Bicuspid Aortic Valve Disease With Early Onset Complications: Characteristics And Aortic Outcomes.

Bicuspid aortic valve (BAV) is the most common congenital heart malformation in adults but can also cause childhood-onset complications. In multicenter study, we found that adults who experience significant complications of BAV disease before age 30 are distinguished from the majority of BAV cases that manifest after age 50 by a relatively severe clinical course, with higher rates of surgical interventions, more frequent second interventions, and a greater burden of congenital heart malformations. These observations highlight the need for prompt recognition, regular lifelong surveillance, and targeted interventions to address the significant health burdens of patients with early onset BAV complications.

rs822336 binding to C/EBPß and NFIC modulates induction of PD-L1 expression and predicts anti-PD-1/PD-L1 therapy in advanced NSCLC.

Efficient predictive biomarkers are needed for immune checkpoint inhibitor (ICI)-based immunotherapy in non-small cell lung cancer (NSCLC). Testing the predictive value of single nucleotide polymorphisms (SNPs) in programmed cell death 1 (PD-1) or its ligand 1 (PD-L1) has shown contrasting results. Here, we aim to validate the predictive value of PD-L1 SNPs in advanced NSCLC patients treated with ICIs as well as to define the molecular mechanisms underlying the role of the identified SNP candidate. rs822336 efficiently predicted response to anti-PD-1/PD-L1 immunotherapy in advanced non-oncogene addicted NSCLC patients as compared to rs2282055 and rs4143815. rs822336 mapped to the promoter/enhancer region of PD-L1, differentially affecting the induction of PD-L1 expression in human NSCLC cell lines as well as their susceptibility to HLA class I antigen matched PBMCs incubated with anti-PD-1 monoclonal antibody nivolumab. The induction of PD-L1 expression by rs822336 was mediated by a competitive allele-specificity binding of two identified transcription factors: C/EBPß and NFIC. As a result, silencing of C/EBPß and NFIC differentially regulated the induction of PD-L1 expression in human NSCLC cell lines carrying different rs822336 genotypes. Analysis by binding microarray further validated the competitive allele-specificity binding of C/EBPß and NFIC to PD-L1 promoter/enhancer region based on rs822336 genotype in human NSCLC cell lines. These findings have high clinical relevance since identify rs822336 and induction of PD-L1 expression as novel biomarkers for predicting anti-PD-1/PD-L1-based immunotherapy in advanced NSCLC patients.

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.

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.

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.

Risk of Recurrence in Uterine Leiomyoma with Bizarre Nuclei: a Systematic Review and Meta-Analysis.

Objective Leiomyoma with bizarre nuclei (LBN) is a variant of uterine leiomyoma, which has replaced the previous category of "atypical leiomyoma" and must be distinguished from smooth muscle tumors of uncertain malignant potential (STUMP). However, previously published series of "atypical leiomyoma" might have included both LBN and STUMP, due to the lack of strict diagnostic criteria. Based on such hypothesis, we aimed to define the risk of recurrence in LBN. Study Design A systematic review and meta-analysis was performed by searching 4 electronic databases for all studies assessing the outcome of patients with "atypical leiomyoma" or LBN. The pooled absolute risk of recurrence was calculated. The included studies were subdivided into two subgroups based on the criteria used: "LBN + STUMP" or "LBN-only". Results Twelve studies with 433 patients were included. The pooled risk of recurrence was 5.5% overall. The funnel plot showed two cluster of studies which superimposed to the two subgroups. In the LBN + STUMP cluster/subgroup, the pooled risk of recurrence was 7.7%. In the LBN-only cluster/subgroup, the pooled risk of recurrence was 1.9%. Statistical heterogeneity was null in all analyses. Conclusion Our results show a risk of recurrence of 1.9% for LBN; higher recurrence rates in older studies are likely due to the inclusion of STUMPs.

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.

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.

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 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.

Diagnostic accuracy of p53 immunohistochemistry as surrogate of TP53 sequencing in endometrial cancer.

Aberrant p53 immunohistochemical expression is used to identify the copy-number-high/TP53-mutant subgroup of endometrial cancer (EC). We aimed to determine the diagnostic accuracy of p53 immunohistochemistry as surrogate for TP53 sequencing through a systematic review and meta-analysis. Electronic databases were searched from their inception to June 2019. All studies assessing p53 expression and TP53 mutations in EC were included. Diagnostic accuracy was assessed based on area under the curve (AUC). Immunohistochemical criteria used to define aberrant p53 expression were "overexpression" and "overexpression or complete absence". Subgroup analysis was based on the sequencing technique adopted (Polymerase Chain Reaction + sequencing, or next generation sequencing, NGS). Thirteen observational studies with 727 endometrial cancers were included. Both "overexpression" and "overexpression or complete absence" showed high diagnostic accuracy (AUC = 0.9088 and 0.9030, respectively). The subgroup with "overexpression" and NGS showed the best results, with very high diagnostic accuracy (AUC = 0.9927). In conclusion, immunohistochemistry for p53 is a highly accurate surrogate of TP53 sequencing. Overexpression of p53 in ≥70-80% showed the best accuracy in predicting TP53 mutations. Further studies in this field should adopt optimized immunohistochemical procedures and take into account less common p53 patterns (e.g. cytoplasmic expression).

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 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.