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Glycogen storage disease type Ia (GSDIa) is an inherited metabolic disorder caused by mutations in the enzyme glucose-6-phosphatase-α (G6Pase-α). Affected individuals develop renal and liver complications, including the development of hepatocellular adenoma/carcinoma and kidney failure. The purpose of this study was to identify potential biomarkers of the evolution of the disease in GSDIa patients. To this end, we analyzed the expression of exosomal microRNAs (Exo-miRs) in the plasma exosomes of 45 patients aged 6 to 63 years. Plasma from age-matched normal individuals were used as controls. We found that the altered expression of several Exo-miRs correlates with the pathologic state of the patients and might help to monitor the progression of the disease and the development of late GSDIa-associated complications.

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c.5713034. Copyright © 2021 The Authors. Amplification of the proto-oncogene MYCN is a key molecular aberration in high-risk neuroblastoma and predictive of poor outcome in this childhood malignancy. We investigated the role of MYCN in regulating the protein cargo of extracellular vesicles (EVs) secreted by tumour cells that can be internalized by recipient cells with functional consequences. Using a switchable MYCN system coupled to mass spectrometry analysis, we found that MYCN regulates distinct sets of proteins in the EVs secreted by neuroblastoma cells. EVs produced by MYCN-expressing cells or isolated from neuroblastoma patients induced the Warburg effect, proliferation and c-MYC expression in target cells. Mechanistically, we linked the cancer-promoting activity of EVs to the glycolytic kinase pyruvate kinase M2 (PKM2) that was enriched in EVs secreted by MYC-expressing neuroblastoma cells. Importantly, the glycolytic enzymes PKM2 and hexokinase II were detected in the EVs circulating in the bloodstream of neuroblastoma patients, but not in those of non-cancer children. We conclude that MYC-activated cancers might spread oncogenic signals to remote body locations through EVs. Neuroblastoma UK to AS and FAPESP SPRINT Award (50356-4); FAPESP (2014/06863-3, 2018/18257-1, 2018/15549-1, 16/50356-4); CNPq “bolsa de produtividade”; Ricerca Finalizzata GR11-172. https://doi.org/10.6084/m9.figshare.c.5713034

Background Tetralogy of Fallot (ToF) and Atrial Septal Defects (ASD) are the most common types of congenital heart diseases and a major cause of childhood morbidity and mortality. Cardiopulmonary bypass (CPB) is used during corrective cardiac surgery to support circulation and heart stabilization. However, this procedure triggers systemic inflammatory and stress response and consequent increased risk of postoperative complications. The aim of this study was to define the molecular bases of ToF and ASD pathogenesis and response to CPB and identify new potential biomarkers. Methods Comparative transcriptome analysis of right atrium specimens collected from 10 ToF and 10 ASD patients was conducted before (Pre-CPB) and after (Post-CPB) corrective surgery. Total RNA isolated from each sample was individually hybridized on Affymetrix HG-U133 Plus Array Strips containing 38,500 unique human genes. Differences in the gene expression profiles and functional enrichment/network analyses were assessed using bioinformatic tools. qRT-PCR analysis was used to validate gene modulation. Results Pre-CPB samples showed significant differential expression of a total of 72 genes, 28 of which were overexpressed in ToF and 44 in ASD. According to Gene Ontology annotation, the mostly enriched biological processes were represented by matrix organization and cell adhesion in ToF and by muscle development and contractility in ASD specimens. GSEA highlighted the specific enrichment of hypoxia gene sets in ToF samples, pointing to a role for hypoxia in disease pathogenesis. The post-CPB myocardium exhibited significant alterations in the expression profile of genes related to transcription regulation, growth/apoptosis, inflammation, adhesion/matrix organization, and oxidative stress. Among them, only 70 were common to the two disease groups, whereas 110 and 24 were unique in ToF and ASD, respectively. Multiple functional interactions among differentially expressed gene products were predicted by network analysis. Interestingly, gene expression changes in ASD samples followed a consensus hypoxia profile. Conclusion Our results provide a comprehensive view of gene reprogramming in right atrium tissues of ToF and ASD patients before and after CPB, defining specific molecular pathways underlying disease pathophysiology and myocardium response to CPB. These findings have potential translational value because they identify new candidate prognostic markers and targets for tailored cardioprotective post-surgical therapies.

Neuroblastoma is the most common extra-cranial pediatric solid tumor, responsible for 13-15% of pediatric cancer death. Its intrinsic heterogeneity makes it difficult to target for successful therapy. The adaptor protein p140Cap/SRCIN1 negatively regulates tumor cell features and limits breast cancer progression. This study wish to assess if p140Cap is a key biological determinant of neuroblastoma outcome. RNAseq profiles of a large cohort of neuroblastoma patients show that SRCIN1 mRNA levels are an independent risk factor inversely correlated to disease aggressiveness. In high-risk patients, CGH+SNP microarray analysis of primary neuroblastoma identifies SRCIN1 as frequently altered by hemizygous deletion, copy-neutral loss of heterozygosity, or disruption. Functional experiments show that p140Cap negatively regulates Src and STAT3 signaling, affects anchorage-independent growth and migration, in vivo tumor growth and spontaneous lung metastasis formation. p140Cap also increases sensitivity of neuroblastoma cells to doxorubicin and etoposide treatment, as well as to a combined treatment with chemotherapy drugs and Src inhibitors. Our functional findings point to a causal role of p140Cap in curbing the aggressiveness of neuroblastoma, due to its ability to impinge on specific molecular pathways, and to sensitize cells to therapeutic treatment. This study provides the first evidence that the SRCIN1/p140Cap adaptor protein is a key player in neuroblastoma as a new independent prognostic marker for patient outcome and treatment. Altogether, these data highlight the potential clinical impact of SRCIN1/p140Cap expression in neuroblastoma tumors, in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.

