay. The 50% inhibiting dose (ID50) value of OBP-702 and OBP-301 for each cell was calculated using cell viability data obtained on day 5 after virus infection. We further evaluated the expression of p53, p21 and cleaved-PARP proteins using western blot analysis in OBP-301-resistant osteosarcoma cells after infection with OBP-702 or Ad-p53, which is a p53-expressing replication-defective adenovirus. OBP-702 showed more cytopathic activity compared to OBP-301. The ID50 value of OBP-702 was much lower than that of OBP-301 in all cell lines. Western blot analysis showed OBP-702 infection induced the cleavage of PARP in a dose-dependent manner and cell cycle analysis also showed that OBP-702 induced the highest percentages of sub-G1 population in MNNG/HOS cells compared to Ad-p53 or OBP-301. These results suggest that OBP-702 induces increased apoptosis compared to Ad-p53 or OBP-301. OBP-702 infection induced more profound p53 expression than Ad-p53, but p53-downstream target p21 proteins were not activated by OBP-702. The expression levels of miR-93 and miR-106b increased after OBP-702 infection. When SaOS-2 cells were infected with Ad-p53 at an MOI of 10 for 48 hours, pre-transfection with miR-93, miR-106b or both efficiently suppressed Ad-p53-induced p21 expression. These results suggest that OBP-702 suppresses p21 expression through E1A-dependent upregulation of miR-93 and miR-106b. In conclusion, we have clearly demonstrated that the p53-expressing oncolytic adenovirus OBP-702 has a much stronger antitumor effect than OBP-301 and Ad-p53. Oncolytic adenovirus-mediated p53 gene transduction would induce profound p53 expression without p21 activation regulated by miR-93 and 106b, resulting in the enhancement of antitumor effect. Citation Format: Joe Hasei, Tsuyoshi Sasaki, Hiroshi Tazawa, Yuuri Hashimoto, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Oncolytic adenovirus-armed p53 induces apoptosis significantly through upregulating miR-93 and 106b in human osteosarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3316. doi:10.1158/1538-7445.AM2013-3316

Abstract 584: Enhanced chemosensitivity of osteosarcoma cells by telomerase-specific oncolytic adenovirus in combination therapy.

Abstract Osteosarcomas are the most common malignant bone tumors. Although the multi-agent chemotherapy has improved the long-term survival rate of osteosarcoma patients, some patients show a poor response to chemotherapy, leading to recurrence and poor prognosis. Therefore, the enhancement of chemosensitivity is required to cure patients with osteosarcomas. We recently developed a telomerase-specific replication-competent oncolytic adenovirus, Telomelysin (OBP-301), and confirmed the antitumor effect of OBP-301 in human osteosarcoma cells. A phase I clinical trial in US has also shown the safety of OBP-301 in cancer patients. In this study, we investigated whether OBP-301 enhances the antitumor effect of chemotherapeutic agents, doxorubicin and cisplatin, that are used for the treatment of osteosarcomas, and the molecular mechanism of chemotherapy- and OBP-301-mediated cell death pathway, autophagy and apoptosis, in human osteosarcoma cell lines. We used four human osteosarcoma cell lines, HOS, MNNG/HOS, 143B and SaOS2. OBP-301 is an attenuated adenovirus, in which the human telomerase reverse transcriptase (hTERT) promoter element drives expression of E1A and E1B genes, and causes tumor-selective lysis in a variety of human malignant tumor cells with high telomerase activity. OBP-301 infection enhanced the cytotoxic effect of chemotherapeutic agents and the calculation of combination index revealed the synergistic effects in all human osteosarcoma cell lines. To analyze the molecular mechanism in the synergistic effect induced by combination therapy, western blot analysis for apoptosis (PARP) and autophagy (LC3, p62) was performed. Combination of OBP-301 increased both apoptosis (the cleavage of PARP) and autophagy (conversion of LC3-I to LC3-II and p62 downregulation) in the chemotherapeutic agents-treated human osteosarcoma cells. In contrast, the replication of OBP-301 was not suppressed by chemotherapeutic agents. Moreover, combination therapy of chemotherapeutic agents with OBP-301 significantly suppressed tumor growth in a subcutaneous xenograft tumor model compared to monotherapy with chemotherapeutic agents or OBP-301. These results suggest that combination therapy of chemotherapeutic agents with OBP-301 provides a novel therapeutic strategy for human osteosarcomas. Citation Format: Shuhei Osaki, Tsuyoshi Sasaki, Hiroshi Tazawa, Joe Hasei, Yasuaki Yamakawa, Yuuri Hashimoto, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Enhanced chemosensitivity of osteosarcoma cells by telomerase-specific oncolytic adenovirus in combination therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 584. doi:10.1158/1538-7445.AM2013-584

