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Smooth muscle cells (SMCs) are dynamic and transition from a contractile to a synthetic phenotype under different circumstances. Plasma factors (fibrin and transforming growth factors, TGFs) are possible components affecting SMCs differentiation and behavior. Thus, the objective of this work was to investigate how the fibrin matrix and TGFs affect SMCs differentiation and motility behavior. SMCs invaded the fibrin gel and adopted a stellate phenotype while reducing the expression of differentiation markers (Acta2, Myh11, and Smtn). At the ultrastructural level, SMCs did not assemble a basal lamina and showed numerous blebs along the entire cell surface. This transition was not associated with changes in focal adhesion kinase (FAK) content and phosphorylation status but reflected a marked change in FAK distribution in the cytoplasm. After 48 h in culture, SMCs caused an active degradation of the fibrin gel. Additionally, we tested the SMCs response to TGFs in a cell layer wound repair assay. TGFα, but not TGFβ1 or TGFβ3, had significantly increased motility. In conclusion, prostatic SMCs present a phenotypical transition when cultured on fibrin, adopting a micro-blebbing based motility behavior and increasing migration in response to TGFα.

Background: Osteoarthritis (OA) is a complex joint disorder characterized by sclerosis of subchondral bone. The knee is one of the most commonly affected joints. Given that the genetic mechanisms u...

Macroautophagy/autophagy is a cellular catabolic process that is implicated in several physiological and pathological processes. However, the role of epidermal autophagy in wound healing remains unknown. Here, using mice with genetic ablation of the essential Atg5 (autophagy related 5) or Atg7 (autophagy related 7) in their epidermis to inhibit autophagy, we show that keratinocyte autophagy regulates wound healing in mice. Wounding induces the expression of autophagy genes in mouse skin. Epidermis-specific autophagy deficiency inhibits wound closure, re-epithelialization, keratinocyte proliferation and differentiation, dermal granulation tissue formation, and infiltration of immune cells including macrophages, neutrophils, and mast cells, while it does not affect angiogenesis. Using cytokine array screening, we found that autophagy deficiency inhibits the transcription and production of the cytokine CCL2/MCP-1 by TNF. At the molecular level, TNF induces autophagic flux and the expression of autophagy genes through NFKB in epidermal keratinocytes. TNF promotes CCL2 transcription through the autophagy-AMPK-BRAF-MAPK1/3/ERK-activator protein 1 (AP1) pathway. Indeed, treating mice with recombinant CCL2 can reverse the effect of autophagy deficiency in keratinocytes. At the cellular level, we found that CCL2 induction via autophagy in keratinocytes is required not only for keratinocyte migration and proliferation but also for dermal fibroblast activation. Our findings demonstrate a critical role of epidermal autophagy in wound healing in vivo and elucidate a critical molecular machinery coordinating keratinocyte-fibroblast interaction in skin repair. Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle; ACTB: β-actin; ADGRE1: adhesion G protein-coupled receptor E1; AMPK: AMP-activated protein kinase; AP1: activator protein 1; AP1-RE: AP1 response element; ATG: autophagy-related; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; BRAF: B-Raf proto-oncogene, serine/threonine kinase; C5: complement C5; CCL2/MCP-1: C-C motif chemokine ligand 2; CCL3: C-C motif chemokine ligand 3; CK: cytokeratin; cKO: conditional knockout; CRTC1: CREB-regulated transcription coactivator 1; CXCL1: C-X-C motif chemokine ligand 1; CXCL2: C-X-C motif chemokine ligand 2; ECM: extracellular matrix; EGF: epidermal growth factor; FGF7: fibroblast growth factor 7; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HBEGF: heparin binding EGF like growth factor; HPRT1: hypoxanthine phosphoribosyltransferase 1; IHC: immunohistochemical; IL1B: interleukin 1 beta; KRT10: keratin 10; KRT14: keratin 14; MAP1LC3B/LC3B-I/II: microtubule-associated protein 1 light chain 3 beta; MAPK1/3/ERK: mitogen-activated protein kinase 1/3; MKI67/Ki-67: marker of proliferation; MPO: myeloperoxidase; NFKB: NF-kappa B, nuclear factor kappa-light-chain-enhancer of activated B cells; NFKB-RE: NFKB response element; PDGF: platelet-derived growth factor; PECAM1: platelet and endothelial cell adhesion molecule 1; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; RELA/p65: RELA proto-oncogene, NFKB subunit; shCON: small hairpin negative control; siNC: negative control; siRNA: small interfering RNA; SP1: sp1 transcription factor; SQSTM1/p62: sequestosome 1; TGFA: transforming growth factor alpha; TGFB1: transforming growth factor beta 1; TIMP1: TIMP metallopeptidase inhibitor 1; TNF/TNF-alpha: tumor necrosis factor; TREM1: triggering receptor expressed on myeloid cells 1; WT: wild-type

