N-Acetyl-L-Cysteine (NAC) Blunts Axitinib-Related Adverse Effects in Preclinical Models of Glioblastoma.

OBJECTIVE: Axitinib is a tyrosine kinase inhibitor characterized by a strong affinity for Vascular Endothelial Growth Factor Receptors (VEGFRs). It was approved in 2012 by Food and Drug Administration and European Medicines Agency as a second line treatment for advanced renal cell carcinoma and is currently under evaluation in clinical trial for the treatment of other cancers. Glioblastoma IDH-wild type (GBM) is a highly malignant brain tumor characterized by diffusely infiltrative growth pattern and by a prominent neo-angiogenesis. In GBM, axitinib has demonstrated a limited effectiveness as a monotherapy, while it was recently shown to significantly improve its efficacy in combination treatments. In preclinical models, axitinib has been reported to trigger cellular senescence both in tumor as well as in normal cells, through a mechanism involving intracellular reactive oxygen species (ROS) accumulation and activation of Ataxia Telangiectasia Mutated kinase (ATM). Limiting axitinib-dependent ROS increase by antioxidants prevents senescence specifically in normal cells, without affecting tumor cells. METHODS: We used brain tumor xenografts obtained by engrafting Glioma Stem Cells (GSCs) into the brain of immunocompromised mice, to investigate the hypothesis that the antioxidant molecule N-Acetyl-L-Cysteine (NAC) might be used to reduce senescence-associated adverse effects of axitinib treatment without altering its anti-tumor activity. RESULTS: We demonstrate that the use of the antioxidant molecule N-Acetyl-Cysteine (NAC) in combination with axitinib stabilizes tumor microvessels in GBM tumor orthotopic xenografts, eventually resulting in vessel normalization, and protects liver vasculature from axitinib-dependent toxicity. CONCLUSION: Overall, we found that NAC co-treatment allows vessel normalization in brain tumor vessels and exerts a protective effect on liver vasculature, therefore minimizing axitinib-dependent toxicity.

Characterization of Glioblastoma Cells Response to Regorafenib.

Glioblastoma (GBM), the most malignant primary brain tumor in adults. Although not frequent, it has a relevant social impact because the peak incidence coincides with the age of professional maturity. A number of novel treatments have been proposed, yet clinical trials have been disappointing. Recently, a phase II clinical trial (REGOMA) demonstrated that the multikinase inhibitor regorafenib significantly increased the median overall survival (OS) of GBM patients when compared to lomustine-treated patients. On this basis, the National Comprehensive Cancer Network (NCCN) 2020 Guidelines included regorafenib as a preferred regimen in relapsed GBM treatment. Despite the use in GBM patients' therapy, little is known about the molecular mechanisms governing regorafenib effectiveness on the GBM tumor. Here we report an in vitro characterization of GBM tumor cells' response to regorafenib, performed both on cell lines and on patient-derived glioma stem cells (GSCs). Overall, regorafenib significantly reduced cell growth of 2D tumor cell cultures and of 3D tumor spheroids. Strikingly, this effect was accompanied by transcriptional regulation of epithelial to mesenchymal transition (EMT) genes and by an increased ability of surviving tumor cells to invade the surrounding matrix. Taken together, our data suggest that regorafenib limits cell growth, however, it might induce an invasive phenotype.

Epitranscriptomics: A New Layer of microRNA Regulation in Cancer.

MicroRNAs are pervasive regulators of gene expression at the post-transcriptional level in metazoan, playing key roles in several physiological and pathological processes. Accordingly, these small non-coding RNAs are also involved in cancer development and progression. Furthermore, miRNAs represent valuable diagnostic and prognostic biomarkers in malignancies. In the last twenty years, the role of RNA modifications in fine-tuning gene expressions at several levels has been unraveled. All RNA species may undergo post-transcriptional modifications, collectively referred to as epitranscriptomic modifications, which, in many instances, affect RNA molecule properties. miRNAs are not an exception, in this respect, and they have been shown to undergo several post-transcriptional modifications. In this review, we will summarize the recent findings concerning miRNA epitranscriptomic modifications, focusing on their potential role in cancer development and progression.

The RNA editing enzyme ADAR2 restricts L1 mobility.

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosines to inosines in double-stranded RNAs (RNA editing A-to-I). ADAR1 and ADAR2 were previously reported as HIV-1 proviral factors. The aim of this study was to investigate the composition of the ADAR2 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 10 non-ribosomal ADAR2-interacting factors. A significant fraction of these proteins was previously found associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons. Considering that we previously demonstrated that ADAR1 is an inhibitor of LINE-1 retrotransposon activity, we investigated whether also ADAR2 played a similar function. To reach this goal, we performed specific cell culture retrotransposition assays in cells overexpressing or ablated for ADAR2. These experiments unveil a novel function of ADAR2 as suppressor of L1 retrotransposition. Furthermore, we showed that ADAR2 binds the basal L1 RNP complex.Overall, these data support the role of ADAR2 as regulator of L1 life cycle.

Post-transcriptional regulation of LINE-1 retrotransposition by AID/APOBEC and ADAR deaminases.

Long interspersed element-1 (LINE-1 or L1) retrotransposons represent the only functional family of autonomous transposable elements in humans and formed 17% of our genome. Even though most of the human L1 sequences are inactive, a limited number of copies per individual retain the ability to mobilize by a process termed retrotransposition. The ongoing L1 retrotransposition may result in insertional mutagenesis that could lead to negative consequences such as genetic disease and cancer. For this reason, cells have evolved several mechanisms of defense to restrict L1 activity. Among them, a critical role for cellular deaminases [activation-induced deaminase (AID)/apolipoprotein B mRNA-editing catalytic polypeptide-like (APOBEC) and adenosine deaminases that act on RNA (ADAR) enzymes] has emerged. The majority of the AID/APOBEC family of proteins are responsible for the deamination of cytosine to uracil (C-to-U editing) within DNA and RNA targets. The ADARs convert adenosine bases to inosines (A-to-I editing) within double-stranded RNA (dsRNA) targets. This review will discuss the current understanding of the regulation of LINE-1 retrotransposition mediated by these enzymes.