Bradykinin drives normal lung fibroblasts into myofibroblasts, induces fibroblast proliferation and activates mitogen activated protein kinase pathways (MAPK) but its effects on bronchial fibroblasts from asthmatics (HBAFb) have not been yet studied. We studied bradykinin-induced fibroblast proliferation and differentiation and the related intracellular mechanisms in HBAFb compared to normal bronchial fibroblasts (HNBFb). Bradykinin-stimulated HBAFb and HNBFb were used to assess: bradykinin B 2 receptor expression by Western blot analysis; cell proliferation by [ 3 H] thymidine incorporation; α-smooth muscle actin (SMA) expression/polymerization by Western blot and immunofluorescence; epidermal growth factor (EGF) receptor, extracellular-regulated kinase (ERK) 1/2 and p38 MAPK activation by immunoprecipitation and Western blot, respectively. Constitutive bradykinin B 2 receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B 2 receptor expression in HBAFb. Bradykinin, via bradykinin B 2 receptor, significantly increased fibroblast proliferation at lower concentration (10 −11 M) and α-SMA expression/polymerization at higher concentration (10 −6 M) in both cells. Bradykinin increased ERK1/2 and p38 phosphorylation via bradykinin B 2 receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B 2 receptor, induced EGF receptor phosphorylation that was suppressed by AG1478. In HBAFb AG1478, GM6001, the ERK1/2-inhibitor U0126 and the p38 inhibitor SB203580 suppressed bradykinin-induced cell proliferation, but only SB203580 reduced myofibroblast differentiation. These data indicate that bradykinin is actively involved in asthmatic bronchial fibroblast proliferation and differentiation, through MAPK pathways and EGF receptor transactivation, by which bradykinin may contribute to airway remodeling in asthma, opening new horizons for potential therapeutic implications in asthmatic patients.

Myeloid dendritic cells (DCs) are professional antigen-presenting cells critical for the orchestration of immunity and maintenance of self-tolerance. DC development and functions are tightly regulated by a complex network of inhibitory and activating signals present in the tissue microenvironment, and dysregulated DC responses may result in amplification of inflammation, loss of tolerance, or establishment of immune escape mechanisms. Generation of mature (m)DCs from monocytic precursors recruited at pathological sites occurs under condition of low partial oxygen pressure (pO(2)). However, the way in which the hypoxic microenvironment modulates the functions of these cells is still not clear. We demonstrate that chronic hypoxia (4 days, 1% O(2)) promotes the onset of a highly proinflammatory gene expression profile in mDCs generated from primary human monocytes, characterized by the modulation of a significant cluster of genes coding for proinflammatory chemokines/cytokines and/or their receptors. Within the chemokine system, strong upregulation of genes encoding proteins chemotactic for neutrophils, such as CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8, and for activated/memory T lymphocytes, monocytes, and immature (i) DCs, e.g. CCL20, CCL3 and CCL5, was observed, concomitant with decreased expression of genes coding for naive/resting T cells chemoattractants, CCL18 and CCL23. Other hypoxia-inducible genes coded for cytokines with a primary role in inflammation and angiogenesis, including osteopontin, vascular endothelial growth factor, and IL-1β. mRNA modulation was paralleled by protein secretion. These results suggest that conditions of reduced O(2) availability reprograms mDCs toward a proinflammatory direction by tuning the cytokine/chemokine repertoire, thus affecting their ability to regulate leukocyte trafficking and activation at pathological sites, with potential implications for the pathogenesis of chronic inflammatory diseases.

Bradykinin (BK) induces fibroblast contraction but the structural changes and intracellular mechanisms involved have not been completely explored. We stimulated HFL-1 fibroblasts with BK to assess: 1) fibroblast contractility; 2) the role of alpha-smooth muscle actin (SMA) in contraction by small interfering RNA (siRNA); 3) alpha-SMA protein expression; 4) alpha-SMA and F-actin structure; 5) intracellular calcium concentration; and 6) phosphorylated myosin light-chain (pMLC) and MLC kinase (MLCK) expression. BK triggered concentration- and time-dependent fibroblast gel contraction in conjunction with alpha-SMA over expression, but not in alpha-SMA-siRNA-treated cells. BK also increased alpha-SMA(+) and F-actin(+) cell number and stress fibre polymerisation (detectable at 5-60 min). These BK-induced changes were associated with an increase in intracellular calcium concentration, which peaked within 15 s, and activation of pMLC, which was detectable at 5-60 min. No MLCK content modification was observed. The different manifestations of the BK-induced fibroblast activation were downregulated at different levels (25-100%) by HOE140, a specific BK B2 receptor (B2R) antagonist and by the Ca(2+) chelator, EGTA. Thus, BK-induced fibroblast contraction, associated with differentiation into alpha-SMA(+) myofibroblasts, is mediated through the activation of the B2R and involves the Ca(2+)/calmodulin pMLC-dependent pathway.

