Overexpression of BAG3 (Bcl2-associated athanogene 3) in serum and skin of patients with systemic sclerosis.

OBJECTIVES: BAG3 (Bcl2-associated athanogene3) is able to induce the transformation of cancer-associated fibroblasts to alpha smooth muscle actin (a-SMA) positive (+) myofibroblasts. In systemic sclerosis (SSc), a-SMA+ myofibroblasts also play an important role in the progression of fibrosis in the skin and involved internal organs. The aim of the study was to investigate whether BAG3 is overexpressed in SSc and may be a biomarker of fibrogenesis. METHODS: BAG3 serum levels were measured in 106 patients with SSc, 47 with the limited (lc) and 59 the diffuse (dc) SSc, and in age- and sex-matched healthy controls (HC). BAG3 levels were then compared according to their clinical subset, nailfold video-capillaroscopic (NVC) patterns, interstitial lung disease (ILD, and correlated with modified Rodnan skin score (mRSS) and global disease activity. BAG3 expression was also investigated in skin biopsies of 8 dcSSc patients. RESULTS: BAG3 serum levels were significantly higher in dcSSc (143.3 pg/mL, 95%CI 78-208.5) than in HC (0.68 pg/mL, 95%CI 0.13-1.23), and were significantly higher in patients with late NVC pattern and ILD but did not correlate with disease activity and mRSS. Of note, BAG3 was strongly expressed in the skin biopsies of dcSSc patients. CONCLUSIONS: BAG3 is overexpressed in dcSSc patients and may contribute to skin and organ fibrosis by prompting the transition of fibroblasts into myofibroblasts and increasing their survival. Thus, BAG3 may play an important role in SSc fibrotic pathogenesis and be a potential biomarker of fibrosis. Further research on its role as a therapeutic target is warranted.

Pancreatic Cancer-Secreted Proteins: Targeting Their Functions in Tumor Microenvironment.

Pancreatic Ductal Adenocarcinoma (PDAC) is a ravaging disease with a poor prognosis, requiring a more detailed understanding of its biology to foster the development of effective therapies. The unsatisfactory results of treatments targeting cell proliferation and its related mechanisms suggest a shift in focus towards the inflammatory tumor microenvironment (TME). Here, we discuss the role of cancer-secreted proteins in the complex TME tumor-stroma crosstalk, shedding lights on druggable molecular targets for the development of innovative, safer and more efficient therapeutic strategies.

The Continuous Adaptive Challenge Played by Arboviruses: An In Silico Approach to Identify a Possible Interplay between Conserved Viral RNA Sequences and Host RNA Binding Proteins (RBPs).

Climate change and globalization have raised the risk of vector-borne disease (VBD) introduction and spread in various European nations in recent years. In Italy, viruses carried by tropical vectors have been shown to cause viral encephalitis, one of the symptoms of arboviruses, a spectrum of viral disorders spread by arthropods such as mosquitoes and ticks. Arboviruses are currently causing alarm and attention, and the World Health Organization (WHO) has released recommendations to adopt essential measures, particularly during the hot season, to restrict the spreading of the infectious agents among breeding stocks. In this scenario, rapid analysis systems are required, because they can quickly provide information on potential virus-host interactions, the evolution of the infection, and the onset of disabling clinical symptoms, or serious illnesses. Such systems include bioinformatics approaches integrated with molecular evaluation. Viruses have co-evolved different strategies to transcribe their own genetic material, by changing the host's transcriptional machinery, even in short periods of time. The introduction of genetic alterations, particularly in RNA viruses, results in a continuous adaptive fight against the host's immune system. We propose an in silico pipeline method for performing a comprehensive motif analysis (including motif discovery) on entire genome sequences to uncover viral sequences that may interact with host RNA binding proteins (RBPs) by interrogating the database of known RNA binding proteins, which play important roles in RNA metabolism and biological processes. Indeed, viral RNA sequences, able to bind host RBPs, may compete with cellular RNAs, altering important metabolic processes. Our findings suggest that the proposed in silico approach could be a useful and promising tool to investigate the complex and multiform clinical manifestations of viral encephalitis, and possibly identify altered metabolic pathways as targets of pharmacological treatments and innovative therapeutic protocols.