Abstract 5455: Differential regulation of E2A (TCF3) by androgens in prostate cancer cells.

Abstract Introduction: E2A (TCF3) is a multifunctional basic helix loop helix (bHLH) transcription factor. E2A promotes cell differentiation, acts as a negative regulator of cell proliferation in normal cells and cancer cell lines and is required for normal B-cell development. Previous studies from our laboratory has shown that E2A expression is highly increased in prostate cancer as compared to normal prostate and that it acts as a tumor promoter in prostate cancer. Given the diverse biological pathways regulated/ influenced by E2A little is known about its regulation in prostate cancer. Experimental design: E2A expression in androgen sensitive LNCaP and insensitive C81 prostate cancer cell lines was determined by western blot after treatments with androgen receptor (AR) agonist R1881 and antagonist casodex. Putative Androgen Response Elements (ARE) were identified in the first intronic region of the E2A gene using some of the online bioinformatics tools and confirmed by Chromatin Immunoprecipitation (ChIP) with AR antibody and Luciferase reporter assays on the above mentioned cell lines after treatments with R1881 and casodex. Results: E2A expression was found to increase with the increasing aggrasiveness of prostate cancer cell lines as compared to normal prostate epithelial cell line RWPE1. When androgen responsive cell line LNCaP was treated with R1881 there was an increased expression of E2A which decreased upon treatment with antiandrogen casodex whereas the E2A expression remained unaltered upon similar treatments in androgen insensitive cell line C81. The first intronic region of the E2A gene was predicted to contain two putative ARE sites. ChIP after treatment of LNCaP and C81 cells with R1881 and casodex showed that the intronic region was bound by AR in LNCaP cells only in the presence of R1881, whereas C81 cells showed a pulldown with AR in presence as well as absence of R1881. Similar results were observed in luciferase reporter assays indicating that E2A is transactivated by AR in LNCaP cell lines whereas it is independent of androgens in C81 cell line. Furthermore, Luciferase reporter assays also confirmed that only one of the two predicted putative AREs was functionally active and responsible for the androgen mediated regulation of E2A. Conclusion: Our results indicate that E2A is differentially regulated in Prostate cancer cell lines. The increased expression of E2A and its role as a tumor promoter in prostate cancer cell lines may be contributed to its loss of androgen dependence as is evident in its progression from androgen dependent LNCaP to independent C81 cells. Acknowledgements: This study was supported by NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12RR03062. Citation Format: Divya Patel, Jaideep Chaudhary. Differential regulation of E2A (TCF3) by androgens in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5455. doi:10.1158/1538-7445.AM2013-5455

Impact of E1 and Cre on Adenovirus Vector Amplification: Developing MDCK CAV-2-E1 and E1-Cre Transcomplementing Cell Lines

