EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

EBV has been reported to impair monocyte in vitro differentiation into dendritic cells (DCs) and reduce cell survival. In this study, we added another layer of knowledge to this topic and showed that these effects correlated with macroautophagy/autophagy, ROS and mitochondrial biogenesis reduction. Of note, autophagy and ROS, although strongly interconnected, have been separately reported to be induced by CSF2/GM-CSF (colony stimulating factor 2) and required for CSF2-IL4-driven monocyte in vitro differentiation into DCs. We show that EBV infects monocytes and initiates a feedback loop in which, by inhibiting autophagy, reduces ROS and through ROS reduction negatively influences autophagy. Mechanistically, autophagy reduction correlated with the downregulation of RAB7 and ATG5 expression and STAT3 activation, leading to the accumulation of SQSTM1/p62. The latter activated the SQSTM1-KEAP1- NFE2L2 axis and upregulated the anti-oxidant response, reducing ROS and further inhibiting autophagy. ROS decrease correlated also with the reduction of mitochondria, the main source of intracellular ROS, achieved by the downregulation of NRF1 and TFAM, mitochondrial biogenesis transcription factors. Interestingly, mitochondria supply membranes and ATP required for autophagy execution, thus their reduction may further reduce autophagy in EBV-infected monocytes. In conclusion, this study shows for the first time that the interconnected reduction of autophagy, intracellular ROS and mitochondria mediated by EBV switches monocyte differentiation into apoptosis, giving new insights into the mechanisms through which this virus reduces immune surveillance. Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CAT: catalase; CSF2: colony stimulating factor 2; CT: control; CYCS (cytochrome C: somatic); DCs: dendritic cells; EBV: Epstein-Barr virus; GSR: glutathione-disulfide reductase; KEAP1: kelch like ECH associated protein 1; IL4: interleukin 4; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MET: metformin; NAC: N-acetylcysteine; NFE2L2/NRF2 nuclear factor: erythroid 2 like 2; NRF1 (nuclear respiratory factor 1); clPARP1: cleaved poly(ADP-ribose) polymerase; Rapa: Rapamycin; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TFAM: (transcription factor A: mitochondrial); TUBA1A: tubulin alpha 1a.

KSHV reduces autophagy in THP-1 cells and in differentiating monocytes by decreasing CAST/calpastatin and ATG5 expression.

We have previously shown that Kaposi sarcoma-associated herpesvirus (KSHV) impairs monocyte differentiation into dendritic cells (DCs). Macroautophagy/autophagy has been reported to be essential in such a differentiating process. Here we extended these studies and found that the impairment of DC formation by KSHV occurs through autophagy inhibition. KSHV indeed reduces CAST (calpastatin) and consequently decreases ATG5 expression in both THP-1 monocytoid cells and primary monocytes. We unveiled a new mechanism put in place by KSHV to escape from immune control. The discovery of viral immune suppressive strategies that contribute to the onset and progression of viral-associated malignancies is of fundamental importance for finding new therapeutic approaches against them.

HCV derived from sera of HCV-infected patients induces pro-fibrotic effects in human primary fibroblasts by activating GLI2.

Hepatitis C virus (HCV) infection is a leading cause of liver fibrosis, especially in developing countries. The process is characterized by the excess accumulation of ECM that may lead, over time, to hepatic cirrhosis, liver failure and also to hepatocarcinoma. The direct role of HCV in promoting fibroblasts trans-differentiation into myofibroblasts, the major fibrogenic cells, has not been fully clarified. In this study, we found that HCV derived from HCV-infected patients infected and directly induced the trans-differentiation of human primary fibroblasts into myofibroblasts, promoting fibrogenesis. This effect correlated with the activation of GLI2, one of the targets of Hedgehog signaling pathway previously reported to be involved in myofibroblast generation. Moreover, GLI2 activation by HCV correlated with a reduction of autophagy in fibroblasts, that may further promoted fibrosis. GLI2 inhibition by Gant 61 counteracted the pro-fibrotic effects and autophagy inhibition mediated by HCV, suggesting that targeting HH/GLI2 pathway might represent a promising strategy to reduce the HCV-induced fibrosis.