Significance Tetraspanin protein (Tspan6) is a member of the tetraspanin family. Using a combination of in vitro and in vivo assays, we demonstrate that Tspan6 functions as a tumor suppressor in colorectal cancer (CRC) by attenuating the epidermal growth factor receptor (EGFR)–based signaling axis. Tspan6 forms a tripartite complex with transmembrane form of TGF-α and an adaptor protein syntenin-1 and negatively regulates secretion of TGF-α. The expression of Tspan6 is frequently decreased in CRC, and this correlates with poor survival. Importantly, the expression of Tspan6 in CRC correlated independently of tumor molecular profile with better patient responses to Cetuximab, an EGFR-targeted therapy. These results identify Tspan6 as a regulator of CRC development and a potential predictive biomarker for EGFR-targeted therapies., Early stages of colorectal cancer (CRC) development are characterized by a complex rewiring of transcriptional networks resulting in changes in the expression of multiple genes. Here, we demonstrate that the deletion of a poorly studied tetraspanin protein Tspan6 in Apcmin/+ mice, a well-established model for premalignant CRC, resulted in increased incidence of adenoma formation and tumor size. We demonstrate that the effect of Tspan6 deletion results in the activation of EGF-dependent signaling pathways through increased production of the transmembrane form of TGF-α (tmTGF-α) associated with extracellular vesicles. This pathway is modulated by an adaptor protein syntenin-1, which physically links Tspan6 and tmTGF-α. In support of this, the expression of Tspan6 is frequently decreased or lost in CRC, and this correlates with poor survival. Furthermore, the analysis of samples from the epidermal growth factor receptor (EGFR)–targeting clinical trial (COIN trial) has shown that the expression of Tspan6 in CRC correlated with better patient responses to EGFR-targeted therapy involving Cetuximab. Importantly, Tspan6-positive patients with tumors in the proximal colon (right-sided) and those with KRAS mutations had a better response to Cetuximab than the patients that expressed low Tspan6 levels. These results identify Tspan6 as a regulator of CRC development and a potential predictive marker for EGFR-targeted therapies in CRC beyond RAS pathway mutations.

By knocking out all four EGFR ligands expressed in MDCK cells, this study shows the redundant and specific roles of each EGFR ligand in the ERK activation waves during collective cell migration., Epidermal growth factor receptor (EGFR) plays a pivotal role in collective cell migration by mediating cell-to-cell propagation of extracellular signal-regulated kinase (ERK) activation. Here, we aimed to determine which EGFR ligands mediate the ERK activation waves. We found that epidermal growth factor (EGF)–deficient cells exhibited lower basal ERK activity than the cells deficient in heparin-binding EGF (HBEGF), transforming growth factor alpha (TGFα) or epiregulin (EREG), but all cell lines deficient in a single EGFR ligand retained the ERK activation waves. Surprisingly, ERK activation waves were markedly suppressed, albeit incompletely, only when all four EGFR ligands were knocked out. Re-expression of the EGFR ligands revealed that all but HBEGF could restore the ERK activation waves. Aiming at complete elimination of the ERK activation waves, we further attempted to knockout NRG1, a ligand for ErbB3 and ErbB4, and found that NRG1-deficiency induced growth arrest in the absence of all four EGFR ligand genes. Collectively, these results showed that EGFR ligands exhibit remarkable redundancy in the propagation of ERK activation waves during collective cell migration.

