Heshan Sam Zhou

Adenoviruses induce autophagy to promote virus replication and oncolysis.

Adenoviruses with deletion of E1b have been used in clinical trials to treat cancers that are resistant to conventional therapies. The efficacy of viral replication within cancer cells determines the results of oncolytic therapy, which remains poorly understood and requires further improvement. In this report, we show that adenoviruses induce autophagy by increasing the conversion of LC3-I to LC3-II and the formation of the Atg12-Atg5 complex. Inhibition of autophagy with 3-methyladenine (3MA) resulted in a decreased synthesis of adenovirus structural proteins, and thereby a poor viral replication; promotion of autophagy with rapamycin increased adenovirus yield. This study indicates that adenovirus-induced autophagy correlates positively with virus replication and oncolytic cell death, and that autophagy may generate nutrients that can be used for building viral progeny particles. These results further suggest that chemotherapeutic agents that increase cancer cell autophagy may improve the efficacy of oncolytic virotherapy.

E1A-induced apoptosis does not prevent replication of adenoviruses with deletion of E1b in majority of infected cancer cells

Apoptotic pathways are initiated as a cellular defense mechanism to eliminate adenovirus-infected cells. We have investigated how E1A-induced apoptosis interferes with viral replication in cancer cells. We found that E1B19K alone can efficiently suppress E1A-induced apoptosis in cancer cells. Viruses deleted for both E1B19K and E1B55K resulted in cellular DNA degradation. However, less than 20% of human lung cancer cells infected with a virus deleted for both E1B19K and E1B55 K had evidence of chromatin condensation and multiple-micronuclei formation (apoptotic hallmarks); these cells could not produce infectious viral particles. The majority of cancer cells infected with viruses deleted for the entire E1b gene did not undergo extended apoptosis and produced abundant viral progeny. Thus, only a fraction of cancer cells underwent apoptosis and did not allow E1b-deleted viruses to replicate, while the majority of cancer cells were resistant to E1A-induced apoptosis and could support virus-selective replication. The results of this study imply that, in addition to inhibiting E1A-induced apoptosis, E1B proteins may contribute other important roles in the viral life cycle. Our results also suggest that combining virus-induced apoptosis and selective viral replication into one vector will be a novel approach to destroy cancer cells.

Molecular Basis for Viral Selective Replication in Cancer Cells: Activation of CDK2 by Adenovirus-Induced Cyclin E

Adenoviruses (Ads) with deletion of E1b55K preferentially replicate in cancer cells and have been used in cancer therapies. We have previously shown that Ad E1B55K protein is involved in induction of cyclin E for Ad replication, but this E1B55K function is not required in cancer cells in which deregulation of cyclin E is frequently observed. In this study, we investigated the interaction of cyclin E and CDK2 in Ad-infected cells. Ad infection significantly increased the large form of cyclin E (cyclin EL), promoted cyclin E/CDK2 complex formation and increased CDK2 phosphorylation at the T160 site. Activated CDK2 caused pRb phosphorylation at the S612 site. Repression of CDK2 activity with the chemical inhibitor roscovitine or with specific small interfering RNAs significantly decreased pRb phosphorylation, with concomitant repression of viral replication. Our results suggest that Ad-induced cyclin E activates CDK2 that targets the transcriptional repressor pRb to generate a cellular environment for viral productive replication. This study reveals a new molecular basis for oncolytic replication of E1b-deleted Ads and will aid in the development of new strategies for Ad oncolytic virotherapies.

Production of helper-dependent adenovirus vector relies on helper virus structure and complementing

The helper‐dependent (HD) adenoviral (Ad) vector relies on a helper virus to provide viral proteins for vector amplification. HD‐Ad vectors can significantly increase therapeutic gene expression and improve safety. However, the yield of an HD‐Ad vector is generally lower than that of an E1‐deleted first‐generation vector, likely due to the alterations in viral E3 or packaging regions of a helper virus that attenuate its replication and complementing for an HD‐Ad vector.

Combined therapy of oncolytic adenovirus and temozolomide enhances lung cancer virotherapy in vitro and in vivo

Oncolytic adenoviruses (OAds) are very promising for the treatment of lung cancer. However, OAd-based monotherapeutics have not been effective during clinical trials. Therefore, the effectiveness of virotherapy must be enhanced by combining OAds with other therapies. In this study, the therapeutic potential of OAd in combination with temozolomide (TMZ) was evaluated in lung cancer cells in vitro and in vivo. The combination of OAd and TMZ therapy synergistically enhanced cancer cell death; this enhanced cancer cell death may be explained via three related mechanisms: apoptosis, virus replication, and autophagy. Autophagy inhibition partially protected cancer cells from this combined therapy. This combination significantly suppressed the growth of subcutaneous H441 lung cancer xenograft tumors in athymic nude mice. In this study, we have provided an experimental rationale to test OAds in combination with TMZ in a lung cancer clinical trial.