Multianalyte Serum Biomarker Panel for Early Detection of Pancreatic Adenocarcinoma.

PURPOSE: We determined whether a large, multianalyte panel of circulating biomarkers can improve detection of early-stage pancreatic ductal adenocarcinoma (PDAC). MATERIALS AND METHODS: We defined a biologically relevant subspace of blood analytes on the basis of previous identification in premalignant lesions or early-stage PDAC and evaluated each in pilot studies. The 31 analytes that met minimum diagnostic accuracy were measured in serum of 837 subjects (461 healthy, 194 benign pancreatic disease, and 182 early-stage PDAC). We used machine learning to develop classification algorithms using the relationship between subjects on the basis of their changes across the predictors. Model performance was subsequently evaluated in an independent validation data set from 186 additional subjects. RESULTS: A classification model was trained on 669 subjects (358 healthy, 159 benign, and 152 early-stage PDAC). Model evaluation on a hold-out test set of 168 subjects (103 healthy, 35 benign, and 30 early-stage PDAC) yielded an area under the receiver operating characteristic curve (AUC) of 0.920 for classification of PDAC from non-PDAC (benign and healthy controls) and an AUC of 0.944 for PDAC versus healthy controls. The algorithm was then validated in 146 subsequent cases presenting with pancreatic disease (73 benign pancreatic disease and 73 early- and late-stage PDAC cases) and 40 healthy control subjects. The validation set yielded an AUC of 0.919 for classification of PDAC from non-PDAC and an AUC of 0.925 for PDAC versus healthy controls. CONCLUSION: Individually weak serum biomarkers can be combined into a strong classification algorithm to develop a blood test to identify patients who may benefit from further testing.

Spike-mediated viral membrane fusion is inhibited by a specific anti-IFITM2 monoclonal antibody.

The early steps of viral infection involve protein complexes and structural lipid rearrangements which characterize the peculiar strategies of each virus to invade permissive host cells. Members of the human immune-related interferon-induced transmembrane (IFITM) protein family have been described as inhibitors of the entry of a broad range of viruses into the host cells. Recently, it has been shown that SARS-CoV-2 is able to hijack IFITM2 for efficient infection. Here, we report the characterization of a newly generated specific anti-IFITM2 mAb able to impair Spike-mediated internalization of SARS-CoV-2 in host cells and, consequently, to reduce the SARS-CoV-2 cytopathic effects and syncytia formation. Furthermore, the anti-IFITM2 mAb reduced HSVs- and RSV-dependent cytopathic effects, suggesting that the IFITM2-mediated mechanism of host cell invasion might be shared with other viruses besides SARS-CoV-2. These results show the specific role of IFITM2 in mediating viral entry into the host cell and its candidacy as a cell target for antiviral therapeutic strategies.

What Have We Learned from Patients Who Have Arboleda-Tham Syndrome Due to a De Novo KAT6A Pathogenic Variant with Impaired Histone Acetyltransferase Function? A Precise Clinical Description May Be Critical for Genetic Testing Approach and Final Diagnosis.

Pathogenic variants in genes are involved in histone acetylation and deacetylation resulting in congenital anomalies, with most patients displaying a neurodevelopmental disorder and dysmorphism. Arboleda-Tham syndrome caused by pathogenic variants in KAT6A (Lysine Acetyltransferase 6A; OMIM 601408) has been recently described as a new neurodevelopmental disorder. Herein, we describe a patient characterized by complex phenotype subsequently diagnosed using the clinical exome sequencing (CES) with Arboleda-Tham syndrome (ARTHS; OMIM 616268). The analysis revealed the presence of de novo pathogenic variant in KAT6A gene, a nucleotide c.3385C>T substitution that introduces a premature termination codon (p.Arg1129*). The need for straight multidisciplinary collaboration and accurate clinical description findings (bowel obstruction/megacolon/intestinal malrotation) was emphasized, together with the utility of CES in establishing an etiological basis in clinical and genetical heterogeneous conditions. Therefore, considering the phenotypic characteristics, the condition's rarity and the reviewed literature, we propose additional diagnostic criteria that could help in the development of future clinical diagnostic guidelines. This was possible thanks to objective examinations performed during the long follow-up period, which permitted scrupulous registration of phenotypic changes over time to further assess this rare disorder. Finally, given that different genetic syndromes are associated with distinct genomic DNA methylation patterns used for diagnostic testing and/or as biomarker of disease, a specific episignature for ARTHS has been identified.

