Carmela Passaro

Aurora B: A New Prognostic Marker and Therapeutic Target in Cancer

Aurora B is a serine-threonine kinase belonging to the highly conserved Aurora family of mitotic kinases. Aurora B is a chromosomal passenger protein involved in chromosome segregation, spindle-checkpoint, and cytokinesis. Alteration of each of these steps could induce aneuploidy, one of main features, and driving force of cancer progression. The overexpression of Aurora B has been observed in several tumor types, and has been linked with a poor prognosis of cancer patients. In this review we will focus on the role of Aurora B in cancer development, its role as a prognostic marker, and the clinical outcome of recently developed Aurora(s) inhibitors.

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.

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.

High Levels of Gpr30 Protein in Human Testicular Carcinoma In Situ and Seminomas Correlate with Low Levels of Estrogen Receptor-Beta and Indicate a Switch in Estrogen Responsiveness

The G protein‐coupled estrogen receptor (GPR30) is suggested to be involved in non‐nuclear estrogen signalling and is expressed in a variety of hormone dependent cancer entities. It is well established that oestrogens are involved in pathological germ cell proliferation including testicular germ cell tumours. This study was performed to further elucidate the role of this receptor and the possible correlation with the estrogen receptor β in human testicular carcinoma in situ (CIS), seminomas and in GC1 and TCam‐2 germ cell lines; in addition, a Tissue Micro‐Array was built using the most representative areas from 25 cases of human testicular seminomas and 20 cases of CIS. The expression of ERβ and GPR30 were observed by using Western blot analysis in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that down regulation of ERβ associates with GPR30 over‐expression both in human testicular CIS and seminomas. In addition, we show that 17β‐oestradiol induces the ERK1/2 activation and increases c‐Fos expression through GPR30 associated with ERβ down‐regulation in TCam‐2 cell line. The present results suggest that exposure to oestrogens or oestrogen‐mimics, in some as of yet undefined manner, diminishes the ERβ‐mediated growth restraint in CIS and in human testicular seminoma, probably due to ERβ down‐regulation associated to GPR30 increased expression indicating that GPR30 could be a potential therapeutic target to design specific inhibitors. J. Cell. Physiol. 230: 1290–1297, 2015.

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.

PARP inhibitor olaparib increases the oncolytic activity of dl922‐947 in in vitro and in vivo model of anaplastic thyroid carcinoma

PARP inhibitors are mostly effective as anticancer drugs in association with DNA damaging agents. We have previously shown that the oncolytic adenovirus dl922‐947 induces extensive DNA damage, therefore we hypothesized a synergistic antitumoral effect of the PARP inhibitor olaparib in association with dl922‐947. Anaplastic thyroid carcinoma was chosen as model since it is a particularly aggressive tumor and, because of its localized growth, it is suitable for intratumoral treatment with oncolytic viruses.

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.

Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles

Glioblastoma can suppress immunity by using surface PD-L1 on extracellular vesicles to block T cell receptor–mediated T cell activation. Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1–dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.

Selenium supplementation modulates apoptotic processes in thyroid follicular cells

Selenium (Se) is an essential micronutrient modulating several physiopathological processes in the human body. The aim of the study is to characterize the molecular effects determined by Se-supplementation in thyroid follicular cells, using as model the well-differentiated rat thyroid follicular cell line FRTL5. Experiments have been performed to evaluate the effects of Se on cell growth, mortality and proliferation and on modulation of pro- and antiapoptotic pathways. The results indicate that Se-supplementation improves FRTL5 growth rate. Furthermore, Se reduces the proportion of cell death and modulates both proapoptotic (p53 and Bim) and antiapoptotic (NF-kB and Bcl2) mRNA levels. In addition, incubation with high doses of Na-Se might prevent the ER-stress apoptosis induced by tunicamycin, as assessed by membrane integrity maintenance, reduction in caspase 3/7 activities, and reduction in Casp-3 and PARP cleavage. Taken together, these results provide molecular evidences indicating the role of Se supplementation on cell death and apoptosis modulation in thyroid follicular cells. These observations may be useful to understand the effects of this micronutrient on the physiopathology of the thyroid gland.

Preclinical investigation of combined gene-mediated cytotoxic immunotherapy and immune checkpoint blockade in glioblastoma

Background Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. Methods The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. Results GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Conclusions Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.