Selective replication of E1B55K-deleted adenoviruses depends on enhanced E1A expression in cancer cells

E1B55K-deleted dl1520 could selectively replicate in cancer cells and has been used in clinical trials as an antitumor agent. The mechanism of virus selective replication in cancer cells, including a possible role of p53, is unclear. Studies with established cancer cell lines have demonstrated that some cancer cells are resistant to dl1520 replication, regardless of the p53 status. Hep3B cells supported the E1b-deleted adenoviruses to replicate, whereas Saos2 cells were resistant to viral replication. We applied p53-null Hep3B and Saos2 cells as models to clarify the replication ability of E1B55K-deleted adenoviruses with different expression levels of E1a. We show that lower E1A expression in Saos2 may be the reason for the poor replication in some cancer cells due to the fact that E1a promoter was less activated in Saos2 than in Hep3B. We also demonstrate that the E1B55K protein can increase E1A expression in Saos2 cells for efficient virus replication. In addition, the upstream regions of the E1a promoter have transcriptional activity in Hep3B cells but not in Saos2 cells. The viral E1B55K protein may activate cancer cellular factor(s) that targets the upstream regions of the E1a gene to increase its expression. This is the first study demonstrating that E1B55K protein affects the E1A production levels that is related to cancer selective replication. Our studies have suggested that increase of E1A expression from E1b-deleted adenoviruses may enhance killing cancer cells that otherwise are resistant to viral replication.

Oncolytic Replication of E1b-Deleted Adenoviruses.

Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viral mRNA export, and cell cycle disruption.

Induction of Apoptosis Signal-Regulating Kinase 1 by E2F-1 May Not Be Essential for E2F-1-Mediated Apoptosis in Melanoma Cells

Objectives: In the present study, we investigate the role of apoptosis signal-regulating kinase 1 (ASK1) mitogen-activated protein (MAP) kinase signal pathways in E2F-1-mediated apoptosis. Methods: A gene expression profile in response to E2F-1 overexpression was performed by cDNA microarray analysis and confirmed by real-time reverse-transcription polymerase chain reaction. Kinase activities were assayed by Western blot analysis or kinase assay. Apoptosis was assessed by morphologic inspection and flow-cytometric analysis. Cytotoxicity was monitored by MTT assay. Results:E2F-1 upregulated the expression of ASK1 8-fold compared to the Ad-LacZ-infected control in SK-MEL-2 melanoma cells, which was confirmed by reverse-transcription polymerase chain reaction. Sequence analysis showed that there are 2 putative E2F-1 DNA binding sites in the ASK1 promoter region. Truncated E2F-1 protein, which lacks the transactivation domain, failed to upregulate ASK1, suggesting that ASK1 was regulated at the transcriptional level by E2F-1. E2F-1 overexpression resulted in the transient activation of c-Jun N-terminal kinase (JNK); however, dominant negative mutant ASK1 had no effect on E2F-1 cytotoxicity and JNK activation. p38 was not activated by E2F-1, and inhibition of p38 had no effect on E2F-1-mediated cell death. The ASK1 kinase assay showed that ASK1 activity was not upregulated in response to E2F1 overexpression. The inhibition of ASK1 upstream kinase-AKT can enhance E2F-1-mediated cell death. Moreover, an adenovirus expressing truncated E2F-1 keeps the ability of inducing apoptosis in melanoma cells. Conclusions:ASK1 expression is upregulated by E2F-1 at the transcription level, but the upregulation of ASK1 expression by E2F-1 was not coordinated with an increased ASK1 activity. The ASK1-JNK/p38 pathway does not appear to play a crucial role in E2F-1-induced apoptosis.

Oncolytic adenovirus targeting cyclin E overexpression repressed tumor growth in syngeneic immunocompetent mice

BackgroundClinical trials have indicated that preclinical results obtained with human tumor xenografts in mouse models may overstate the potential of adenovirus (Ad)-mediated oncolytic therapies. We have previously demonstrated that the replication of human Ads depends on cyclin E dysregulation or overexpression in cancer cells. ED-1 cell derived from mouse lung adenocarcinomas triggered by transgenic overexpression of human cyclin E may be applied to investigate the antitumor efficacy of oncolytic Ads.MethodsAd-cycE was used to target cyclin E overexpression in ED-1 cells and repress tumor growth in a syngeneic mouse model for investigation of oncolytic virotherapies.ResultsMurine ED-1 cells were permissive for human Ad replication and Ad-cycE repressed ED-1 tumor growth in immunocompetent FVB mice. ED-1 cells destroyed by oncolytic Ads in tumors were encircled in capsule-like structures, while cells outside the capsules were not infected and survived the treatment.ConclusionAd-cycE can target cyclin E overexpression in cancer cells and repress tumor growth in syngeneic mouse models. The capsule structures formed after Ad intratumoral injection may prevent viral particles from spreading to the entire tumor.

Indole-3-carbinol (I3C) increases apoptosis, represses growth of cancer cells, and enhances adenovirus-mediated oncolysis.

Epidemiological studies suggest that high intake of cruciferous vegetables is associated with a lower risk of cancer. Experiments have shown that indole-3-carbinol (I3C), a naturally occurring compound derived from cruciferous vegetables, exhibits potent anticarcinogenic properties in a wide range of cancers. In this study, we showed that higher doses of I3C (≥400 μM) induced apoptotic cancer cell death and lower doses of I3C (≤200 μM) repressed cancer cell growth concurrently with suppressed expression of cyclin E and its partner CDK2. Notably, we found that pretreatment with low doses of I3C enhanced Ad-mediated oncolysis and cytotoxicity of human carcinoma cells by synergistic upregulation of apoptosis. Thus, the vegetable compound I3C as a dietary supplement may benefit cancer prevention and improve Ad oncolytic therapies.