Concerted BAG3 and SIRPα blockade impairs pancreatic tumor growth.

The BAG3- and SIRPα- mediated pathways trigger distinct cellular targets and signaling mechanisms in pancreatic cancer microenvironment. To explore their functional connection, we investigated the effects of their combined blockade on cancer growth in orthotopic allografts of pancreatic cancer mt4-2D cells in immunocompetent mice. The anti-BAG3 + anti-SIRPα mAbs treatment inhibited (p = 0.007) tumor growth by about the 70%; also the number of metastatic lesions was decreased, mostly by the effect of the anti-BAG3 mAb. Fibrosis and the expression of the CAF activation marker α-SMA were reduced by about the 30% in animals treated with anti-BAG3 mAb compared to untreated animals, and appeared unaffected by treatment with the anti-SIRPα mAb alone; however, the addition of anti-SIRPα to anti-BAG3 mAb in the combined treatment resulted in a > 60% (p < 0.0001) reduction of the fibrotic area and a 70% (p < 0.0001) inhibition of CAF α-SMA positivity. Dendritic cells (DCs) and CD8+ lymphocytes, hardly detectable in the tumors of untreated animals, were modestly increased by single treatments, while were much more clearly observable (p < 0.0001) in the tumors of the animals subjected to the combined treatment. The effects of BAG3 and SIRPα blockade do not simply reflect the sum of the effects of the single blockades, indicating that the two pathways are connected by regulatory interactions and suggesting, as a proof of principle, the potential therapeutic efficacy of a combined BAG3 and SIRPα blockade in pancreatic cancer.

BAG3 induces α-SMA expression in human fibroblasts and its over-expression correlates with poorer survival in fibrotic cancer patients.

Hypoxia and angiogenesis in solid tumors are often strictly linked to the development of fibrotic tissues, a detrimental event that compromises the antitumor immunity. As a consequence, tumor aggressiveness and poor patient prognosis relate to higher incidence of tissue fibrosis and stromal stiffness. The molecular pathways through which normal fibroblasts are converted in cancer-associated fibroblasts (CAFs) have a central role in the onset of fibrosis in tumor stroma, thus emerging as a strategic target of novel therapeutic approaches for cancer disease. Several studies addressed the role of BAG3 in sustaining growth and survival of cancer cell and also shed light on the different mechanisms in which the intracellular protein is involved. More recently, new pieces of evidence revealed a pivotal role of extracellular BAG3 in pro-tumor cell signaling in the tumor microenvironment, as well as its involvement in the development of fibrosis in tumor tissues. Here we report further data showing the presence of the BAG3 receptor (Interferon-induced transmembrane protein [IFITM]-2) on the plasma membrane of normal dermal fibroblasts and the activity of BAG3 as a factor able to induce the expression of α-smooth muscle actin and the phosphorylation of AKT and focal adhesion kinase, that sustain CAF functions in tumor microenvironment. Furthermore, in agreement with these findings, bag3 gene expression has been analyzed by high throughput RNA sequencing databases from patients-derived xenografts. A strong correlation between bag3 gene expression and patients' survival was found in several types of fibrotic tumors. The results obtained provide encouraging data that identify BAG3 as a promising therapeutic target to counteract fibrosis in tumors.

BAG3 induces fibroblasts to release key cytokines involved in pancreatic cell migration.