RNF11 is a GGA protein cargo and acts as a molecular adaptor for GGA3 ubiquitination mediated by Itch.

Ring finger protein 11 (RNF11) is a RING (really interesting new gene)-H2 E3 ligase that is overexpressed in several human tumor tissues. The mature protein, which is anchored to membranes via a double acylation, localizes to early endosome and recycling compartments. Apart from its subcellular localization, additional lines of evidence implicate RNF11 in the mechanisms underlying vesicle traffic. Here we identify two acidic-cluster dileucine (Ac-LL) motifs, which are recognized by the VHS domains of Golgi-localized, gamma adaptin era-containing, ADP-ribosylation factor-binding protein (GGA) adaptors, as the molecular determinants governing RNF11 sorting at the trans-Golgi network and its internalization from the plasma membrane. We also show that RNF11 recruits itch to drive the ubiquitination of GGA3. This function is experimentally detectable only in cells overexpressing an RNF11 variant that is inactivated in the RING domain, indicating that RNF11 recruits GGA3 and controls its ubiquitination by regulating itch activity. Accordingly, our data demonstrate the involvement of itch in regulating GGA3 stability. Indeed, we observe that the endogenous levels of GGA3 are increased in cells knocked down for itch and endogenous GGA3 is hyperubiquitinated in an itch-dependent manner in a cell line expressing catalytically inactive RNF11. Our data are consistent with a model whereby the RING E3 ligase RNF11 is a novel GGA cargo actively participating in regulating the ubiquitination of the GGA protein family. The results that we are presenting put RNF11 at the center of a finally regulated system where it acts both as an adaptor and a modulator of itch-mediated control of ubiquitination events underlying membrane traffic.

Exosomal HIF1α supports invasive potential of nasopharyngeal carcinoma-associated LMP1-positive exosomes.

It has emerged recently that exosomes are potential carriers of pro-tumorigenic factors that participate in oncogenesis. However, whether oncogenic transcription factors are transduced by exosomes is unknown. Hypoxia-inducible factor-1α (HIF1α) transcriptionally regulates numerous key aspects of tumor development and progression by promoting a more aggressive tumor phenotype, characterized by increased proliferation and invasiveness coupled with neoangiogenesis. It has been shown that the principal oncoprotein of Epstein-Barr virus (EBV), latent membrane protein 1 (LMP1), drives oncogenic processes and tumor progression of the highly invasive EBV malignancy, nasopharyngeal carcinoma (NPC). We now demonstrate that endogenous HIF1α is detectable in exosomes and that LMP1 significantly increases levels of HIF1α in exosomes. HIF1 recovered from exosomes retains DNA-binding activity and is transcriptionally active in recipient cells after exosome uptake. We also show that treatment of EBV-negative cells with LMP1-exosomes increases migration and invasiveness of NP cell lines in functional assays, which correlates with the phenotype associated with epithelial-mesenchymal transition (EMT). In addition, we provide evidence that HIF1α itself participates in exosome-mediated pro-metastatic effects in recipient cells, as exosome-mediated delivery of active and inactive forms of HIF1α results in reciprocal changes in the expression of E- and N-cadherins associated with EMT. Further, immunohistochemical analysis of NPC tumor tissues revealed direct correlation between protein levels of LMP1 and of the endosome/exosome marker tetraspanin, CD63, which suggests an increase in exosome formation in this EBV-positive malignancy. We hypothesize that exosome-mediated transfer of functional pro-metastatic factors by LMP1-positive NPC cells to surrounding tumor cells promotes cancer progression.

Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells.

In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.

Evaluation of E6 and E7 mRNA expression in HPV DNA positive breast cancer.

