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.

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.

What's in the BAGs? Intrinsic disorder angle of the multifunctionality of the members of a family of chaperone regulators.

In humans, the family of Bcl-2 associated athanogene (BAG) proteins includes six members characterized by exceptional multifunctionality and engagement in the pathogenesis of various diseases. All of them are capable of interacting with a multitude of often unrelated binding partners. Such binding promiscuity and related functional and pathological multifacetedness cannot be explained or understood within the frames of the classical "one protein-one structure-one function" model, which also fails to explain the presence of multiple isoforms generated for BAG proteins by alternative splicing or alternative translation initiation and their extensive posttranslational modifications. However, all these mysteries can be solved by taking into account the intrinsic disorder phenomenon. In fact, high binding promiscuity and potential to participate in a broad spectrum of interactions with multiple binding partners, as well as a capability to be multifunctional and multipathogenic, are some of the characteristic features of intrinsically disordered proteins and intrinsically disordered protein regions. Such functional proteins or protein regions lacking unique tertiary structures constitute a cornerstone of the protein structure-function continuum concept. The aim of this paper is to provide an overview of the functional roles of human BAG proteins from the perspective of protein intrinsic disorder which will provide a means for understanding their binding promiscuity, multifunctionality, and relation to the pathogenesis of various diseases.

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.

Modulation of BAG3 expression in human normal urothelial cells by Diuron.

Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] is a substituted urea herbicide, carcinogenic for the rat urinary bladder. It has been hypothesized that Diuron cytotoxicity, resulting in regenerative proliferation, leads to urothelial hyperplasia and, finally, to bladder tumors, but molecular mechanisms of carcinogenesis have not still fully investigated. Here, we report the results of a study aimed at verifying the involvement of BAG3, an intracellular protein expressed in several tumors, in the Diuron-induced carcinogenesis. For this purpose, we analyzed the effect of Diuron on human primary urothelial cells and on human dermal fibroblasts. We found that while high concentrations of Diuron have a cytotoxic effect in human primary urothelial cells, in the same cells, noncytotoxic concentrations of the herbicide induce BAG3 expression. These findings show that BAG3 is a molecular target of Diuron and unravel the possible involvement of BAG3 protein in bladder carcinogenesis induced by the herbicide. In addition, these results suggest that BAG3 might be a potential early biomarker of damage induced by chronic exposure to Diuron.

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.

Role of BAG3 in cancer progression: A therapeutic opportunity.

BAG3 is a multifunctional protein that can bind to heat shock proteins (Hsp) 70 through its BAG domain and to other partners through its WW domain, proline-rich (PXXP) repeat and IPV (Ile-Pro-Val) motifs. Its intracellular expression can be induced by stressful stimuli, while is constitutive in skeletal muscle, cardiac myocytes and several tumour types. BAG3 can modulate the levels, localisation or activity of its partner proteins, thereby regulating major cell pathways and functions, including apoptosis, autophagy, mechanotransduction, cytoskeleton organisation, motility. A secreted form of BAG3 has been identified in studies on pancreatic ductal adenocarcinoma (PDAC). Secreted BAG3 can bind to a specific receptor, IFITM2, expressed on macrophages, and induce the release of factors that sustain tumour growth and the metastatic process. BAG3 neutralisation therefore appears to constitute a novel potential strategy in the therapy of PDAC and, possibly, other tumours.

IKK{gamma} protein is a target of BAG3 regulatory activity in human tumor growth.

BAG3, a member of the BAG family of heat shock protein (HSP) 70 cochaperones, is expressed in response to stressful stimuli in a number of normal cell types and constitutively in a variety of tumors, including pancreas carcinomas, lymphocytic and myeloblastic leukemias, and thyroid carcinomas. Down-regulation of BAG3 results in cell death, but the underlying molecular mechanisms are still elusive. Here, we investigated the molecular mechanism of BAG3-dependent survival in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show that bag3 overexpression in tumors promotes survival through the NF-kappaB pathway. Indeed, we demonstrate that BAG3 alters the interaction between HSP70 and IKKgamma, increasing availability of IKKgamma and protecting it from proteasome-dependent degradation; this, in turn, results in increased NF-kappaB activity and survival. These results identify bag3 as a potential target for anticancer therapies in those tumors in which this gene is constitutively expressed. As a proof of principle, we show that treatment of a mouse xenograft tumor model with bag3siRNA-adenovirus that down-regulates bag3 results in reduced tumor growth and increased animal survival.

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.

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.

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.

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.

Copper binds the carboxy-terminus of trefoil protein 1 (TFF1), favoring its homodimerization and motogenic activity.

Trefoil protein 1 (TFF1) is a small secreted protein belonging to the trefoil factor family of proteins, that are present mainly in the gastrointestinal (GI) tract and play pivotal roles as motogenic factors in epithelial restitution, cell motility, and other incompletely characterized biological processes. We previously reported the up-regulation of TFF1 gene in copper deficient rats and the unexpected property of the peptide to selectively bind copper. Following the previous evidence, here we report the characterization of the copper binding site by fluorescence quenching spectroscopy and mass spectrometric analyses. We demonstrate that Cys58 and at least three Glu surrounding residues surrounding it, are essential to efficiently bind copper. Moreover, copper binding promotes the TFF1 homodimerization, thus increasing its motogenic activity in in vitro wound healing assays. Copper levels could then modulate the TFF1 functions in the GI tract, as well as its postulated role in cancer progression and invasion.