We studied the expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate limiting enzyme in the hexosamine biosynthetic pathway controlling protein glycosylation. We obtained the first evidence that the GFAT mRNA and protein are constitutively expressed in murine mononuclear phagocytes (Mf) and inducible by picolinic acid (PA), a catabolite of tryptophan, hypoxia and desferrioxamine (DFX). These stimuli share the property to transactivate gene expression through the Hypoxia Responsive Element (HRE). The promoter of GFAT contains the consensus sequence of HRE in position −74/-65 (GFAT-HRE), and we studied the role of HRE on the activation of the promoter utilizing appropriate expression vectors. We found that GFAT-HRE is essential for the response to hypoxia, PA or DFX and that Hypoxia Inducible Factor-1α (HIF-1α) can augment this response. Finally, we demonstrate that iron chelation is part of the mechanism by which PA and DFX activate GFAT expression. Our results provide the first indication that hypoxia, PA or DFX induce the transcription of GFAT gene in murine Mf cell lines and that the HRE of the promoter is essential for this response.

Langerhans cells (LCs) are a specialized dendritic cell subset that resides in the epidermis and mucosal epithelia and is critical for the orchestration of skin immunity. Recent evidence suggest that LCs are involved in aberrant wound healing and in the development of hypertrophic scars and chronic wounds, which are characterized by a hypoxic environment. Understanding LCs biology under hypoxia may, thus, lead to the identification of novel pathogenetic mechanisms of wound repair disorders and open new therapeutic opportunities to improve wound healing. In this study, we characterize a previously unrecognized role for hypoxia in significantly affecting the phenotype and functional properties of human monocyte-derived LCs, impairing their ability to stimulate naive T cell responses, and identify the triggering receptor expressed on myeloid (TREM)-1, a member of the Ig immunoregulatory receptor family, as a new hypoxia-inducible gene in LCs and an activator of their proinflammatory and Th1-polarizing functions in a hypoxic environment. Furthermore, we provide the first evidence of TREM-1 expression in vivo in LCs infiltrating hypoxic areas of active hypertrophic scars and decubitous ulcers, pointing to a potential pathogenic role of this molecule in wound repair disorders.Hypoxia modulates surface molecule expression and cytokine profile in Langerhans cells. Hypoxia impairs human Langerhans cell stimulatory activity on naive T cells. Hypoxia selectively induces TREM-1 expression in human Langerhans cells. TREM-1 engagement stimulates Langerhans cell inflammatory and Th1-polarizing activity. TREM-1 is expressed in vivo in Langerhans cells infiltrating hypoxic skin lesions.

Neuroblastoma (NB) is the most common solid tumor during infancy and the first cause of death among the preschool age diseases. The availability of several NB genomic profiles improves the prognostic ability, but the outcome prediction for this pathology remains imperfect. We previously produced a novel prognostic gene signature based on the response of NB cells to hypoxia, a condition of tumor microenvironment strictly connected with cancer aggressiveness. Here we attempted to further define the expression of hypoxia-modulated specific genes, looking at their protein level in NB specimens, considering in particular the hypoxia inducible factor-1α (HIF-1α), the mitochondrial pyruvate dehydrogenase kinase 1 (PDK1), and the HIF-prolyl hydroxylase domain 3 (PHD3). The evaluation of expression was performed by Western blot and immunocytochemistry on NB cell lines and by immunohistochemistry on tumor specimens. Stimulation of both HIF-1α and PDK1 and inhibition of PHD3 expression were observed in NB cell lines cultured under prolonged hypoxic conditions as well as in most of the tumors with poor outcome. Our results indicate that the immunohistochemistry analysis of the protein expression of PDK1, PHD3, and HIF-1α defines the hypoxic status of NB tumors and can be used as a simple and relevant tool to stratify high-risk patients.

The Rho guanine nucleotide exchange factor protoDbl is involved in different biochemical pathways affecting cell proliferation and migration. The N-terminal sequence of protoDbl contains negative regulatory elements that restrict the catalytic activity of the DH-PH module. Here, we report the identification of a new mouse protoDbl splice variant lacking exon 3. We found that the splice variant mRNA is expressed in the spleen and bone marrow lymphocytes, adrenal gland, gonads and brain. The protoDbl variant protein was detectable in the brain. The newly identified variant displays the disruption of the SEC14 domain, positioned on exons 2 and 3 in the protoDbl N-terminal region. We show here that an altered SEC14 sequence leads to enhanced Dbl translocation to the plasma membrane and to augmented transforming and exchange activity.

