Ionizing radiation enhances dl922-947-mediated cell death of anaplastic thyroid carcinoma cells.

dl922-947 is an oncolytic adenovirus potentially suitable for the treatment of aggressive localized tumors, such as anaplastic thyroid carcinoma (ATC). In this study, we have analyzed the effects of dl922-947 in combination with ionizing radiations, testing different schedules of administration and observing synergistic effects only when ATC cells were irradiated 24 h prior to viral infection. Cells undergoing combined treatment exhibited a marked increase in cell death and viral replication, suggesting that irradiation blocks cells in a more permissive state for viral life cycle. We also show that dl922-947 triggers a DNA damage response, characterized by mobilization of the MRN complex (composed by Mre11-Rad50-Nbs1), accumulation of γH2AX, and activation of the checkpoint kinases ataxia telangiectasia mutated (ATM) and Chk1. Based on these observations, we speculate that the DNA damage response acts as a cellular protective mechanism to hinder viral infection and replication. To confirm this hypothesis, we demonstrate that the ATM inhibitor KU55933 increased the oncolytic activity of dl922-947 and its replication. Finally, we validate the potential therapeutic use of this approach by showing in vivo that the combined treatment slows tumor xenograft growth more potently than either irradiation or infection alone.

Inhibition of autophagy enhances the effects of E1A-defective oncolytic adenovirus dl922-947 against glioma cells in vitro and in vivo.

Oncolytic viruses represent a novel therapeutic approach for aggressive tumors, such as glioblastoma multiforme, which are resistant to available treatments. Autophagy has been observed in cells infected with oncolytic viruses; however, its role in cell death/survival is unclear. To elucidate the potential therapeutic use of autophagy modulators in association with viral therapy, we analyzed autophagy induction in human glioma cell lines U373MG and U87MG infected with the oncolytic adenovirus dl922-947. dl922-947 infection triggered an autophagic cellular response, as shown by the development of acidic vesicular organelles, LC3-I→LC3-II conversion, and reduction of p62 levels. However, on infection, the Akt/mTOR/p70s6k pathway, which negatively regulates autophagy, was activated, whereas the ERK1/2 pathway, a positive regulator of autophagy, was inhibited. Accordingly, MEK inhibition by PD98059 sensitized glioma cells to dl922-947 effects, whereas autophagy induction by rapamycin protected cells from dl922-947-induced death. Treatment with two inhibitors of autophagy, chloroquine and 3-methyladenine, increased the cytotoxic effects of dl922-947 in vitro. In vivo, the growth of U87MG-induced xenografts was further reduced by adding chloroquine to the dl922-947 treatment. In conclusion, autophagy acts as a survival response in glioma cells infected with dl922-947, thus suggesting autophagy inhibitors as adjuvant/neoadjuvant drugs in oncolytic virus-based treatments.

AZD1152 negatively affects the growth of anaplastic thyroid carcinoma cells and enhances the effects of oncolytic virus dl922-947.

Novel therapeutic approaches are required for the treatment of anaplastic thyroid carcinoma (ATC), an incurable disease resistant to current available therapies. Aurora B is an important mitotic kinase involved in chromosome segregation and cytokinesis. It is overexpressed in many cancers including ATC and represents a potential target for chemotherapy. The effects of AZD1152, a specific Aurora B kinase inhibitor, have been evaluated against ATC, showing G(2)/M accumulation, polyploidy and subsequent cell death by mitotic catastrophe upon drug treatment. Only three administrations of AZD1152 significantly reduced the growth of ATC tumour xenogratfs. Oncolytic viruses in association with other forms of treatment have proven highly promising in preclinical and clinical reports. The oncolytic adenovirus dl922-947 is active against ATC cells, and we have evaluated the effects of the association between AZD1152 and dl922-947. In cells treated with virus and drug, we report additive/synergistic killing effects. Interestingly, the phosphorylation of histone H3 (Ser10), the main Aurora B substrate, is inhibited by dl922-947 in a dose-dependent manner, and completely abolished in association with AZD1152. The combined treatment significantly inhibited the growth of ATC tumour xenografts with respect to single treatments. Our data demonstrate that the Aurora B inhibitor AZD1152, alone or in combination with oncolytic virus dl922-947, could represent a novel therapeutic option for the treatment of ATC.

Aurora A and B kinases--targets of novel anticancer drugs.

The Aurora Kinases are highly related serine-threonine kinases, essential for accurate and equal segregation of genomic material during mitosis. A large number of studies have linked the aberrant expression of Aurora kinases to cancer, leading to the development of specific Aurora kinases inhibitors. Several small molecules inhibit with a similar efficacy both Aurora A and Aurora B, however, in most cases the effects resemble Aurora B disruption by genetic methods, indicating that Aurora B represents an effective therapeutic target. These drugs are currently under preclinical or clinical evaluation and are reviewed in this article. The relevant patents are discussed.

PED/PEA-15 modulates coxsackievirus-adenovirus receptor expression and adenoviral infectivity via ERK-mediated signals in glioma cells.

Glioblastoma multiforme (GBM) is the most aggressive human brain tumor, and is highly resistant to chemo- and radiotherapy. Selectively replicating oncolytic viruses represent a novel approach for the treatment of neoplastic diseases. Coxsackievirus-adenovirus receptor (CAR) is the primary receptor for adenoviruses, and loss or reduction of CAR greatly decreases adenoviral entry. Understanding the mechanisms regulating CAR expression and localization will contribute to increase the efficacy of oncolytic adenoviruses. Two glioma cell lines (U343MG and U373MG) were infected with the oncolytic adenovirus dl922-947. U373MG cells were more susceptible to cell death after viral infection, compared with U343MG cells. The enhanced sensitivity was paralleled by increased adenoviral entry and CAR mRNA and protein levels in U373MG cells. In addition, U373MG cells displayed a decreased ERK1/2 (extracellular signal-regulated kinase-1/2) nuclear-to-cytosolic ratio, compared with U343MG cells. Intracellular content of PED/PEA-15, an ERK1/2-interacting protein, was also augmented in these cells. Both ERK2 overexpression and genetic silencing of PED/PEA-15 by antisense oligonucleotides increased ERK nuclear accumulation and reduced CAR expression and adenoviral entry. Our data indicate that dl922-947 could represent an useful tool for the treatment of GBM and that PED/PEA-15 modulates CAR expression and adenoviral entry, by sequestering ERK1/2.

Aurora B expression in post-puberal testicular germ cell tumours.

Aurora/Ipl1-related kinases are a conserved family of proteins that are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumours and have been implicated in mechanisms leading to mitotic spindle aberrations, aneuploidy, and genomic instability. Previous studies of our group have shown that Aurora B expression is restricted to specific germinal cells. In this study, we have evaluated by immunohistochemical analysis Aurora B expression in post-puberal testicular germ cell tumours (22 seminomas, 2 teratomas, 15 embryonal carcinomas, 5 mixed germinal tumours with a prominent yolk sac tumour component and 1 choriocarcinoma). The Aurora B protein expression was detected in all intratubular germ cell tumours, seminomas and embryonal carcinomas analysed but not in teratomas and yolk sac carcinomas. The immunohistochemical data were further confirmed by Western blot analysis. In addition, the kinase Aurora B was vigorously expressed in GC-1 cells line derived from murine spermatogonia. The block of Aurora B function induced by a pharmacological inhibitor significantly reduced the growth of GC-1 cells suggesting that Aurora B is a potential therapeutic target.