A disintegrin and metalloproteinase 17 (Adam17), also known as TNFα-converting enzyme (Tace), is a membrane-anchored protein involved in shedding of TNF, IL-6 receptor, ligands of epidermal growth factor receptor (EGFR), and Notch receptor. This study aimed to examine the role of Adam17 in adult articular cartilage and osteoarthritis (OA) pathophysiology. Adam17 expression was examined in mouse knee joints during OA development. We analyzed OA development in tamoxifen-inducible chondrocyte-specific Adam17 knockout mice of a resection of the medial meniscus and medial collateral ligament (medial) model, destabilization of the medial meniscus (DMM) model, and aging model. We analyzed downstream pathways by in vitro experiments, and further performed intra-articular administration of an Adam17 inhibitor TAPI-0 for surgically induced mouse OA. Adam17 expression in mouse articular cartilage was increased by OA progression. In all models, Adam17 knockout mice showed ameliorated progression of articular cartilage degradation. Adam17 knockout decreased matrix metallopeptidase 13 (Mmp13) expression in both in vivo and in vitro experiments, whereas Adam17 activation by phorbol-12-myristate-13-acetate (PMA) increased Mmp13 and decreased aggrecan in mouse primary chondrocytes. Adam17 activation enhanced release of soluble TNF and transforming growth factor alpha, a representative EGF ligand, from mouse primary chondrocytes, while it did not change release of soluble IL-6 receptor or nuclear translocation of Notch1 intercellular domain. Intra-articular administration of the Adam17 inhibitor ameliorated OA progression. This study demonstrates regulation of OA development by Adam17, involvement of EGFR and TNF pathways, and the possibility of Adam17 as a therapeutic target for OA.

Early growth response-1 (EGR1) is a transcription factor correlated with prostate cancer (PC) progression in a variety of contexts. For example, EGR1 levels increase in response to suppressed androgen receptor signaling or loss of the tumor suppressor, PTEN. EGR1 has been shown to regulate genes influencing proliferation, apoptosis, immune cell activation, and matrix degradation, among others. Despite this, the impact of EGR1 on PC metastatic colonization is unclear. We demonstrate using a PC model (DU145/RasB1) of bone and brain metastasis that EGR1 expression regulates angiogenic and osteoclastogenic properties of metastases. We have shown previously that FN14 (TNFRSF12A) and downstream NF-κB signaling is required for metastasis in this model. Here we demonstrate that FN14 ligation also leads to NF-κB-independent, MEK-dependent EGR1 expression. EGR1-depletion in DU145/RasB1 cells reduced both the number and size of metastases but did not affect primary tumor growth. Decreased EGR1 expression led to reduced blood vessel density in brain and bone metastases as well as decreased osteolytic bone lesion area and reduced numbers of osteoclasts at the bone-tumor interface. TWEAK (TNFSF12) induced several EGR1-dependent angiogenic and osteoclastogenic factors (e.g., PDGFA, TGFB1, SPP1, IL6, IL8, and TGFA, among others). Consistent with this, in clinical samples of PC, the level of several genes encoding angiogenic/osteoclastogenic pathway effectors correlated with EGR1 levels. Thus, we show here that EGR1 has a direct effect on prostate cancer metastases. EGR1 regulates angiogenic and osteoclastogenic factors, informing the underlying signaling networks that impact autonomous and microenvironmental mechanisms of cancer metastases.

Lipofibromatosis is a rare pediatric soft tissue tumor with predilection for the hands and feet. Previously considered to represent "infantile fibromatosis", lipofibromatosis has distinctive morphological features, with mature adipose tissue, short fascicles of bland fibroblastic cells, and lipoblast-like cells. Very little is known about the genetic underpinnings of lipofibromatosis. Prompted by our finding of the FN1-EGF gene fusion, previously shown to be a characteristic feature of calcifying aponeurotic fibroma (CAF), in a morphologically typical case of lipofibromatosis that recurred showing features of CAF, we studied a cohort of 20 cases of lipofibromatosis for this and other genetic events. The cohort was composed of 14 males and 6 females (median age 3 years; range 1 month-14 years). All primary tumors showed classical lipofibromatosis morphology. Follow-up disclosed three local recurrences, two of which contained calcifying aponeurotic fibroma-like nodular calcifications in addition to areas of classic lipofibromatosis, and no metastases. By FISH and RNA sequencing, four cases were positive for FN1-EGF and one case each showed an EGR1-GRIA1, TPR-ROS1, SPARC-PDGFRB, FN1-TGFA, EGFR-BRAF, VCL-RET, or HBEGF-RBM27 fusion. FN1-EGF was the only recurrent fusion, suggesting that some cases of "lipofibromatosis" may represent calcifying aponeurotic fibroma lacking hallmark calcifications. Several of the genes involved in fusions (BRAF, EGFR, PDGFRB, RET, and ROS1) encode receptor tyrosine kinases (RTK), or ligands to the RTK EGFR (EGF, HBEGF, TGFA), suggesting a shared deregulation of the PI3K-AKT-mTOR pathway in a large subset of lipofibromatosis cases.

Epidermal growth factor receptor (EGFR) plays a pivotal role in collective cell migration by mediating cell-to-cell propagation of extracellular signal-regulated kinase (ERK) activation. Here, we aimed to determine which EGFR ligands mediate the ERK activation waves by gene knockout. Four of the seven known EGFR ligands are expressed in MDCK cells. We found that epidermal growth factor (EGF)-deficient cells exhibited lower basal ERK activity than the cells deficient in heparin-binding EGF (HBEGF), transforming growth factor alpha (TGFα) or epiregulin (EREG), but all cell lines deficient in a single EGFR ligand retained the ERK activation waves. Therefore, we knocked out the EGFR ligand genes in decreasing order of expression. ERK activation waves were markedly suppressed, albeit incompletely, only when all four EGFR ligands were knocked-out. Re-expression of the EGFR ligands revealed that all but HBEGF could restore the ERK activation waves. Aiming at complete elimination of the ERK activation waves, we further attempted to knockout Nrg1, a ligand for ErbB3 and ErbB4, and found that Nrg1 deficiency induced growth arrest in the absence of all four EGFR ligand genes expressed in MDCK cells. Collectively, these results showed that EGFR ligands exhibit remarkable redundancy in the propagation of ERK activation waves during collective cell migration.

Preclinical studies conducted to date suggest that depression could be elicited by the elevated expression of proinflammatory molecules: these play a key role in the mediation of neurochemical, neuroendocrine and behavioral changes. Thus, this study investigates the effect of chronic mild stress (CMS) and administration of venlafaxine (SSRI) on the expression and methylation status of new target inflammatory genes: TGFA, TGFB, IRF1, PTGS2 and IKBKB, in peripheral blood mononuclear cells (PMBCs) and in selected brain structures of rats. Adult male Wistar rats were subjected to the CMS and further divided into matched subgroups to receive vehicle or venlafaxine. TaqMan gene expression assay and methylation-sensitive high-resolution melting (MS-HRM) were used to evaluate the expression of the genes and the methylation status of their promoters, respectively. Our results indicate that both CMS and chronic treatment with venlafaxine were associated with changes in expression of the studied genes and their promoter methylation status in PMBCs and the brain. Moreover, the effect of antidepressant administration clearly differed between brain structures. Summarizing, our results confirm at least a partial association between TGFA, TGFB, IRF1, PTGS2 and IKBKB and depressive disorders.