Several studies have suggested a possible role for HPV in the pathogenesis of the breast cancer. We investigated the presence of the HPV DNA in breast cancers and non malignant disease breast tissues by the use of a standard HPV detection method (INNO-Lipa HPV), in order to detect HPV DNA in metastatic nodes, to investigate a possible cervical HPV co-infection, and to evaluate the E6/E7 mRNA expression in HPV DNA positive breast cancer tissues. The rate of HPV infection was significantly higher in the cancer group than in controls (9/31 vs. 0/12, p = 0.04). One out of eight metastatic axillary nodes was positive for HPV infection; 2/3 of the positive HPV breast cancer patients were co-infected at the cervical site. The role of the virus in breast oncogenesis is still unclear, since our analysis failed in demonstrating the expression of viral E6 and E7 in positive HPV positive breast tumor tissues.

Ehlers-Danlos syndrome: case report and an electron microscopy study.

Ehlers-Danlos syndrome (EDS) type III is a inherited connective tissue disorders characterized by extensibility of the skin, hypermobility of the joints, chronic pain, tissue fragility, easy bruising, and delayed wound healing with result of atrophic scars. The patients report commonly a history of recurrent dislocations of the shoulders and knees after low-impact trauma, chronic joint pain, and early osteoarthritis, which lead to diagnosis. The pathogenesis of this condition is unknown, and the diagnosis is generally made in adult age, based only on clinical criteria. In this report, we describe a case of a 50-year-old woman with a 30-year history of recurrent dislocations and atrophic scars. We performed diagnosis of EDS type III after a complete clinical and instrumental evaluation, comprising of histological and electron microscopic studies, that highlighted collagen abnormalities.

HPV16 E5 affects the KGFR/FGFR2b-mediated epithelial growth through alteration of the receptor expression, signaling and endocytic traffic.

The E5 oncoprotein of the human papillomavirus type 16 (HPV16 E5) cooperates in cervical carcinogenesis and in epithelial transformation deregulating cell growth, survival and differentiation through the modulation of growth factor receptors. Among the epithelial receptor tyrosine kinases, the keratinocyte growth factor receptor/fibroblast growth factor receptor 2b (KGFR/FGFR2b) is a major paracrine mediator of epithelial homeostasis and appears to have an unique and unusual role in epithelial tissues, exerting a tumor-suppressive function in vitro and in vivo. With the aim to better elucidate the molecular events involved in the pathological activity of 16E5, we investigated if the viral protein would be able to affect the KGFR expression, signaling and turnover by interference with its degradative and recycling endocytic pathways. Quantitative reverse transcriptase-PCR and biochemical approaches on human keratinocytes transfected with 16E5-HA showed that E5 protein is able to induce KGFR down-modulation at both transcript and protein levels. Immunofluorescence microscopy in double-transfected cells expressing both E5 and KGFR revealed that the viral protein alters the receptor endocytic trafficking and triggers its endosomal sorting to the indirect juxtanuclear recycling pathway. The shift from lysosomal degradation to recycling at the plasma membrane correlates with a reduced phosphorylation of the fibroblast growth factor receptor substrate-2α tyrosine 196, the major docking site for Grb2-Cbl complexes responsible for receptor ubiquitination and degradation. 5'-Bromo-deoxyuridine incorporation assay demonstrated that expression of 16E5 induces a decrease in the growth response to the receptor ligands as a consequence of KGFR down-modulation, suggesting that 16E5 might have a role on HPV infection in perturbing the KGFR-mediated physiological behavior of confluent keratinocytes committed to differentiation.

Prognostic implication of high risk human papillomavirus E6 and E7 mRNA in patients with intraepithelial lesions of the cervix in relationship to age.