Beckwith-Wiedemann syndrome is associated with an increased risk of tumors in the pediatric age. We report the case of a newborn with Beckwith-Wiedemann syndrome presenting the simultaneous occurrence of a cystic pancreatoblastoma and an adrenal neuroblastoma. Surgery was required to treat the pancreatoblastoma, and a "wait and see" policy was adopted for the neuroblastoma, which spontaneously regressed within a few months.

Peripheral blood monocytes are recruited to inflammatory and tumor lesions where they undergo terminal differentiation into macrophages. Monocytes/macrophages integrate stimulatory and inhibitory signals present in the pathologic microenvironment through a defined repertoire of cell surface receptors, and deregulated expression of these molecules may result in amplification of inflammation or establishment of immune escape mechanisms. Characterization of the expression and function of these receptors is required for a better understanding of the regulation of monocyte/macrophage activity at pathologic sites. Hypoxia is a common feature of many pathological situations and an important regulator of monocyte/macrophage pro-inflammatory responses. In this study, we identify the leukocyte membrane antigen, CD300a, a member of the CD300 superfamily of immunoregulatory receptors, as a new hypoxia-inducible gene in primary human monocytes and monocyte-derived macrophages. CD300a mRNA up-regulation by hypoxia was rapid and reversible, paralleled by increased surface protein expression, and mediated by hypoxia-inducible factor-1α. CD300a induction was also triggered by the hypoxia-mimetic agent, desferrioxamine. CD300a exhibited both activating and inhibitory potential, differentially regulating CCL20 and vascular endothelial growth factor pro-inflammatory cytokine production by monocytes/macrophages upon triggering by an agonist Ab. These results suggest that CD300a induction by the hypoxic environment represents a mechanism of regulation of monocyte/macrophage pro-inflammatory responses at pathologic sites.

Background Neuroblastoma is the most common pediatric solid tumor of the sympathetic nervous system. Development of improved predictive tools for patients stratification is a crucial requirement for neuroblastoma therapy. Several studies utilized gene expression-based signatures to stratify neuroblastoma patients and demonstrated a clear advantage of adding genomic analysis to risk assessment. There is little overlapping among signatures and merging their prognostic potential would be advantageous. Here, we describe a new strategy to merge published neuroblastoma related gene signatures into a single, highly accurate, Multi-Signature Ensemble (MuSE)-classifier of neuroblastoma (NB) patients outcome. Methods Gene expression profiles of 182 neuroblastoma tumors, subdivided into three independent datasets, were used in the various phases of development and validation of neuroblastoma NB-MuSE-classifier. Thirty three signatures were evaluated for patients' outcome prediction using 22 classification algorithms each and generating 726 classifiers and prediction results. The best-performing algorithm for each signature was selected, validated on an independent dataset and the 20 signatures performing with an accuracy > = 80% were retained. Results We combined the 20 predictions associated to the corresponding signatures through the selection of the best performing algorithm into a single outcome predictor. The best performance was obtained by the Decision Table algorithm that produced the NB-MuSE-classifier characterized by an external validation accuracy of 94%. Kaplan-Meier curves and log-rank test demonstrated that patients with good and poor outcome prediction by the NB-MuSE-classifier have a significantly different survival (p < 0.0001). Survival curves constructed on subgroups of patients divided on the bases of known prognostic marker suggested an excellent stratification of localized and stage 4s tumors but more data are needed to prove this point. Conclusions The NB-MuSE-classifier is based on an ensemble approach that merges twenty heterogeneous, neuroblastoma-related gene signatures to blend their discriminating power, rather than numeric values, into a single, highly accurate patients' outcome predictor. The novelty of our approach derives from the way to integrate the gene expression signatures, by optimally associating them with a single paradigm ultimately integrated into a single classifier. This model can be exported to other types of cancer and to diseases for which dedicated databases exist.

The Rho guanine nucleotide exchange factor (GEF) Dbl binds to the N-terminal region of ezrin, a member of the ERM (ezrin, radixin, moesin) proteins known to function as linkers between the plasma membrane and the actin cytoskeleton. Here we have characterized the interaction between ezrin and Dbl. We show that binding of Dbl with ezrin involves positively charged amino acids within the region of the pleckstrin homology (PH) domain comprised between β1 and β2 sheets. In addition, we show that Dbl forms a complex with the tuberous sclerosis-1 (TSC-1) gene product hamartin and with ezrin. We demonstrate that hamartin and ezrin are both required for activation of Dbl. In fact, the knock-down of ezrin and hamartin, as well as the expression of a mutant hamartin, unable to bind ezrin, inhibit Dbl transforming and exchange activity. These results suggest that Dbl is regulated by hamartin through association with ezrin.