Adenovirus vectors have been extensively studied through the manipulation of viral genome. However, little attention is being paid to their producer cell-lines; cells are selected according to virus yields, neglecting the expression profile of transcomplementing gene products underlying cell performance. This work evaluates the impact of E1 (E1A and E1B) and Cre recombinase levels in the production of E1-deleted and helper-dependent canine adenovirus type 2 (CAV-2) vectors using MDCK cells. E1A and E1B gene expression and Cre activity were evaluated in different cell clones and compared with the corresponding cell productivity and susceptibility to oxidative stress injury. CAV-2 production was proportional to E1A expression (the highest levels of E1A corresponding to productivities of 3000–5000 I.P./cell), while E1B prolonged host cell viability after infection, conferring protection against apoptosis. Cre recombinase counteracted E1B anti-apoptotic properties, however viral production was maintained under high levels of Cre. Yet, Cre recombinase side effects can be reduced using cell lines with lower Cre-activities, without compromising the excision efficiency of helper vector packaging signal. These results highlight the influence of transcomplementing gene products on CAV-2 producer cell line performance, and the ability to express high levels of E1A and E1B as an important feature for cell line establishment and high adenovirus titers.

Oncolytic adenoviral therapy enhanced by targeting cyclin E overexpression and inducing autophagy

Oncolytic virotherapy is a new potential approach in cancer therapy. Here we aim to enhance the efficacy of this drug platform with two approaches. First, we used the cyclin E promoter to control the E1a gene of a tumor‐specific oncolytic adenovirus (Ad). So far, most tumor‐specific promoters are relatively weak and only Ad dl1520 with its native E1a promoter has been approved for the cancer treatment. We have shown that the activity of cyclin E promoter is high in cancer cells and further enhanced by Ad infection. Thus, we constructed a novel oncolytic Ad‐cycE which specifically targets cancer cells and self‐enhance during the viral replication process. Ad‐cycE is competent to induce oncolysis in multiple cancer cells comparable to the wild‐type Ad and Ad dl1520. Our animal studies also illustrated the promising efficacy of Ad‐cycE on repressing lung tumors growth. Secondly, we applied autophagy to improve the oncolytic Ad‐cycE replication. We have demonstrated that Ad‐induced autophagy helps Ad replication and is related to cell death. Here we further revealed that an autophagy inducer rapamycin increased adenoviral E1A expression of Ad‐cycE and increases virus production in the human lung cancer cells. Our studies provide a new insight in vector development and demonstrate the novel roles of autophagy in adenovirus replication that may benefit future combination virotherapy. Supported by NIH Grant R01 CA129975.

E47 and Id1 Interplay in Epithelial-Mesenchymal Transition

E12/E47 proteins (encoded by E2A gene) are members of the class I basic helix-loop-helix (bHLH) transcription factors (also known as E proteins). E47 has been described as repressor of E-cadherin and inducer of epithelial-mesenchymal transition (EMT). We reported previously that EMT mediated by E47 in MDCK cells occurs with a concomitant overexpression of Id1 and Id3 proteins. Id proteins belong to class V of HLH factors that lack the basic domain; they dimerise with E proteins and prevent their DNA interaction, thus, acting as dominant negative of E proteins. Here, we show that E47 interacts with Id1 in E47 overexpressing MDCK cells that underwent a full EMT as well as in mesenchymal breast carcinoma and melanoma cell lines. By conducting chromatin immunoprecipitation assays we demonstrate that E47 binds directly to the endogenous E-cadherin promoter of mesenchymal MDCK-E47 cells in a complex devoid of Id1. Importantly, our data suggest that both E47 and Id1 are required to maintain the mesenchymal phenotype of MDCK-E47 cells. These data support the collaboration between E47 and Id1 in the maintenance of EMT by mechanisms independent of the dominant negative action of Id1 on E47 binding to E-cadherin promoter. Finally, the analysis of several N0 breast tumour series indicates that the expression of E47 and ID1 is significantly associated with the basal-like phenotype supporting the biological significance of the present findings.

