p62/SQSTM1 promotes mitophagy and activates the NRF2-mediated antioxidant and anti-inflammatory response restraining EBV-driven B lymphocyte proliferation.

Reactive oxygen species (ROS) and DNA repair, respectively, promote and limit oncogenic transformation of B cells driven by Epstein-Barr virus (EBV). We have previously shown that EBV infection reduced autophagy in primary B lymphocytes and enhanced ROS and interleukin 6 (IL-6) release, promoting B-cell proliferation and immortalization. In this study, we explored the role of p62/SQSTM1, accumulated as a consequence of autophagy reduction in EBV-infected B lymphocytes, and found that it exerted a growth-suppressive effect in these cells. At the molecular level, we found that p62 counteracted IL-6 production and ROS increase by interacting with NRF2 and promoting mitophagy. Moreover, p62/NRF2 axis sustained the expression level of H2AX and ataxia-telangiectasia mutated (ATM), whose activation has been shown to have growth-suppressive effects during the first steps of EBV infection, before latency is established. In conclusion, this study shows for the first time that the accumulation of p62 and the activation of p62/axis counteracted EBV-driven proliferation of primary B lymphocytes.

The cross-talk between STAT1/STAT3 and ROS up-regulates PD-L1 and promotes the release of pro-inflammatory/immune suppressive cytokines in primary monocytes infected by HHV-6B.

Programmed death ligand 1 (PD-L1) up-regulation on antigen presenting cells induces T cell dysfunction, strongly impairing immune response. Human Herpesviruses (HHV) 6B is a ß-herpesvirus that, although displays a higher tropism for T cells, can infect other immune cells including monocytes and dendritic cells (DCs) and neuronal cells. We have previously shown that HHV-6B infection of primary monocytes reduced autophagy and induced Endoplasmic Reticulum (ER) stress/ Unfolded Protein Response (UPR), impairing their survival and differentiation into DCs. In this study, we found that PD-L1 expression was up-regulated by HHV-6B on the surface of infected monocytes and that its extracellular release also increased, effects known to lead to an impairment of anti-viral immune response. At molecular level, PD-L1 up-regulation correlated with the activation of a positive regulatory circuit between the increase of intracellular ROS and the activation of STAT1 and STAT3 induced by HHV-6B, accompanied by a high release of pro-inflammatory/immune suppressive cytokines. In conclusion, this study unveils new strategies put in place by HHV-6B to induce immune dysfunction and the underlying molecular pathways that could be targeted to counteract such immune suppressive effects.

Viral Infection and Autophagy Dysregulation: The Case of HHV-6, EBV and KSHV.

Human Herpes Virus-6 (HHV-6), Epstein-Barr Virus (EBV) and Kaposi Sarcoma Herpes Virus (KSHV) are viruses that share with other member of the Herpesvirus family the capacity to interfere with the autophagic process. In this paper, mainly based on the findings of our laboratory, we describe how, through different mechanisms, these viruses converge in reducing autophagy to impair DC immune function and how, by infecting and dysregulating autophagy in different cell types, they promote the pathologies associated with their infection, from the neurodegenerative diseases such Alzheimer's disease to cancer.

BFRF1 protein is involved in EBV-mediated autophagy manipulation.

Viral egress and autophagy are two mechanisms that seem to be strictly connected in Herpesviruses's biology. Several data suggest that the autophagic machinery facilitates the egress of viral capsids and thus the production of new infectious particles. In the Herpesvirus family, viral nuclear egress is controlled and organized by a well conserved group of proteins named Nuclear Egress Complex (NEC). In the case of EBV, NEC is composed by BFRF1 and BFLF2 proteins, although the alterations of the nuclear host cell architecture are mainly driven by BFRF1, a multifunctional viral protein anchored to the inner nuclear membrane of the host cell. BFRF1 shares a peculiar distribution with several nuclear components and with them it strictly interacts. In this study, we investigated the possible role of BFRF1 in manipulating autophagy, pathway that possibly originates from nucleus, regulating the interplay between autophagy and viral egress.

KSHV dysregulates bulk macroautophagy, mitophagy and UPR to promote endothelial to mesenchymal transition and CCL2 release, key events in viral-driven sarcomagenesis.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of KS, an aggressive neoplasm that mainly occurs in immune-compromised patients. Spindle cells represent the main feature of this aggressive malignancy and arise from KSHV-infected endothelial cells undergoing endothelial to mesenchymal transition (EndMT), which changes their cytoskeletal composition and organization. As in epithelial to mesenchymal transition (EMT), EndMT is driven by transcription factors such as SNAI1 and ZEB1 and implies a cellular reprogramming mechanism regulated by several molecular pathways, particularly PI3K/AKT/MTOR. Here we found that KSHV activated MTOR and its targets 4EBP1 and ULK1 and reduced bulk macroautophagy and mitophagy to promote EndMT, activate ER stress/unfolded protein response (UPR), and increase the release of the pro-angiogenic and pro-inflammatory chemokine CCL2 by HUVEC cells. Our study suggests that the manipulation of macroautophagy, mitophagy and UPR and the interplay between the three could be a promising strategy to counteract EndMT, angiogenesis and inflammation, the key events of KSHV-driven sarcomagenesis.

KSHV infection skews macrophage polarisation towards M2-like/TAM and activates Ire1 α-XBP1 axis up-regulating pro-tumorigenic cytokine release and PD-L1 expression.

BACKGROUND: Kaposi's Sarcoma Herpesvirus (KSHV) is a gammaherpesvirus strongly linked to human cancer. The virus is also able to induce immune suppression, effect that contributes to onset/progression of the viral-associated malignancies. As KSHV may infect macrophages and these cells abundantly infiltrate Kaposi's sarcoma lesions, in this study we investigated whether KSHV-infection could affect macrophage polarisation to promote tumorigenesis. METHODS: FACS analysis was used to detect macrophage markers and PD-L1 expression. KSHV infection and the molecular pathways activated were investigated by western blot analysis and by qRT-PCR while cytokine release was assessed by Multi-analyte Kit. RESULTS: We found that KSHV infection reduced macrophage survival and skewed their polarisation towards M2 like/TAM cells, based on the expression of CD163, on the activation of STAT3 and STAT6 pathways and the release of pro-tumorigenic cytokines such as IL-10, VEGF, IL-6 and IL-8. We also found that KSHV triggered Ire1 α-XBP1 axis activation in infected macrophages to increase the release of pro-tumorigenic cytokines and to up-regulate PD-L1 surface expression. CONCLUSIONS: The findings that KSHV infection of macrophages skews their polarisation towards M2/TAM and that activate Ire1 α-XBP1 to increase the release of pro-tumorigenic cytokines and the expression of PD-L1, suggest that manipulation of UPR could be exploited to prevent or improve the treatment of KSHV-associated malignancies.

HHV-6A infection dysregulates autophagy/UPR interplay increasing beta amyloid production and tau phosphorylation in astrocytoma cells as well as in primary neurons, possible molecular mechanisms linking viral infection to Alzheimer's disease.

HHV-6A and HHV-6B are neurotropic viruses able to dysregulate autophagy and activate ER stress/UPR in several cell types. The appropriate functioning of these processes is required for cell homeostasis, particularly in post-mitotic cells such as neuronal cells. Interestingly, neurodegenerative diseases such as Alzheimer's disease (AD) are often accompanied by autophagy dysregulation and abnormal UPR activation. This study demonstrated for the first time that HHV-6A infection of astrocytoma cells and primary neurons reduces autophagy, increases Aß production and activates ER stress/UPR promoting tau protein hyper-phosphorylation. Our results support previous studies suggesting that HHV-6A infection may play a role in AD and unveil the possible underlying molecular mechanisms involved.

Quercetin Interrupts the Positive Feedback Loop Between STAT3 and IL-6, Promotes Autophagy, and Reduces ROS, Preventing EBV-Driven B Cell Immortalization.

The oncogenic gammaherpesvirus Epstein-Barr virus (EBV) immortalizes in vitro B lymphocytes into lymphoblastoid cell lines (LCLs), a model that gives the opportunity to explore the molecular mechanisms driving viral tumorigenesis. In this study, we addressed the potential of quercetin, a widely distributed flavonoid displaying antioxidant, anti-inflammatory, and anti-cancer properties, in preventing EBV-driven B cell immortalization. The results obtained indicated that quercetin inhibited thectivation of signal transducer and activator of transcription 3 (STAT3) induced by EBV infection and reduced molecules such as interleukin-6 (IL-6) and reactive oxidative species (ROS) known to be essential for the immortalization process. Moreover, we found that quercetin promoted autophagy and counteracted the accumulation of sequestosome1/p62 (SQSTM1/p62), ultimately leading to the prevention of B cell immortalization. These findings suggest that quercetin may have the potential to be used to counteract EBV-driven lymphomagenesis, especially if its stability is improved.

HHV-6B reduces autophagy and induces ER stress in primary monocytes impairing their survival and differentiation into dendritic cells.

HHV-6A and HHV-6B are ubiquitous human betaherpesviruses sharing more than 80% homology. HHV-6B is the most common cause of encephalitis in transplant patients and its primary infection may cause the exanthema subitum and febrile seizures in infants. HHV-6A and HHV-6B are able to infect several immune cell types such as T cells, monocytes and dendritic cells (DCs). In this study we found that HHV-6 B derived from patients affected by exanthema subitum impaired monocyte differentiation into DCs, as the infected cells acquired less CD1a DC marker and retained more CD14 monocyte marker. In agreement with the previous finding that HHV-6B dysregulated autophagy and induced endoplasmic reticulum (ER) stress in cells in which it replicated, here we found that these effects occurred also in differentiating monocytes and that ER stress relief, by using the chemical chaperone sodium 4-phenylbutirate (PBA), partially restored DC formation. This suggests that the induction of ER stress, likely exacerbated by autophagy inhibition, could contribute to the immune suppression induced by HHV-6B derived from exanthema subitem patients.

Autophagy manipulation as a strategy for efficient anticancer therapies: possible consequences.

Autophagy is a catabolic process whose activation may help cancer cells to adapt to cellular stress although, in some instances, it can induce cell death. Autophagy stimulation or inhibition has been considered an opportunity to treat cancer, especially in combination with anticancer therapies, although autophagy manipulation may be viewed as controversial. Thus, whether to induce or to inhibit autophagy may be the best option in the different cancer patients is still matter of debate. Her we will recapitulate the possible advantages or disadvantages of manipulating autophagy in cancer, not only with the aim to obtain cancer cell death and disable oncogenes, but also to evaluate its interplay with the immune response which is fundamental for the success of anticancer therapies.

Kaposi Sarcoma Herpes Virus (KSHV) infection inhibits macrophage formation and survival by counteracting Macrophage Colony-Stimulating Factor (M-CSF)-induced increase of Reactive Oxygen Species (ROS), c-Jun N-terminal kinase (JNK) phosphorylation and autophagy.

Kaposi Sarcoma Herpes Virus (KSHV) is an oncovirus belonging to the human gammaherpesvirus family, able to infect several immune cell types including B cells, dendritic cells (DCs) and monocytes. In this study, we found that KSHV infection of monocytes counteracted the Reactive Oxygen Species (ROS) increase induced by Macrophage Colony-Stimulating Factor (M-CSF), prevented c-Jun N-terminal kinase (JNK) and B-cell lymphoma-2 (Bcl-2) phosphorylation and inhibited autophagy, leading to an impairment of cell survival and differentiation into macrophages. We also show that, to further dysregulate immune response in monocytes, KSHV reduced the production of pro-inflammatory cytokines such as Tumor necrosis factor alpha (TNF α) while increased the release of the immune suppressive cytokine Interleukin-10 (IL-10). These results unveils new strategies put in place by KSHV to induce immune suppression and to persist into the infected host.

Could autophagy dysregulation link neurotropic viruses to Alzheimer's disease?

Neurotropic herpesviruses have been associated with the onset and progression of Alzheimer's disease, a common form of dementia that afflicts a large percentage of elderly individuals. Interestingly, among the neurotropic herpesviruses, herpes simplex virus-1, human herpesvirus-6A, and human herpesvirus-6B have been reported to infect several cell types present in the central nervous system and to dysregulate autophagy, a process required for homeostasis of cells, especially neurons. Indeed autophagosome accumulation, indicating an unbalance between autophagosome formation and autophagosome degradation, has been observed in neurons of Alzheimer's disease patients and may play a role in the intracellular and extracellular accumulation of amyloid ß and in the altered protein tau metabolism. Moreover, herpesvirus infection of central nervous system cells such as glia and microglia can increase the production of oxidant species through the alteration of mitochondrial dynamics and promote inflammation, another hallmark of Alzheimer's disease. This evidence suggests that it is worth further investigating the role of neurotropic herpesviruses, particularly human herpesvirus-6A/B, in the etiopathogenesis of Alzheimer's disease.

Mutant p53, Stabilized by Its Interplay with HSP90, Activates a Positive Feed-Back Loop Between NRF2 and p62 that Induces Chemo-Resistance to Apigenin in Pancreatic Cancer Cells.

Pancreatic cancer is one of the most aggressive cancers whose prognosis is worsened by the poor response to the current chemotherapies. In this study, we investigated the cytotoxic effect of Apigenin, against two pancreatic cell lines, namely Panc1 and PaCa44, harboring different p53 mutations. Apigenin is a flavonoid widely distributed in nature that displays anti-inflammatory and anticancer properties against a variety of cancers. Here we observed that Apigenin exerted a stronger cytotoxic effect against Panc1 cell line in comparison to PaCa44. Searching for mechanisms responsible for such different effect, we found that the higher cytotoxicity of Apigenin correlated with induction of higher level of intracellular ROS, reduction of mutant (mut) p53 and HSP90 expression and mTORC1 inhibition. Interestingly, we found that mutp53 was stabilized by its interplay with HSP90 and activates a positive feed-back loop between NRF2 and p62, up-regulating the antioxidant response and reducing the cytotoxicity of Apigenin. These results suggest that targeting the molecules involved in the mTOR-HSP90-mutp53-p62-NRF2-antioxidant response axis could help to overcome the chemo-resistance of pancreatic cancer to Apigenin.

STAT3 phosphorylation affects p53/p21 axis and KSHV lytic cycle activation.

The Tyr705 STAT3 constitutive activation, besides promoting PEL cell survival, contributes to the maintenance of viral latency. We found indeed that its de-phosphorylation by AG490 induced KSHV lytic cycle. Moreover, Tyr705 STAT3 de-phosphorylation, mediated by the activation of tyrosine phosphatases, together with the increase of Ser727 STAT3 phosphorylation contributed to KSHV lytic cycle induction by TPA. We then observed that p53-p21 axis, essential for the induction of KSHV replication, was activated by the inhibition of Tyr705 and by the increase of Ser727 STAT3 phosphorylation. As a possible link between STAT3, p53-p21 and KSHV lytic cycle, we found that TPA and AG490 reduced the expression of KAP-1, promoting p53 stability, p21 transcription and KSHV lytic cycle activation in PEL cells.

Epstein-Barr virus-encoded EBNA2 alters immune checkpoint PD-L1 expression by downregulating miR-34a in B-cell lymphomas.

Cancer cells subvert host immune surveillance by altering immune checkpoint (IC) proteins. Some Epstein-Barr virus (EBV)-associated tumors have higher Programmed Cell Death Ligand, PD-L1 expression. However, it is not known how EBV alters ICs in the context of its preferred host, the B lymphocyte and in derived lymphomas. Here, we found that latency III-expressing Burkitt lymphoma (BL), diffuse large B-cell lymphomas (DLBCL) or their EBNA2-transfected derivatives express high PD-L1. In a DLBCL model, EBNA2 but not LMP1 is sufficient to induce PD-L1. Latency III-expressing DLBCL biopsies showed high levels of PD-L1. The PD-L1 targeting oncosuppressor microRNA miR-34a was downregulated in EBNA2-transfected lymphoma cells. We identified early B-cell factor 1 (EBF1) as a repressor of miR-34a transcription. Short hairpin RNA (shRNA)-mediated knockdown of EBF1 was sufficient to induce miR-34a transcription, which in turn reduced PD-L1. MiR-34a reconstitution in EBNA2-transfected DLBCL reduced PD-L1 expression and increased its immunogenicity in mixed lymphocyte reactions (MLR) and in three-dimensional biomimetic microfluidic chips. Given the importance of PD-L1 inhibition in immunotherapy and miR-34a dysregulation in cancers, our findings may have important implications for combinatorial immunotherapy, which include IC inhibiting antibodies and miR-34a, for EBV-associated cancers.

Impact of HHV-6A and HHV-6B lytic infection on autophagy and endoplasmic reticulum stress.

Herpesviruses are known to manipulate autophagy to optimize their replication, counteract immune response and probably to promote tumourigenesis. This study explored, for the first time, the impact of human herpesvirus (HHV)-6 lytic infection on autophagy and demonstrated that HHV-6A and B (viruses sharing more than 80 % homology) differently affected this cellular process. Indeed, while HHV-6A (GS) infection of HSB2 cells promoted autophagy, HHV-6B (Z29) or the virus isolated from the serum of roseola infantum-affected patient-inhibited autophagy in Molt-3 cells or in PBMCs, respectively. Interestingly, the different behaviour of HHV-6A and B on the autophagic process was accompanied by different effects on endoplasmic reticulum stress, unfolded protein response and cell survival that was more strongly reduced by HHV-6B infection. We hypothesize that the ability to inhibit autophagy displayed by HHV-6B could be due to the fact that it contains gene homologues of those encoding for TRS1; the protein responsible for the block of autophagy by human cytomegalovirus. Understanding how HHV-6A/B infection regulates autophagy could be of particular interest, as it has been recently shown that this virus may be involved in Alzheimer's disease in which a dysregulation of autophagy may also play a role.

Cytotoxic Drugs Activate KSHV Lytic Cycle in Latently Infected PEL Cells by Inducing a Moderate ROS Increase Controlled by HSF1, NRF2 and p62/SQSTM1.

Previous studies have indicated that cytotoxic treatments may induce or not activate viral lytic cycle activation in cancer cells latently infected by Kaposi's sarcoma-associated herpesvirus (KSHV). To investigate the molecular mechanisms responsible for such an effect, we compared two cytotoxic treatments able to induce the viral lytic cycle, named 12-O-tetradecanoylphorbol 13-acetate (TPA) (T) in combination with sodium butyrate (B) and bortezomib (BZ), with two cytotoxic treatments that did not activate this process, named metformin (MET) and quercetin (Q). Our results indicated that TB and bortezomib increased levels of oxygen reactive species (ROS) while metformin and quercetin reduced them. The finding that N-acetylcysteine (NAC), a reactive oxigen species (ROS) scavenger, counteracted K-bZIP expression induced by TB or bortezomib, confirmed that an ROS increase played a role in KSHV lytic cycle activation. Moreover, we found that TB and bortezomib up-regulated p62/Sequestosome1(p62/SQSTM1) protein, while metformin and quercetin down-regulated it. p62/SQSTM1 silencing or the inhibition of NF-E2-related factor 2 (NRF2) or Heat Shock Factor 1 (HSF1), that mediate p62/SQSTM1 transcription, also reduced KSHV lytic antigen expression induced by TB or bortezomib. Interestingly, such combination treatments further increased intracellular ROS and cytotoxicity induced by the single TB or bortezomib treatment, suggesting that NRF2, HSF1 and p62/SQSTM1 keep the ROS level under control, allowing primary effusion lymphoma (PEL) cells to continue to survive and KSHV to replicate.

EBV up-regulates PD-L1 on the surface of primary monocytes by increasing ROS and activating TLR signaling and STAT3.

Programmed death ligand 1 (PD-L1) (also called B7-H1) is a membrane immune-modulatory protein whose overexpression on the surface of tumor cells as well as APCs impairs T-cell-mediated killing. Viruses that establish chronic infections have developed a number of strategies to escape from immune recognition including the up-regulation of PD-L1. This study shows for the first time that the human oncovirus EBV infects human primary monocytes using HLA-DR and induced a strong up-regulation of PD-L1 expression on their surface. Searching for the underlying mechanism/s leading to this immune suppressive effect, we found that EBV activated TLR signaling, increased intracellular ROS, and phosphorylated STAT3. Targeting these molecules partially reverted PD-L1 up-regulation that correlated with an altered cytokine production and a reduction of monocyte cell survival, strongly impairing the antiviral immune response.

The Nefarious Nexus of Noncoding RNAs in Cancer.

The past decade has witnessed enormous progress, and has seen the noncoding RNAs (ncRNAs) turn from the so-called dark matter RNA to critical functional molecules, influencing most physiological processes in development and disease contexts. Many ncRNAs interact with each other and are part of networks that influence the cell transcriptome and proteome and consequently the outcome of biological processes. The regulatory circuits controlled by ncRNAs have become increasingly more relevant in cancer. Further understanding of these complex network interactions and how ncRNAs are regulated, is paving the way for the identification of better therapeutic strategies in cancer.

Apigenin, by activating p53 and inhibiting STAT3, modulates the balance between pro-apoptotic and pro-survival pathways to induce PEL cell death.

BACKGROUND: Apigenin is a flavonoid widely distributed in plant kingdom that exerts cytotoxic effects against a variety of solid and haematological cancers. In this study, we investigated the effect of apigenin against primary effusion lymphoma (PEL), a KSHV-associated B cell lymphoma characterized by a very aggressive behavior, displaying constitutive activation of STAT3 as well as of other oncogenic pathways and harboring wtp53. METHODS: Cell death was assessed by trypan blue exclusion assay, FACS analysis as well as by biochemical studies. The latter were also utilized to detect the occurrence of autophagy and the molecular mechanisms leading to the activation of both processes by apigenin. FACS analysis was used to measure the intracellular ROS utilizing DCFDA. RESULTS: We show that apigenin induced PEL cell death and autophagy along with reduction of intracellular ROS. Mechanistically, apigenin activated p53 that induced catalase, a ROS scavenger enzyme, and inhibited STAT3, the most important pro-survival pathway in PEL, as assessed by p53 silencing. On the other hand, STAT3 inhibition by apigenin resulted in p53 activation, since STAT3 negatively influences p53 activity, highlighting a regulatory loop between these two pathways that modulates PEL cell death/survival. CONCLUSION: The findings of this study demonstrate that apigenin may modulate pro-apoptotic and pro-survival pathways representing a valid therapeutic strategy against PEL.