Restricting retrotransposons: ADAR1 is another guardian of the human genome.

ADAR1 is an enzyme that belongs to the Adenosine Deaminases Acting on RNA (ADARs) family. These enzymes deaminate adenosines to inosines (RNA editing A-to-I) within double-stranded RNA regions in transcripts. Since inosines are recognized as guanosines by the cellular machinery, RNA editing mediated by ADARs can either lead to the formation of an altered protein (recoding) or affect different aspects of RNA metabolism. Recently, a proteomic analysis led to the identification of novel ADAR1-associated factors and found that a good fraction of them is shared with the Long Interspersed Element 1 (LINE-1 or L1) ribonucleoparticles (RNPs). This evidence suggested a possible role of ADAR1 in regulating the L1 life cycle. By taking advantage of the use of cell culture retrotransposition assays, a novel function of this deaminase as an inhibitor of L1 retrotransposition was demonstrated. These results pave the way toward a better comprehension of the mechanisms of restriction of retrotransposons.

Novel HBsAg mutations correlate with hepatocellular carcinoma, hamper HBsAg secretion and promote cell proliferation in vitro.

BACKGROUND: An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation. METHODS: This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry. RESULTS: Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001). CONCLUSIONS: Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.

ADAR1 restricts LINE-1 retrotransposition.

Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosines to inosines in double-stranded RNAs. ADAR1 was demonstrated to be functional on different viruses exerting either antiviral or proviral effects. Concerning HIV-1, several studies showed that ADAR1 favors viral replication. The aim of this study was to investigate the composition of the ADAR1 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 14 non-ribosomal ADAR1-interacting proteins, most of which are novel. A significant fraction of these proteins were previously demonstrated to be associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons that continuously re-enter host-genome.Hence, we investigated the function of ADAR1 in the regulation of L1 activity.By using different cell-culture based retrotransposition assays in HeLa cells, we demonstrated a novel function of ADAR1 as suppressor of L1 retrotransposition. Apparently, this inhibitory mechanism does not occur through ADAR1 editing activity. Furthermore, we showed that ADAR1 binds the basal L1 RNP complex. Overall, these data support the role of ADAR1 as regulator of L1 life cycle.

Resetting cancer stem cell regulatory nodes upon MYC inhibition.

MYC deregulation is common in human cancer and has a role in sustaining the aggressive cancer stem cell populations. MYC mediates a broad transcriptional response controlling normal biological programmes, but its activity is not clearly understood. We address MYC function in cancer stem cells through the inducible expression of Omomyc-a MYC-derived polypeptide interfering with MYC activity-taking as model the most lethal brain tumour, glioblastoma. Omomyc bridles the key cancer stemlike cell features and affects the tumour microenvironment, inhibiting angiogenesis. This occurs because Omomyc interferes with proper MYC localization and itself associates with the genome, with a preference for sites occupied by MYC This is accompanied by selective repression of master transcription factors for glioblastoma stemlike cell identity such as OLIG2, POU3F2, SOX2, upregulation of effectors of tumour suppression and differentiation such as ID4, MIAT, PTEN, and modulation of the expression of microRNAs that target molecules implicated in glioblastoma growth and invasion such as EGFR and ZEB1. Data support a novel view of MYC as a network stabilizer that strengthens the regulatory nodes of gene expression networks controlling cell phenotype and highlight Omomyc as model molecule for targeting cancer stem cells.

The ADAR1 editing enzyme is encapsidated into HIV-1 virions.

Adenosine deaminase acting on RNA1 (ADAR1) was previously reported to affect HIV-1 replication. We report data showing that ADAR1 interacts with the HIV-1 p55 Gag protein, the major structural protein of the immature virus capsid. Furthermore, we found that the endogenous ADAR1 is incorporated into virions purified from the supernatant of primary HIV-1-infected CD4(+) T lymphocytes. Additional experiments demonstrated that the expression of the p55 Gag protein is sufficient for ADAR1 incorporation into virus-like particles (VLPs). Overall, our data originally support the evidence that ADAR1 can be part of the cell protein array uploaded in HIV-1 particles.

HIV-1 infection causes a down-regulation of genes involved in ribosome biogenesis.

HIV-1 preferentially infects CD4+ T cells, causing fundamental changes that eventually lead to the release of new viral particles and cell death. To investigate in detail alterations in the transcriptome of the CD4+ T cells upon viral infection, we sequenced polyadenylated RNA isolated from Jurkat cells infected or not with HIV-1. We found a marked global alteration of gene expression following infection, with an overall trend toward induction of genes, indicating widespread modification of the host biology. Annotation and pathway analysis of the most deregulated genes showed that viral infection produces a down-regulation of genes associated with the nucleolus, in particular those implicated in regulating the different steps of ribosome biogenesis, such as ribosomal RNA (rRNA) transcription, pre-rRNA processing, and ribosome maturation. The impact of HIV-1 infection on genes involved in ribosome biogenesis was further validated in primary CD4+ T cells. Moreover, we provided evidence by Northern Blot experiments, that host pre-rRNA processing in Jurkat cells might be perturbed during HIV-1 infection, thus strengthening the hypothesis of a crosstalk between nucleolar functions and viral pathogenesis.

The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222.

Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells.