Background: Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with asbestos exposure that urgently requires effective therapeutic strategies. Current treatments are unable to increase significantly patient survival, which is often limited to <1 year from diagnosis. Virotherapy, based on the use of oncolytic viruses that exert anti-cancer effects by direct cell lysis and through the induction of anti-tumor immune response, represents an alternative therapeutic option for rare tumors with limited life expectancy. In this study, we propose the use of the adenovirus dl 922-947, engineered to allow selective replication in cancer cells, to counteract MPM. Methods: We performed a thorough preclinical assessment of dl 922-947 effects in a set of MPM cell lines and xenografts. Cytotoxicity of dl 922-947 alone and in combination assays was evaluated by sulforhodamine B assay. Cell cycle, calreticulin expression, and high mobility group box protein 1 (HMGB1) secretion were determined by flow cytometry, whereas ATP content was determined by a luminescence-based bioassay. The modulation of angiogenic factors in MPM-infected cells was evaluated through ELISA. Results: We found that dl 922-947 infection exhibits cytotoxic effects in MPM cell lines, affecting cell viability, cell cycle progression, and regulating main hallmarks of immunogenic cell death inducing calreticulin surface exposure, HMGB1 and ATP release. Our results also suggest that dl 922-947 may affect angiogenic signals by regulation of VEGF-A and IL-8 secretion. Furthermore, dl 922-947 shows anti-tumor efficacy in murine xenograft models reducing tumor growth and enhancing survival. Finally, the combination with cisplatin potentiated the cytotoxic effect of dl 922-947. Conclusions: Overall our data identify virotherapy, based on the use of dl 922-947, as a new possible therapeutic strategy against MPM, which could be used alone, in combination with standard chemotherapy drugs, as shown here, or other approaches also aimed at enhancing the antitumoral immune response elicited by the virus.

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The retinoblastoma (RB) protein family includes RB1/p105, RBL1/p107, and RBL2/p130, which are key factors in cell-cycle regulation and stand at the crossroads of multiple pathways dictating cell fate decisions. The role of RB proteins in apoptosis is controversial because they can inhibit or promote apoptosis depending on the context, on the apoptotic stimuli and on their intrinsic status, impacting on the response to antitumoral treatments. Here we identified RBL2/p130 as a direct substrate of the AKT kinase, a key antiapoptotic factor hyperactive in multiple cancer types. We showed that RBL2/p130 and AKT1 physically interact and AKT phosphorylates RBL2/p130 Ser941, located in the pocket domain, but not when this residue is mutated into Ala. We found that pharmacological inhibition of AKT, through the highly selective AKT inhibitor VIII (AKTiVIII), impairs RBL2/p130 Ser941 phosphorylation and increases RBL2/p130 stability, mRNA expression and nuclear levels in both lung cancer and mesothelioma cell lines, mirroring the more extensively studied effects on the p27 cell-cycle inhibitor. Consistently, AKT inhibition reduced cell viability, induced cell accumulation in G0/G1, and triggered apoptosis, which proved to be largely dependent on RBL2/p130 itself, as shown upon RBL2/p130 silencing. AKT inhibition induced RBL2/p130-dependent apoptosis also in HEK-293 cells, in which re-expression of a short hairpin-resistant RBL2/p130 was able to rescue AKTiVIII-induced apoptosis upon RBL2/p130 silencing. Our data also showed that the combination of AKT and cyclin-dependent kinases (CDK) inhibitors, which converge on the re-activation of RBL2/p130 antitumoral potential, could be a promising anticancer strategy.

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