Since the introduction of the cytological screening programs, a significant reduction in the incidence of cervical cancer has been achieved. Almost all of these cancers are related to high-risk (HR) Human Papillomavirus (HPV) cervical infections. However, the natural history of HPV infection seems to be different in younger patients, resulting in a higher rate of regression. There is, therefore, the need to identify HPV-related biomarkers in order to enhance the effectiveness of screening of high-risk cytological lesions, in particular in women over 35 years of age. This study aims to evaluate the prognostic value of the HR HPV E6 and E7 mRNA expression in women with intraepithelial lesions of the cervix, older or younger than 35 years of age. One hundred and eighty-four HR HPV DNA positive patients with a low squamous intraepithelial lesion (LSIL) were tested for mRNA expressions, included in an observational study, and evaluated at follow-up with standard cytology up to 24 months from the mRNA test. The frequency of HSIL/LSIL cytology in the older cohort of mRNA positive patients was significantly higher compared to mRNA-negative patients, both at 1 and 2 years of follow-up (Chi-square: p 0.007 and p 0.009), but this difference was not found in the younger cohort. According to our results, the E6/E7 mRNA test could be a biomarker for viral activity, useful in identifying patients at higher risk of abnormal cytology, and in implementing the management of HR HPV DNA-positive women over 35 years of age.

Anetoderma: evidence of the relationship with autoimmune disease and a possible role of macrophages in the etiopathogenesis.

Anetoderma is a benign condition characterized by round or oval macular lesions with focal loss of dermal elastic tissue resulting in localized areas of flaccid or herniated saclike skin. Often, the anetoderma is associated with immuno-mediated pathogenetic mechanism. In this article, we describe the association between anetoderma and autoimmune diseases, by underlining the role and the action of macrophages as a possible etiopathogenesis.

Multiple modification and protein interaction signals drive the Ring finger protein 11 (RNF11) E3 ligase to the endosomal compartment.

Ring finger protein 11 (RNF11) is a small RING E3-ligase overexpressed in numerous human prostate, colon and invasive breast cancers. Although functional studies have implicated RNF11 in a variety of biological processes, including signal transduction and apoptosis, the molecular mechanisms underlying its function are still poorly understood. In this study we show that RNF11 is a membrane-associated E3 ligase co-localizing with markers of both the early and the recycling endosomes. Several modification and protein interaction signals in the RNF11 sequence are shown to affect its compartmentalization. Membrane binding requires two acylation motifs driving the myristoylation of Gly2 and the S-palmitoylation of Cys4. Accordingly, genetic removal of the myristoylating signal results in diffuse staining, whereas an RNF11 protein mutated in the palmitoylation signal is retained in compartments of the early secretory pathway. However, amino-terminal fusion to green fluorescent protein of a 10-residue peptide containing both acylation signals re-localizes the chimera to the plasma membrane, but it is not sufficient to direct it to the recycling compartment suggesting that additional signals contribute to the correct localization. In addition, we show that membrane anchoring through acylation is necessary for RNF11 to be post-translationally modified by the addition of several ubiquitin moieties and that loss of acylation severely impairs the in vivo ubiquitination mediated by the HECT E3-ligases Itch and Nedd4. Finally, in cells transfected with RNF11 we observe a correlation between high RNF11 expression, as in tumor cells, and a swelling of the endosomal compartment suggesting a possible role of the dysregulation of the endosome compartment in tumorigenesis.

Role of fibroblast-derived growth factors in regulating hyperpigmentation of solar lentigo.