Pancreatic ductal adenoma carcinoma (PDAC) is considered one of the deadliest solid cancers as it is usually diagnosed in advanced stages and has a poor response to treatment. The enormous effort made in the last 2 decades in the oncology field has not led to significant progress in improving early diagnosis or therapy for PDAC. The stroma of PDAC plays an active role in tumour initiation and progression and includes immune cells and stromal cells. We previously reported that Bcl2-associated athanogene (BAG3) secreted by PDAC cells activates tumour-associated macrophages to promote tumour growth. The disruption of this tumour-stroma axis by the anti-BAG3 H2L4 therapeutic antibody is sufficient to delay tumour growth and limit metastatic spreading in different PDAC preclinical models. In the present study, we examined the role of BAG3 to activate human fibroblasts (HF) in releasing cytokines capable of supporting tumour progression. Treatment of fibroblasts with recombinant BAG3 induced important changes in the organisation of the cytoskeleton of these cells and stimulated the production of interleukin-6, monocyte chemoattractant protein-1/C-C motif chemokine ligand 2, and hepatocyte growth factor. Specifically, we observed that BAG3 triggered a depolymerisation of microtubules at the periphery of the cell while they were conserved in the perinuclear area. Conversely, the vimentin-based intermediate filaments increased and spread to the edges of the cells. Finally, the conditioned medium (CM) collected from BAG3-treated HF promoted the survival, proliferation, and migration of the PDAC cells. Blocking of the PDAC-fibroblast axis by the H2L4 therapeutic anti-BAG3 antibody, resulted in inhibition of cytokine release and, consequently, the inhibition of the migratory phenotype conferred by the CM to PDAC cells.

An emerging role for BAG3 in gynaecological malignancies.

BAG3, a member of the BAG family of co-chaperones, is a multidomain protein with a role in several cellular processes, including the control of apoptosis, autophagy and cytoskeletal dynamics. The expression of bag3 is negligible in most cells but can be induced by stress stimuli or malignant transformation. In some tumours, BAG3 has been reported to promote cell survival and resistance to therapy. The expression of BAG3 has been documented in ovarian, endometrial and cervical cancers, and studies have revealed biochemical and functional connections of BAG3 with proteins involved in the survival, invasion and resistance to therapy of these malignancies. BAG3 expression has also been shown to correlate with the grade of dysplasia in squamous intraepithelial lesions of the uterine cervix. Some aspects of BAG3 activity, such as its biochemical and functional interaction with the human papillomavirus proteins, could help in our understanding of the mechanisms of oncogenesis induced by the virus. This review aims to highlight the potential value of BAG3 studies in the field of gynaecological tumours.

A SARS-CoV-2 host infection model network based on genomic human Transcription Factors (TFs) depletion.

In December 2019 a new beta-coronavirus was isolated and characterized by sequencing samples from pneumonia patients in Wuhan, Hubei Province, China. Coronaviruses are positive-sense RNA viruses widely distributed among different animal species and humans in which they cause respiratory, enteric, liver and neurological symptomatology. Six species of coronavirus have been described (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1) that cause cold-like symptoms in immunocompetent or immunocompromised subjects and two strains of sometimes fatal zoonotic origin that cause severe acute respiratory syndrome (SARS-CoV and MERS-CoV). The SARS-CoV-2 strain is the emerging seventh member of the coronavirus family, which is actually determining a global emergency. In silico analysis is a promising approach for understanding biological events in complex diseases and due to serious worldwide emergency and serious threat to global health, it is extremely important to use bioinformatics methods able to study an emerging pathogen like SARS-CoV-2. Herein, we report on in silico comparative analysis between complete genome of SARS-CoV, MERS-CoV, HCoV-OC43 and SARS-CoV-2 strains, to identify the occurrence of specific conserved motifs on viral genomic sequences which should be able to bind and therefore induce a subtraction of host's Transcription Factors (TFs) which lead to a depletion, an effect comparable to haploinsufficiency (a genetic dominant condition in which a single copy of wild-type allele at a locus, in heterozygous combination with a variant allele, is insufficient to produce the correct quantity of transcript and, therefore, of protein, for a correct standard phenotypic expression). In this competitive scenario, virus versus host, the proposed in silico protocol identified the TFs same as the distribution of TFBSs (Transcription Factor Binding Sites) on analyzed viral strains, potentially able to influence genes and pathways with biological functions confirming that this approach could brings useful insights regarding SARS-CoV-2. According to our results obtained by this in silico approach it is possible to hypothesize that TF-binding motifs could be of help in the explanation of the complex and heterogeneous clinical presentation in SARS-CoV-2 and subsequently predict possible interactions regarding metabolic pathways, and drug or target relationships.