Dbl is the prototype of a large family of GDP–GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho, Rac, and Cdc42 and to induce a transformed phenotype in murine fibroblasts. We previously reported that Dbl-null mice are viable and fertile but display defective dendrite elongation of distinct subpopulations of cortical neurons, suggesting a role of Dbl in controlling dendritic growth. To gain deeper insights into the role of Dbl in development and disease, we attempted a knock-in approach to create an endogenous allele that encodes a missense-mutation-mediated loss of function in the DH domain. We generated, by gene targeting technology, a mutant mouse strain by inserting a mutagenized human proto-Dbl cDNA clone expressing only the Dbl N terminus regulatory sequence at the starting codon of murine exon 1. Animals were monitored over a 21-month period, and necropsy specimens were collected for histological examination and immunohistochemistry analysis. Dbl knock-in mice are viable and did not manifest either decreased reproductive performances or gross developmental phenotype but revealed a reduced lifespan compared to wild-type (w.t.) mice and showed, with aging, a B cell lymphoproliferation that often has features of a frank diffuse large B cell lymphoma. Moreover, Dbl knock-in male mice displayed an increased incidence of lung adenoma compared to w.t. mice. These data indicate that Dbl is a tumor susceptibility gene in mice and that loss of function of Dbl DH domain by genetic missense mutations is responsible for induction of diffuse large B cell lymphoma.

Dendritic cells (DCs) are a heterogeneous group of professional antigen-presenting cells functioning as sentinels of the immune system and playing a key role in the initiation and amplification of innate and adaptive immune responses. DC development and functions are acquired during a complex differentiation and maturation process influenced by several factors present in the local milieu. A common feature at pathologic sites is represented by hypoxia, a condition of low pO2, which creates a unique microenvironment affecting cell phenotype and behavior. Little is known about the impact of hypoxia on the generation of mature DCs (mDCs). In this study, we identified by gene expression profiling a significant cluster of genes coding for immune-related cell surface receptors strongly up-regulated by hypoxia in monocyte-derived mDCs and characterized one of such receptors, TREM-1, as a new hypoxia-inducible gene in mDCs. TREM-1 associated with DAP12 in hypoxic mDCs, and its engagement elicited DAP12-linked signaling, resulting in ERK-1, Akt, and IκBα phosphorylation and proinflammatory cytokine and chemokine secretion. Finally, we provided the first evidence that TREM-1 is expressed on mDCs infiltrating the inflamed hypoxic joints of children affected by juvenile idiopathic arthritis, representing a new in vivo marker of hypoxic mDCs endowed with proinflammatory properties.

Background Hypoxia is a condition of low oxygen tension occurring in the tumor microenvironment and it is related to poor prognosis in human cancer. To examine the relationship between hypoxia and neuroblastoma, we generated and tested an in vitro derived hypoxia gene signature for its ability to predict patients' outcome. Results We obtained the gene expression profile of 11 hypoxic neuroblastoma cell lines and we derived a robust 62 probesets signature (NB-hypo) taking advantage of the strong discriminating power of the l1-l2 feature selection technique combined with the analysis of differential gene expression. We profiled gene expression of the tumors of 88 neuroblastoma patients and divided them according to the NB-hypo expression values by K-means clustering. The NB-hypo successfully stratifies the neuroblastoma patients into good and poor prognosis groups. Multivariate Cox analysis revealed that the NB-hypo is a significant independent predictor after controlling for commonly used risk factors including the amplification of MYCN oncogene. NB-hypo increases the resolution of the MYCN stratification by dividing patients with MYCN not amplified tumors in good and poor outcome suggesting that hypoxia is associated with the aggressiveness of neuroblastoma tumor independently from MYCN amplification. Conclusions Our results demonstrate that the NB-hypo is a novel and independent prognostic factor for neuroblastoma and support the view that hypoxia is negatively correlated with tumors' outcome. We show the power of the biology-driven approach in defining hypoxia as a critical molecular program in neuroblastoma and the potential for improvement in the current criteria for risk stratification., Foundation KiKa, Children's Neuroblastoma Cancer Foundation, SKK Foundation, Dutch Cancer Society

The ErbB2 oncogene is often overexpressed in breast tumors and associated with poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration, and proliferation in normal and pathological cells. The functional role of p130Cas in ErbB2-dependent breast tumorigenesis was assessed by its silencing in breast cancer cells derived from mouse mammary tumors overexpressing ErbB2 (N202-1A cells), and by its reexpression in ErbB2-transformed p130Cas-null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2-dependent foci formation, anchorage-independent growth, and in vivo growth of orthotopic N202-1A tumors. Moreover, intranipple injection of p130Cas-stabilized siRNAs in the mammary gland of Balbc-NeuT mice decreases the growth of spontaneous tumors. In ErbB2-transformed cells, p130Cas is a crucial component of a functional molecular complex consisting of ErbB2, c-Src, and Fak. In human mammary cells, MCF10A.B2, the concomitant activation of ErbB2, and p130Cas overexpression sustain and strengthen signaling, leading to Rac1 activation and MMP9 secretion, thus providing invasive properties. Consistently, p130Cas drives N202-1A cell in vivo lung metastases colonization. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers.

