Targeting of Prosurvival Pathways as Therapeutic Approaches against Primary Effusion Lymphomas: Past, Present, and Future

Constitutively activated prosurvival pathways render cancer cells addicted to their effects. Consequently they turn out to be the Achilles’ heels whose inhibition can be exploited in anticancer therapy. Primary effusion lymphomas (PELs) are very aggressive non-Hodgkin’s B cell lymphomas, whose pathogenesis is strictly linked to Kaposi’s sarcoma herpesvirus (KSHV) infection. Here we summarized previous studies from our and other laboratories exploring the cytotoxic effect of drugs inhibiting the main prosurvival pathways activated in PEL cells. Moreover, the immunogenicity of cell death, in terms of dendritic cell (DC) activation and their potential side effect on DCs, is discussed

Hyperglycemia triggers HIPK2 protein degradation

Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies

Interplay between endoplasmic reticulum (ER) stress and autophagy induces mutant p53H273 degradation

The unfolded protein response (UPR) is an adaptive response to intrinsic and external stressors, and it is mainly activated by the accumulation of misfolded proteins at the endoplasmic reticulum (ER) lumen producing ER stress. The UPR signaling network is interconnected with autophagy, the proteolytic machinery specifically devoted to clearing misfolded proteins in order to survive bioenergetic stress and/or induce cell death. Oncosuppressor TP53 may undergo inactivation following missense mutations within the DNA-binding domain (DBD), and mutant p53 (mutp53) proteins may acquire a misfolded conformation, often due to the loss of the DBD-bound zinc ion, leading to accumulation of hyperstable mutp53 proteins that correlates with more aggressive tumors, resistance to therapies, and poorer outcomes. We previously showed that zinc supplementation induces mutp53 protein degradation by autophagy. Here, we show that mutp53 (i.e., Arg273) degradation following zinc supplementation is correlated with activation of ER stress and of the IRE1α/XBPI arm of the UPR. ER stress inhibition with chemical chaperone 4-phenyl butyrate (PBA) impaired mutp53 downregulation, which is similar to IRE1α/XBPI specific inhibition, reducing cancer cell death. Knockdown of mutp53 failed to induce UPR/autophagy activation indicating that the effect of zinc on mutp53 folding was likely the key event occurring in ER stress activation. Recently discovered small molecules targeting components of the UPR show promise as a novel anticancer therapeutic intervention. However, our findings showing UPR activation during mutp53 degradation indicate that caution is necessary in the design of therapies that inhibit UPR components

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

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

DENDRITIC CELL DIFFERENTIATION BLOCKED BY PRIMARY EFFUSION LYMPHOMA-RELEASED FACTORS IS PARTIALLY RESTORED BY INHIBITION OF P38 MAPK

To better understand the molecular mechanisms underlying the dendritic cell (DC) defects in cancer, we analyzed which signaling pathway is implicated in the abnormal monocyte differentiation into DC determined by the presence of Primary effusion lymphoma (PEL) released factors. Our results indicate that the DC, obtained in this condition, together with phenotypic abnormalities and reduced allostimulatory function, showed hyperphosphorylation of signal transducer and activator of transcription 3 (STAT3) and p38 mitogen-activated protein kinase (MAPK) molecules, in comparison to the DC differentiated in the absence of PEL-released factors. The inhibition of p38 MAPK but not of STAT3 phosphorylation, with specific inhibitors, was able to revert the effect of the PEL-released factors on the DC phenotype. This study suggests that p38 MAPK signaling pathway is an important contributor to the abnormal differentiation of DC in PEL

Reduced chemotherapeutic sensitivity in high glucose condition: implication of antioxidant response

Resistance to chemotherapy represents a major obstacle to successful treatment. The generation of reactive oxygen species (ROS) has been directly linked to the cytotoxic effects of several antitumor agents, including Adriamycin (ADR), and modulation of the oxidative balance has been implicated in the development and/or regulation of resistance to chemotherapeutic drugs. We recently showed that high glucose (HG) markedly diminished the cancer cell death induced by anticancer agents such as ADR. In the present study we attempted to evaluate the mechanism that impaired the cytotoxic effect of ADR in HG. We found that, in colon cancer cells, HG attenuated ADR-induced ROS production that consequently diminished ADR-induced H2AX phosphorylation and micronuclei (MN) formation. Mechanistically, HG attenuation of ADR-induced ROS production correlated with increased antioxidant response promoted by NRF2 activity. Thus, pharmacologic inhibition of NRF2 pathway by brusatol re-established the ADR cytotoxic effect impaired by HG. Together, the data provide new insights into chemotherapeutic-resistance mechanisms in HG condition dictated by increased NRF2-induced antioxidant response and how they may be overcome in order to restore chemosensitivity and ADR-induced cell death

The activation of KSHV lytic cycle blocks autophagy in PEL cells

This study confirms that autophagy is activated concomitantly with KSHV lytic cycle induction, and that autophagy inhibition by BECN1 knockdown reduces viral lytic gene expression. In addition, we extend previous observations and show that autophagy is blocked at late steps, during viral replication. This is indicated by the lack of colocalization of autophagosomes and lysosomes and by the LC3-II level that does not increase in the presence of bafilomycin A1 in primary effusion lymphoma (PEL) cells induced to enter the lytic cycle, either by TPA/sodium butyrate (BC3 and BCBL1) or by doxycycline (TRExBCBL1-Rta). The autophagic block correlates with the downregulation of RAB7, whose silencing with specific siRNA results in an autophagic block in the same cells. Finally, by electron microscopy analysis, we observed viral particles inside autophagic vesicles in the cytoplasm of PEL cells undergoing viral replication, suggesting that they may be involved in viral transpor

Bortezomib promotes KHSV and EBV lytic cycle by activating JNK and autophagy

KSHV and EBV are gammaherpesviruses strictly linked to human cancers. Even if the majority of cancer cells harbor a latent infection, the few cells that undergo viral replication may contribute to the pathogenesis and maintenance of the virus-associated malignancies. Cytotoxic drugs used for the therapies of cancers harboring virus-infection often have, as side effect, the activation of viral lytic cycle. Therefore it is important to investigate whether they affect viral reactivation and understand the underlying mechanisms involved. In this study, we found that proteasome inhibitor bortezomib, a cytotoxic drug that efficiently target gammaherpesvirus-associated B cell lymphomas, triggered KSHV or EBV viral lytic cycle by activating JNK, in the course of ER stress, and inducing autophagy. These results suggest that the manipulation of these pathways could limit viral spread and improve the outcome of bortezomib treatment in patients affected by gammaherpesvirus-associated lymphomas

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 β 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