A dual-regulated oncolytic adenovirus expressing interleukin-24 sensitizes melanoma cells to temozolomide via the induction of apoptosis

Malignant melanoma is one of the most lethal and aggressive human malignancies. Suppressed apoptosis and extraordinary invasiveness are the distinctive features that contribute to malignant melanoma. The alkylating agent temozolomide (TMZ) is one of the most effective single chemotherapeutic agents for patients with malignant melanoma, but resistance develops quickly and with high frequency. We constructed a dual-regulated oncolytic adenovirus expressing interleukin 24 (IL-24) gene (Ki67-ZD55-IL-24) by utilizing the Ki67 promoter to replace the native viral promoter of E1A gene. We investigated whether a combination of Ki67-ZD55-IL-24-mediated gene virotherapy and chemotherapy using TMZ produces increased cytotoxicity against human melanoma cells via the induction of apoptosis. Our data indicate that this novel strategy thus holds promising potentials for further developing an effective approach to treat malignant melanoma.

A Microarray Dataset of Genes Expressed by the R28 Retinal Precursor Cell Line

The R28 rat retinal progenitor cell line was developed from postnatal day six rat retina immortalized with the 12S E1A gene of adenovirus. R28 cells have been distributed to over 100 laboratories worldwide, with over 60 publications on topics that include in vitro toxicology, cellular physiology, gene expression analysis, and experimental transplantation. In this paper, we present a microarray dataset of R28 cells that describes the presence or absence of 8799 genes and ESTs that may be relevant to current and future studies of R28 retinal precursor cells.

MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3′ untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

Tat-PTD-Modified Oncolytic Adenovirus Driven by the SCG3 Promoter and ASH1 Enhancer for Neuroblastoma Therapy

Secretogranin III (SGC3) belongs to the granin family and is highly expressed in endocrine and neural tissues. The human SCG3 promoter has not yet been characterized. We identified that a 0.5-kb DNA fragment upstream of the SCG3 gene can selectively drive transgene expression in neuroblastoma cell lines. The strength of transgene expression was further increased, with specificity maintained, by addition of the human achaete-scute complex homolog 1 (ASH1) enhancer. We developed an oncolytic serotype 5-based adenovirus, in which the SCG3 promoter and ASH1 enhancer drive E1A gene expression. The virus was further modified with a cell-penetrating peptide (Tat-PTD) in the viral capsid, which we have previously shown results in increased adenovirus transduction efficiency of many neuroblastoma cell lines. The virus, Ad5PTD(ASH1-SCG3-E1A), shows selective and efficient killing of neuroblastoma cell lines in vitro, including cisplatin-, etoposide-, and doxorubicin-insensitive neuroblastoma cells. Furthermore, it delays tumor growth and thereby prolonged survival for nude mice harboring subcutaneous human neuroblastoma xenograft. In conclusion, we report a novel oncolytic adenovirus with potential use for neuroblastoma therapy.

Development of a Novel Adenovirus Vector Exhibiting MicroRNA-mediated Suppression of the Leaky Expression of Adenovirus Genes

Replication-incompetent adenovirus (Ad) vectors are widely used in gene therapy studies because they are beneficial as a gene delivery vehicle enabling high-titer production and highly efficient gene transfer into a wide spectrum of dividing and non-dividing cells in vitro and in vivo. Theoretically, Ad genes should not be expressed following transduction with a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with a conventional Ad vector, which leads to a cellular immunity against Ad proteins as well as Ad protein-induced toxicity. Such Ad protein-induced cellular immunity and toxicity frequently cause both an elimination of Ad vector-transduced cells and tissue damage, leading to short-lived transgene expression. To date, no detailed analysis of the leaky expression profile of Ad genes has been performed. First, we systematically examined the expression profiles of Ad genes in cells using real-time RT-PCR following transduction with a conventional Ad vector. The results revealed that significant expression was found for E2A, E4, and pIX genes. Next, in order to suppress the leaky expression of Ad genes, complementary sequences for microRNA (miRNA) were inserted into the 3'-untranslated region of the E2A, E4, or pIX genes. miRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3'-untranslated region of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. Incorporation of the miRNA-targeted sequences significantly suppressed the leaky expression of Ad genes in an miRNA-dependent manner.