The dbl oncogene is generated by substitution of the 5ae portion of its normal counterpart with an unrelated human sequence. To analyze the genomic structure and transcriptional regulation of the dbl proto-oncogene, we have isolated human genomic clones containing the entire human proto-dbl gene, localized in Xq26. Restriction mapping of a 600 kb YAC clone (yWXD311) placed proto-dbl about 50 kb telomeric to the coagulation Factor IX gene. The genomic DNA fragment containing the 5ae end of proto-dbl was subcloned into plasmid vectors and the nucleotide sequences of exon 1, the flanking intronic region and genomic DNA 5ae of the first codon were determined. Sequence analysis of 85 119 bp from the region revealed the genomic structure of proto-dbl. It contains 25 exons coding for a 4.7 kb transcript including large 5ae- and 3ae- (1218 bp and 701 bp, respectively) untranslated regions ( UTRs). RNase protection and primer extension assays on RNA from medullary thyroid carcinoma (TT ) cells, which normally express dbl, revealed a transcription start site 1218 bp upstream of the ATG of the first exon. A 1.6 kb genomic 5ae of the translation start sites drives the expression of a CAT-reporter in transient transfections in the TT cell line, though lacking TATA or CAAT boxes. © 2000 Elsevier Science B.V. All rights reserved.

The dbl oncogene was initially isolated from a human diffuse B-cell lymphoma. Antisera from mice bearing tumors induced by this oncogene specifically detected a protein of about 66 kDa (p66) in dbl transformants. dbl cDNA-selected poly(A)+ RNA isolated from a transfectant clone expressing p66 directed the in vitro synthesis of this protein, establishing that it is encoded by dbl. Subcellular localization studies revealed that p66 is a cytoplasmic protein distributed between cytosol and crude membrane fractions. Moreover, p66 was shown to be a phosphoprotein, with phosphorylation specific to serine residues. Our characterization of the dbl-encoded protein appears to distinguish this transforming gene product from those of other known oncogenes.

We isolated cDNA clones representing the human dbl proto-oncogene transcript. Nucleotide sequence analysis revealed an open reading frame encoding a predicted protein of 925 amino acids. Using peptide antisera directed against specific proto-dbl peptides, a 115-kd protein was detected in COS cells transfected with an expression vector containing the entire coding region of proto-dbl. This mol. wt is consistent with that predicted from the open reading frame. We have previously shown that the dbl oncogene was generated by substitution of the 5' portion of proto-dbl with an unrelated human sequence. In this study we show that this rearrangement resulted in the loss of the 497 amino-terminal codons of the dbl proto-oncogene. Under the influence of a strong promoter proto-dbl could readily transform NIH/3T3 cells but its transforming activity was less than that of the dbl oncogene driven by the same promoter. Proto-dbl overexpression is, therefore, sufficient to transform NIH/3T3 cells, but specific structural alterations of its coding region significantly enhance its transforming activity. No apparent similarity was detected between the predicted proto-dbl product and other known proto-oncogenes. However, a stretch of 300 amino acids within the N-terminal half of proto-dbl showed structural similarity to the intermediate filament vimentin. This region in proto-dbl contains a heptad repeat motif characteristic of an alpha-helical coiled-coil structure. Taken together, these findings indicate that the human proto-dbl represents a new class of cellular oncogenes that may be related to cytoskeletal elements of the cell.

Polyadenylated RNAs of certain human tumour cell lines are shown to contain transcripts related to the cell-derived transforming onc genes of molecularly cloned primate, murine or avian transforming retrovirus genomes. Thus, analogues of retroviral transforming genes are both present and frequently expressed in human neoplastic cells.

Utilizing DNA transfection analysis1 with the continuous NIH 3T3 cell line as assay cell2, we3 and others4,5 have observed that as many as 10–50% of human haematopoietic tumours contain oncogenes, the vast majority of which are members of the ras proto-oncogene family. In addition, Cooper and co-workers6–8 have reported the detection and isolation of specific oncogenes, B-lym and T-lym, which appear to be activated in human and rodent tumours of certain B and T lymphoid cells, respectively. In surveying human haematopoietic malignancies, we observed that DNA of a primary human diffuse B-cell lymphoma induced an unusual transformed focus on transfection of NIH 3T3 cells. Here, we report the molecular cloning and physical characterization of this human oncogene, whose transforming activity was shown to reside within a human DNA sequence of 45 kilobases (kb) cloned in a cosmid vector. Its properties distinguish it from previously reported retroviral or nonretroviral oncogenes.

The DBL transforming gene was originally identified by transfection of NIH 3T3 cells with DNA from a human B-cell lymphoma. This gene was found to have arisen as a result of recombination of the 3' portion of the DBL protooncogene coding sequences with an unrelated segment of human DNA. It encodes a cytoplasmic protein that is equally distributed between cytosol and crude membrane fractions. To further characterize this transforming gene, a biologically active cDNA clone of the DBL transforming gene mRNA was isolated. Analysis of the sequence of the DBL oncogene cDNA revealed a long open reading frame that encodes a hybrid protein whose first 50 amino acids (at least) derive from a complete exon of a different locus. No significant homology with known oncogenes or any known protein sequences was demonstrated. The computer analysis of the predicted DBL protein indicated it is highly hydrophilic with no hydrophobic domains characteristic of a membrane-spanning region or signal peptide. Thus, the DBL oncoprotein is distinct among known transforming gene products.