Prognostic significance of atypical mitotic figures in smooth muscle tumors of uncertain malignant potential (STUMP) of the uterus and uterine adnexa.

We aimed to assess whether the presence of atypical mitotic figures (AMF) in smooth muscle tumors of uncertain malignant potential (STUMP) of the uterus and uterine adnexa is associated with increased risk of recurrence, and the association of AMF with the Stanford criteria, that is, significant cytologic atypia, mitotic index ≥ 10/10HPF, and coagulative tumor cell necrosis (CTCN). A systematic review was performed to identify all studies reporting the presence of AMF and oncologic outcomes in STUMP series. Fisher's exact test was used to assess the association of AMF with the three Stanford parameters. Kaplan-Meier and Cox regression survival analyses with hazard ratio (HR) calculation were performed to assess the association between AMF and STUMP recurrence. A p-value < 0.05 was considered significant. Five studies with 80 STUMPs were included, out of which 23.8% had AMF. AMF were significantly associated with the presence of significant atypia (p = 0.023), but not with the presence of a mitotic index ≥ 10/10HPF (p = 0.769), CTCN (p = 1), or more than one Stanford parameter (p = 0.171). AMF was not significantly associated with the risk of STUMP recurrence at both univariate (HR = 0.366; p = 0.188) and multivariate analyses (HR = 0.528; p = 0.463). In STUMP of the uterus and uterine adnexa, AMF are more common in the case of significant cytologic atypia, but do not seem to increase the risk of recurrence. Further studies are necessary in this regard.

EV20­sss­vc/MMAF, an HER­3 targeting antibody­drug conjugate displays antitumor activity in liver cancer.

Liver cancer (LC) is an aggressive disease with a markedly poor prognosis. Therapeutic options are limited, and, until recently the only FDA­approved agent for first­line treatment of patients with LC was the multi­kinase inhibitor sorafenib, which exhibits limited activity and an increased overall survival (OS) of only 3 months over placebo. Therefore, the development of alternative therapeutic molecules for the treatment of LC is an urgent medical need. Antibody­drug conjugates (ADCs) are an emerging class of novel anticancer agents, which have been developed recently for the treatment of malignant conditions, including LC, and are being studied in preclinical and clinical settings. Our group has recently generated an ADC [EV20/monomethyl auristatin F (MMAF)] by coupling the HER3 targeting antibody (EV20) to MMAF via a non­cleavable maleimidocaproyl linker. This ADC was revealed to possess potent therapeutic activity in melanoma and breast carcinoma. In the present study, using western blot and flow cytometric analysis, it was reported that HER­3 receptor was highly expressed in LC and activated by its ligand NRG­1ß in a panel of LC cell lines, thus indicating that this receptor may serve as a suitable target for ADC therapy. A novel ADC [EV20­sss­valine­citrulline (vc)/MMAF] was generated, in which the cytotoxic payload MMAF was site­specifically coupled to an engineered variant of EV20 via a vc cleavable linker. Cytotoxicity assays were performed to investigate in vitro antitumor activity of EV20­sss­vc/MMAF and it was compared to EV20/MMAF, which revealed only modest activity in LC.EV20­sss­vc/MMAF exhibited a significant cell killing activity in several LC cell lines. Additionally, in vivo xenograft experiments revealed that EV20­sss­vc/MMAF inhibited growth of LC tumors. The present data indicated that EV20­sss­vc/MMAF is a worthy candidate for the treatment of HER­3 positive LC.

The multiple activities of BAG3 protein: Mechanisms.

BACKGROUND: BAG3 was identified as a co-chaperone of the heat shock protein (Hsp) 70, which helps, through the binding to the ATPase domain, the ADP release from the chaperone and the nucleotide cycling. By interacting with Hsp70, BAG3 modulates the activities of this chaperone, including the delivery of client proteins to proteasome. BAG3 can also carry out Hsp70- independent functions, through its interactions with other proteins involved in apoptosis, cytoskeleton dynamics and other pathways. SCOPE OF REVIEW: Here we provide a summary of the main mechanisms which encompass BAG3 as an intracellular factor involved in different pathways which regulate and modulate the physiological cell response. Furthermore, it has been shown that BAG3 can be secreted by some cell types and is able to activate the monocytes through the binding on a membrane cell receptor, indicating that the protein can act like an alarmin with different functions inside and outside the cell. MAJOR CONCLUSIONS: Whereas intracellularly BAG3 sustains the levels of anti-apoptotic factors and other molecules, participates in protein quality control, drives the cytoskeleton dynamics, exerts structural and functional roles in myocytes, the discovery of a secreted BAG3 opened a new field of investigation in tumor development and progression, revealing its role in a new signaling pathway, mediated by the BAG3/BAG3R axis, which also includes monocytes and other stromal cells. GENERAL SIGNIFICANCE: BAG3 is a multifunctional protein that is involved cell stress response through its participation in several regulatory pathways which control cell homeostatic response in physiological and pathological conditions.

BAG3 in Tumor Resistance to Therapy.

BAG3 is highly expressed across cancer types and its intracellular activity is critical for cancer cell survival. However, recent findings suggest that BAG3 can also modulate the tumor microenvironment to promote cancer progression and resistance to therapies, suggesting new ways to target this protein in cancer therapy.