Different mechanisms underlie IL-6 release in chemosensitive and chemoresistant ovarian carcinoma cells.

Interleukin (IL)-6 has been detected in serum and ascites from patients affected by epithelial ovarian cancers, and also in some human ovarian cancer cell lines. To investigate the role of IL-6 in ovarian lesions, we first measured its levels in serum samples of 24 healthy donors and in 17 and 9 patients affected by ovarian carcinomas and ovarian benign cysts respectively. IL-6 levels were significantly higher than healthy donors in serum samples from ovarian cancer patients, but not in benign ovarian cysts. We then investigated the mechanism of IL-6 production in two cell lines obtained from the same patient with high grade serous ovarian carcinoma before (PEA1) and after (PEA2) development of cisplatinum resistance. The levels of intracellular IL-6, analysed by western blotting, did not relevantly differ in the two cell lines, and they did not change after the cell treatment with an AKT inhibitor. Although the interleukin was present in supernatants from both cell lines, its concentration in the supernatant of chemoresistant cells was significantly higher than chemosensitive cells. Interestingly, exposure to the AKT inhibitor resulted in a reduced IL-6 release in PEA1, but not in PEA2 cells. These results let infer different mechanisms of IL-6 release in chemoresistant and chemosensitive cell lines, and contribute new insights in ovarian cancer biology that suggest more in depth studies about the role of AKT in IL-6 release and in development of chemoresistance.

BAG3 expression correlates with the grade of dysplasia in squamous intraepithelial lesions of the uterine cervix.

INTRODUCTION: Bcl-2-associated athanogene 3 (BAG3) is a protein involved in apoptosis and stress response, which is overexpressed in invasive cervical cancer. However, nothing is known about BAG3 expression in precancerous lesions of the uterine cervix. We aimed to evaluate the expression of BAG3 in cervical intraepithelial neoplasia/squamous intraepithelial lesions (CIN/SIL). MATERIAL AND METHODS: Forty patients (16 CIN1/L-SIL, 11 CIN2/H-SIL and 13 CIN3/H-SIL) were assessed by immunohistochemistry for BAG3. The intensity of BAG3 expression was categorized as null, minimal, weak, moderate or strong. The association of BAG2 intensity of expression with the grade of dysplasia was assessed using Chi-square test (significant P value <0.05). RESULTS: In all normal controls, BAG3 expression was negative. In L-SIL specimens, BAG3 expression was confined to the basal third of the epithelium, with an intensity minimal in nine cases (56.3%), weak in six (37.5%) and strong in one (6.3%). In H-SIL specimens, BAG3 expression involved also the two upper thirds of the epithelium, with an intensity moderate in 13 cases (54.2%; 8 CIN2 and 5 CIN3) and strong in 11 cases (45.8%; 3 CIN2 and 8 CIN3). The distribution of BAG3 expression correlated perfectly with the grade of dysplasia (P = 0.0); a moderate/strong expression of BAG3 was significantly associated with H-SIL (P < 0.0001), with no significant difference between CIN2 and CIN3 (P = 0.1228). CONCLUSIONS: In CIN/SIL, both distribution and intensity of BAG3 expression correlate directly with the grade of dysplasia, supporting the involvement of BAG3 in all phases of cervical carcinogenesis and its possible diagnostic and prognostic role in cervical premalignant lesions.