Total cellular RNAs from a variety of fresh and culture-derived human hematopoietic neoplastic cell types at various stages of differentiation and human sarcoma, carcinoma, melanoma, and glioblastoma cell lines were enriched for poly(A)- containing sequences, fractionated by gel electrophoresis, and blot hybridized to a cloned DNA probe containing the transforming sequences (v-amv) of avian myeloblastosis virus (AMV), a virus known to cause myeloid leukemias in chickens. Expression of RNA sequences homologous to AMV was detected in all immature myeloid and lymphoid T cells in addition to the single erythroid cell line examined, but not in mature T cells or in B cells, including lymphoblast cell lines derived from patients with Burkitt lymphoma. In addition, induction of the cell line HL60, a promyelocytic leukemia line, to differentiate with dimethyl sulfoxide or retinoic acid resulted in a reduction of the level of expression of the human cellular gene c-amv homologous to v-amv. There was no detectable c-amv mRNA in any of the solid tumor cell lines examined. Thus, expression of the human c-amv gene could be correlated with the stage of differentiation of different hematopoietic cell types determined by morphologic and marker studies. Expression of c-amv could not be correlated with the extent of methylation in HL60 and in HL60 induced to differentiate with dimethyl sulfoxide.

The DBL oncogene was generated by rearrangements involving three discontinuous regions of the human genome. Analyses of panels of human × rodent somatic cell hybrids demonstrated that the DBL proto-oncogene located on the X chromosome (just proximal or distal to bands q26–27.2) underwent recombination at its 5′ and 3′ ends with sequences derived from chromosomes 3 (p13q-ter) and 16 (p13–q22), respectively. DBL was localized to chromosome Xq27–q28 by in situ hybridization. Another oncogene, MCF2, was previously shown to contain sequences derived from Xq27 as well. Comparison of the restriction maps and nucleotide sequences of genomic and cDNA clones representing the chromosome X-specific sequences of the DBL oncogene and MCF2, taken together with their chromosomal localization, indicates that these oncogenes were derived from the same genetic locus.

We surveyed 22 human hematopoietic tumors and tumor cell lines for sequences capable of transforming NIH 3T3 cells by DNA transfection. A primary human acute myelogenous leukemia, a chronic myelogenous leukemia cell line, and cell lines derived from three independent acute lymphocytic leukemias demonstrated oncogenes capable of conferring the transformed phenotype to NIH 3T3 cells through serial cycles of transfection. One of three transforming genes associated with acute lymphocytic leukemia cells (classified as thymocyte developmental stage II) was identified as the activated cellular homologue of the Kirsten murine sarcoma virus onc gene, kis, a member of the ras family of onc genes. A transforming gene, which was demonstrated to be common to several human myeloid and lymphoid tumor cells, was shown to be a distantly related member of the ras gene family. Thus, the NIH 3T3 transfection assay commonly detects related oncogenes in human hematopoietic tumor cells. Moreover, the activation of these oncogenes appears to be independent of the specific stage of cell differentiation or tumor phenotype.

Total cellular poly(A)-enriched RNA from a variety of fresh human leukemic blood cells and hematopoietic cell lines was analyzed for homology with molecularly cloned DNA probes containing the onc sequence of Abelson murine leukemia virus (Ab-MuLV), Harvey murine sarcoma virus (Ha-MuSV), simian sarcoma virus (SSV), and avian myelocytomatosis virus strain MC29. Results with the fresh blood cells paralleled those obtained with the cell lines. With Ab-MuLV and Ha-MuSV, multiple RNA bands were visualized in all cell types examined without significant variation in the relative intensities of the bands. When SSV was used as the probe, expression of related onc sequences was absent in all of the hematopoietic cell types examined except for one neoplastic T-cell line (HUT 102), which produces the human T-cell leukemia (lymphoma) retrovirus HTLV. In this cell line, a single band (4.2 kilobases) was observed. With MC29 as the probe, a single band of 2.7 kilobases was visualized in all cell types examined with only a 10 to 2-fold variation in intensity of hybridization. An exception was the promyelocytic cell line, HL60, which expressed approximately 10-fold more MC29-related onc sequences. With induction of differentiation of HL60 with either dimethyl sulfoxide or retinoic acid, a marked diminution in the amount of the MC29-related, but not the Ab-MuLV-related, onc message was observed.