BACKGROUND: Cutaneous pigmentation is regulated by a complex melanogenic network in which both keratinocytes and fibroblasts synthesize growth factors and cytokines. Solar lentigo (SL) is characterized by hyperpigmented lesions occurring on photodamaged skin areas. Despite the association of SL to ultraviolet (UV) exposure, the mechanisms underlying the development of these spots are not completely defined. OBJECTIVES: To analyse the involvement of the fibroblast-derived growth factors, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and stem cell factor (SCF) in SL hyperpigmentation; to evaluate whether the photoageing process occurring in fibroblasts could be responsible for the altered expression of these cytokines; and to investigate a new possible role of KGF in regulating pigmentation through the specific induction of melanogenic cytokines by keratinocytes. METHODS: We performed immunohistochemical analysis of HGF, KGF and SCF on SL biopsies. We analysed the mRNA expression of these cytokines using an in vitro model of photoageing induced on fibroblasts. Finally, we evaluated the effects of KGF on the expression of melanogenic cytokines at the mRNA and protein levels on keratinocytes. RESULTS: We found positive staining for HGF, KGF and SCF in the upper dermis of SL lesions and a significant induction of the three cytokines in photoaged fibroblasts. We also demonstrated the contribution of KGF to pigmentation, showing its ability specifically to modulate the expression of SCF in keratinocytes. CONCLUSIONS: Fibroblasts may be persistently activated by UV exposure to release melanogenic growth factors; this inducible cytokine network acts both directly and indirectly through keratinocytes and may contribute to the hyperpigmentation of SL.

Keratinocyte growth factor receptor (KGF-R) in cholesteatoma tissue.

CONCLUSION: Distribution of the receptor for epidermal growth factor (EGF-R) and of the receptor for the keratinocyte growth factor (KGF-R) in cholesteatoma was found to differ in analogy with other epithelial tissues and accordingly to epidermal differentiation and intensity of paracrine stimulation. Moreover, both EGF-R and KGF-R expression was increased, suggesting a fair correlation with aggressiveness and recurrence rate of this pathology. OBJECTIVES: To obtain information on the biological behaviour of cholesteatoma by assessing the expression and localization of EGF-R and KGF-R and correlating their tissue distribution with that of cytokeratins as a marker of differentiation. MATERIALS AND METHODS: Cholesteatoma tissue was taken during tympanoplasty surgery and processed for indirect immunofluorescence. Murine monoclonal antibodies were tested for the different growth factor receptors and pancytokeratins analysed. Fluorescence intensity signal was measured on randomly captured digital images, using FISH 2000/HI software, with a pseudocolours generation module. RESULTS: EGF-R was mostly expressed at the level of keratinocytes of the basal layer, while KGF-R signal was mainly distributed on the spinous and granular suprabasal layers that were also highly positive for cytokeratins. Significant correlation between the immunofluorescence signals was found for KGF-R and cytokeratins only, demonstrating that KGF-R expression is increased in more differentiated areas of the cholesteatoma tissue, while EGF-R is associated with proliferative and migratory portions of the lesion.

Identification and characterization of the product encoded by ORF69 of Kaposi's sarcoma-associated herpesvirus.

We report the identification and characterization of p33, the product of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.

Involvement of hTERT in apoptosis induced by interference with Bcl-2 expression and function.

Here, we investigated the role of telomerase on Bcl-2-dependent apoptosis. To this end, the 4625 Bcl-2/Bcl-xL bispecific antisense oligonucleotide and the HA14-1 Bcl-2 inhibitor were used. We found that apoptosis induced by 4625 oligonucleotide was associated with decreased Bcl-2 protein expression and telomerase activity, while HA14-1 triggered apoptosis without affecting both Bcl-2 and telomerase levels. Interestingly, HA14-1 treatment resulted in a profound change from predominantly nuclear to a predominantly cytoplasmic localization of hTERT. Downregulation of endogenous hTERT protein by RNA interference markedly increased apoptosis induced by both 4625 and HA14-1, while overexpression of wild-type hTERT blocked Bcl-2-dependent apoptosis in a p53-independent manner. Catalytically and biologically inactive hTERT mutants showed a similar behavior as the wild-type form, indicating that hTERT inhibited the 4625 and HA14-1-induced apoptosis regardless of telomerase activity and its ability to lengthening telomeres. Finally, hTERT overexpression abrogated 4625 and HA14-1-induced mitochondrial dysfunction and nuclear translocation of hTERT. In conclusion, our results demonstrate that hTERT is involved in mitochondrial apoptosis induced by targeted inhibition of Bcl-2.