Humanin (HN) is a mitochondrial-derived peptide with robust cytoprotective effects in many cell types. Although the administration of HN analogs has been proposed to treat degenerative diseases, its role in the pathogenesis of cancer is poorly understood. Here, we evaluated whether HN affects the chemosensitivity of glioblastoma (GBM) cells. We found that chemotherapy upregulated HN expression in GBM cell lines and primary cultures derived from GBM biopsies. An HN analog (HNGF6A) boosted chemoresistance, increased the migration of GBM cells and improved their capacity to induce endothelial cell migration and proliferation. Chemotherapy also upregulated FPR2 expression, an HN membrane-bound receptor, and the HNGF6A cytoprotective effects were inhibited by an FPR2 receptor antagonist (WRW4). These effects were observed in glioma cells with heterogeneous genetic backgrounds, i.e., glioma cells with wild-type (wtIDH) and mutated (mIDH) isocitrate dehydrogenase. HN silencing using a baculoviral vector that encodes for a specific shRNA for HN (BV.shHN) reduced chemoresistance, and impaired the migration and proangiogenic capacity of GBM cells. Taken together, our findings suggest that HN boosts the hallmark characteristics of GBM, i.e., chemoresistance, migration and endothelial cell proliferation. Thus, strategies that inhibit the HN/FPR2 pathway may improve the response of GBM to standard therapy

We aimed to assess the potential of baculoviral vectors (BV) for brain cancer gene therapy. We compared them with adenoviral vectors (AdV), which are used in neuro-oncology, but for which there is pre-existing immunity. We constructed BVs and AdVs encoding fluorescent reporter proteins and evaluated their transduction efficiency in glioma cells and astrocytes. Naïve and glioma-bearing mice were intracranially injected with BVs to assess transduction and neuropathology. Transgene expression was also assessed in the brain of BV-preimmunized mice. While the expression of BVs was weaker than AdVs in murine and human glioma cell lines, BV-mediated transgene expression in patient-derived glioma cells was similar to AdV-mediated transduction and showed strong correlation with clathrin expression, a protein that interacts with the baculovirus glycoprotein GP64, mediating BV endocytosis. BVs efficiently transduced normal and neoplastic astrocytes in vivo, without apparent neurotoxicity. BV-mediated transgene expression was stable for at least 21 days in the brain of naïve mice, but it was significantly reduced after 7 days in mice systemically preimmunized with BVs. Our findings indicate that BVs efficiently transduce glioma cells and astrocytes without apparent neurotoxicity. Since humans do not present pre-existing immunity against BVs, these vectors may constitute a valuable tool for the delivery of therapeutic genes into the brain.

Tandem repeats (TRs) in protein sequences are consecutive, highly similar sequence motifs. Some types of TRs fold into structural units that pack together in ensembles, forming either an (open) elongated domain or a (closed) propeller, where the last unit of the ensemble packs against the first one. Here, we examine TR proteins (TRPs) to see how their sequence, structure, and evolutionary properties favor them for a function as mediators of protein interactions. Our observations suggest that TRPs bind other proteins using large, structured surfaces like globular domains; in particular, open-structured TR ensembles are favored by flexible termini and the possibility to tightly coil against their targets. While, intuitively, open ensembles of TRs seem prone to evolve due to their potential to accommodate insertions and deletions of units, these evolutionary events are unexpectedly rare, suggesting that they are advantageous for the emergence of the ancestral sequence but are early fixed. We hypothesize that their flexibility makes it easier for further proteins to adapt to interact with them, which would explain their large number of protein interactions. We provide insight into the properties of open TR ensembles, which make them scaffolds for alternative protein complexes to organize genes, RNA and proteins.

Baculoviruses are insect-specific pathogens widely used in biotechnology. In particular, the Autographa californica nucleopolyhedrovirus (AcMNPV) has been exploited as a platform for bio-inputs production. This is why the improvement of the technologies used for the production of recombinant baculoviruses takes on particular relevance. To achieve this goal, we developed a highly versatile baculoviral transfer vector generation system called PluriBAC. The PluriBAC system consists of three insert entry levels using Golden Gate assembly technology. The wide availability of vectors and sticky ends allows enough versatility to combine more than four different promoters, genes of interest, and terminator sequences. Here, we report not only the rational design of the PluriBAC system but also its use for the generation of baculoviral reporter vectors applied to different fields of biotechnology. We demonstrated that recombinant AcMNPV baculoviruses generated with the PluriBAC system were capable of infecting Spodoptera frugiperda larvae. On the other hand, we found that the recombinant budded virions (BV) generated using our system were capable of transducing different types of tumor and normal cells both in vitro and in vivo. Our findings suggest that the PluriBAC system could constitute a versatile tool for the generation of insecticide and gene therapy vectors.

Diffuse gliomas (DGs) are malignant primary brain tumors originating from glial cells. This study aimed to investigate the role of Receptor-interacting protein kinase 1 (RIPK1) in DG pathology. The RIPK1 mRNA expression was analyzed in DG databases from The Cancer Genome Atlas (TCGA) containing clinical, genomic, and transcriptomic information from 670 patients. Transcriptomic studies were carried out using USC Xena and R, while in vitro assays were performed with the glioblastoma human cell line U251 and the commercial RIPK1 inhibitor GSK2982772. The results showed that high RIPK1 expression was linked to a lower survival probability in patients. Additionally, the RIPK1 expression was higher in the wtIDH samples compared to that in the mIDH samples. Significant differences in the expression of genes related to cellular dedifferentiation, proinflammatory cell death pathways, and tumor-infiltrating immune cells were found between high- and low-RIPK1 expression groups. To further characterize the role of RIPK1 in DG, the effects of the RIPK1 inhibitor were evaluated, alone or combined with cisplatin, on glioblastoma cell proliferation and apoptosis. The combined treatments effectively reduced cell proliferation and increased apoptosis. The overexpression of RIPK1 was associated with a poor prognosis for DG, suggesting that RIPK1 plays a critical role in glioma pathogenesis and should be considered in therapeutic decision-making.

BIRC6, a member of the inhibitor of apoptosis protein family (IAP), regulates apoptosis, autophagy and cytokinesis. IAPs are often overexpressed in tumors, contributing to oncogenesis, therapy resistance and worse prognosis. In particular, BIRC6 overexpression has been found in several tumor tissues. The aim of this study was to evaluate the effect of BIRC6 silencing on the apoptotic response of breast and lung tumor cells. We used RNA interference based on short hairpin RNA (shRNA) to knock down gene expression encoded by a recombinant baculovirus (BV), an insect-specific virus unable to replicate in mammalian hosts, to carry out preclinical validation tests in experimental models both in vitro and in vivo. Our results indicate that BIRC6 plays an antiapoptotic role in both breast and lung tumor cells. In vivo, treatment with BV-shBRIC6 reduced breast and lung tumor progression and increased overall survival. After histological analysis, BV-shBRIC6 was able to increase tumor necrosis. In addition, we demonstrated that BIRC6 expression correlates with antiapoptotic and tumor progression-relevant markers in lung and breast cancer patients. BV-based silencing of BIRC6 may have therapeutic value for the treatment of lung and breast tumors. Further translational studies of BV-shBIRC6 in lung and breast cancer are warranted.