Background More than fifty percent of neuroblastoma (NB) patients with adverse prognosis do not benefit from treatment making the identification of new potential targets mandatory. Hypoxia is a condition of low oxygen tension, occurring in poorly vascularized tissues, which activates specific genes and contributes to the acquisition of the tumor aggressive phenotype. We defined a gene expression signature (NB-hypo), which measures the hypoxic status of the neuroblastoma tumor. We aimed at developing a classifier predicting neuroblastoma patients’ outcome based on the assessment of the adverse effects of tumor hypoxia on the progression of the disease. Methods Multi-layer perceptron (MLP) was trained on the expression values of the 62 probe sets constituting NB-hypo signature to develop a predictive model for neuroblastoma patients’ outcome. We utilized the expression data of 100 tumors in a leave-one-out analysis to select and construct the classifier and the expression data of the remaining 82 tumors to test the classifier performance in an external dataset. We utilized the Gene set enrichment analysis (GSEA) to evaluate the enrichment of hypoxia related gene sets in patients predicted with “Poor” or “Good” outcome. Results We utilized the expression of the 62 probe sets of the NB-Hypo signature in 182 neuroblastoma tumors to develop a MLP classifier predicting patients’ outcome (NB-hypo classifier). We trained and validated the classifier in a leave-one-out cross-validation analysis on 100 tumor gene expression profiles. We externally tested the resulting NB-hypo classifier on an independent 82 tumors’ set. The NB-hypo classifier predicted the patients’ outcome with the remarkable accuracy of 87 %. NB-hypo classifier prediction resulted in 2 % classification error when applied to clinically defined low-intermediate risk neuroblastoma patients. The prediction was 100 % accurate in assessing the death of five low/intermediated risk patients. GSEA of tumor gene expression profile demonstrated the hypoxic status of the tumor in patients with poor prognosis. Conclusions We developed a robust classifier predicting neuroblastoma patients’ outcome with a very low error rate and we provided independent evidence that the poor outcome patients had hypoxic tumors, supporting the potential of using hypoxia as target for neuroblastoma treatment. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1194-3) contains supplementary material, which is available to authorized users.

We have investigated the expression of the dbl protooncogene in a wide variety of human tumors of different embryological derivation. We found that proto-dbl mRNA could be detected preferentially in a few neoplastic histiotypes of neuroectodermal origin. The common, normal 5-kilobase size of the proto-dbl transcript detected indicated that the proto-dbl in these tumors was not rearranged. These data, in agreement with our previous reports, suggest that the dbl protooncogene is expressed in a highly tissue-specific manner and indicate that this gene may be involved in the growth and differentiation of some cells of neuroectodermal origin. We have investigated the expression of the dbl protooncogene in a wide variety of human tumors of different embryologicai derivation. We found that proio-dbl mRNA could be detected preferentially in a few neoplastic histiotypes of neuroectodermal origin. The common, normal 5-kilobase size of the proto-dbl transcript detected indicated that the proto-dbl in these tumors was not rearranged. These data, in agreement with our previous reports, suggest that the dbl protooncogene is expressed in a highly tissue-specific manner and indicate that this gene may be involved in the growth and differentiation of some cells of neuroectodermal origin. © 1991, American Association for Cancer Research. All rights reserved.

The dbl oncogene was initially isolated following transfection of NIH3T3 cells with DNA of a human diffuse B cell lymphoma. Its transcribed sequences were shown to be distributed over a 30-kb span within a molecularly cloned 45-kb segment of human DNA which contained the transforming gene. By restriction mapping, its transcribed region corresponded to that of its normal allele, except at the 5' end where a rearrangement involved transcribed dbl oncogene sequences from another locus. An independent isolate of a dbl-related transforming gene was obtained following transfection of NIH3T3 cells with DNA of a human nodular poorly differentiated lymphoma (NPDL). Physical mapping indicated that this transforming gene, designated NPDL-dbl, shared considerable homology with the dbl oncogene, but differed at both 5' and 3' termini. Its point of divergence from the normal allele at the 5' end was at least 10 kb upstream from that of the dbl oncogene. The oncogenes each expressed truncated transcripts compared to the 5.3-kb normal transcript. The dbl and NPDL-dbl oncogene translational products of 66 and 76 kDa, respectively, were consistent with their corresponding major 2.8- and 3.5-kb transcripts. It was not possible to detect evidence of the 5' structural rearrangements associated with these oncogenes in either of the original tumors. Thus, if these rearrangements were critical to their activation, they occurred in the process of gene transfer or in vivo in only a minority of tumor cells.

The dbl oncogene belongs to a unique class of human transforming genes. The dbl proto-oncogene is activated by substitution of the 5' portion of the gene with an unrelated human sequence. The proto-oncogene product is distributed between the soluble and membrane fractions of the cytoplasm and its function remains still unknown. In order to understand the biological role of dbl in human malignancies or during cell differentiation we have investigated the expression of the dbl oncogene in a wide number of human tumors of different embryological derivation. We found that dbl is preferentially expressed in a few neoplastic histiotypes of neuroectodermal origin. The transcript size of 5.3 Kb strongly suggests that the gene is not truncated in these tumors. These data, together with the information that the proto-oncogene has been found expressed in normal brain and adrenal medulla, indicate that dbl expression may be involved in cell differentiation of some tissues of neuroectodermic origin.

The DNA's from two of four methylcholanthrene-induced mouse fibrosarcomas contained transforming genes that were identical in their pattern of restriction endonuclease resistance to inactivation of biologic activity. This transforming gene was identified as the activated homolog of the Kirsten murine sarcoma virus onc gene, v-kis. The finding that a defined carcinogen reproducibly leads to activation of kis as a transforming gene should be of value in elucidating the role of oncogenes in the neoplastic process.