Background Epithelial-mesenchymal transition (EMT) is an important characteristic in the remodeling of airways that occurs in chronic obstructive pulmonary disease (COPD). Cigarette smoke is a potential driving factor of this EMT in COPD. However, the mechanisms by which cigarette smoke induce EMT remain uncertain. Cathelicidin has been implicated as a causal factor of airway inflammation and mucus hypersecretion in smoking-related COPD. This study aimed to investigate whether cathelicidin induces EMT to promote airway remodeling in this disease. Methods Human lung tissue was collected from smokers with COPD and smokers without COPD. The EMT markers E-cadherin and vimentin were examined by immunohistochemistry. Mouse models of COPD were established by taking mice with airway cathelin-related antimicrobial peptide (CRAMP), the murine homologue of cathelicidin, either upregulated or downregulated by intranasal introduction of lentiviral vectors and then exposing them to cigarette smoke. E-cadherin and vimentin expression in the airways of the model mice was examined using immunofluorescence. Tumor necrosis factor alpha (TNF-α) converting enzyme (TACE), transforming growth factor alpha (TGF-α), and epidermal growth factor receptor (EGFR) expression was analyzed by Western blot. Additionally, NCI-H292 human airway epithelial cells, both with and without cathelicidin downregulation, were stimulated with cigarette smoke extract (CSE) and LL-37 synthetic peptide, a bioactive fragment of cathelicidin. This was done to confirm that the TACE/TGF-α/EGFR signaling pathway is activated in humans exposed to cigarette smoke. Results Significant EMT was found in the small airways of smokers both with and without COPD, as well as in the airways of COPD model mice. Downregulation of CRAMP in COPD mice, however, ameliorated airway EMT induced by cigarette smoke. Conversely, upregulation of CRAMP enhanced airway EMT in vivo; TACE, TGF-α, and EGFR were found to be involved in this process. In vitro, EMT induced by CSE and LL-37 was inhibited by blocking TACE, TGF-α, and EGFR expression. Conclusions Cathelicidin promotes airway EMT by activating the TACE/TGF-α/EGFR signaling pathway. This mediates smoking-induced airway remodeling in the pathogenesis of COPD.

Emerging evidence indicates that microRNAs (miRNAs) play a critical role in breast cancer development. We recently reported that a higher expression of miR-374b in tumor tissues was associated with a better disease-free survival of triple-negative breast cancer (TNBC). However, the functional significance and molecular mechanisms underlying the role of miR-374b in breast cancer are largely unknown. In this current study, we evaluated the biological functions and potential mechanisms of miR-374b in both TNBC and non-TNBC. We found that miR-374b was significantly downregulated in breast cancer tissues, compared to adjacent tissues. MiR-374b levels were also lower in breast cancer cell lines, as compared to breast epithelial cells. In vitro and in vivo studies demonstrated that miR-374b modulates the malignant behavior of breast cancer cells, such as cell proliferation in 2D and 3D, cell invasion ability, colony-forming ability and tumor growth in mice. By using bioinformatics tools, we predicted that miR-374b plays a role in breast cancer cells through negatively regulating cyclin D1 (CCND1) and transforming growth factor alpha (TGFA). We further confirmed that CCND1 and TGFA contribute to the malignant behavior of breast cancer cells in vitro and in vivo. Our rescue experiments showed that overexpressing CCND1 or TGFA reverses the phenotypes caused by miR-374b overexpression. Taken together, our studies suggest that miR-374b modulates malignant behavior of breast cancer cells by negatively regulating CCND1 and TGFA genes. The newly identified miR-374b-mediated CCND1 and TGFA gene silencing may facilitate a better understanding of the molecular mechanisms of breast cancer progression.

Despite the recent introduction of new effective systemic agents, the survival of patients with hepatocellular carcinoma (HCC) at advanced stages remains dismal. This underscores the need for new therapies, which has spurred extensive research on the identification of the main drivers of pathway de-regulation as a source of novel therapeutic targets. Frequently altered pathways in HCC involve growth factor receptors (e.g., VEGFR, FGFR, TGFA, EGFR, IGFR) and/or its cytoplasmic intermediates (e.g., PI3K-AKT-mTOR, RAF/ERK/MAPK) as well as key pathways in cell differentiation (e.g., Wnt/β-catenin, JAK/STAT, Hippo, Hedgehog, Notch). Somatic mutations, chromosomal aberrations and epigenetic changes are common mechanisms for pathway deregulation in HCC. Aberrant pathway activation has also been explored as a biomarker to predict response to specific therapies, but currently, these strategies are not implemented when deciding systemic therapies in HCC patients. Beyond the well-established molecular cascades, there are numerous emerging signaling pathways also deregulated in HCC (e.g., tumor microenvironment, non-coding RNA, intestinal microbiota), which have opened new avenues for therapeutic exploration.

ObjectiveTo evaluate the aryl hydrocarbon receptor (AHR)-dependent transforming growth factor alpha (TGF-α)/vascular endothelial growth factor B (VEGF-B) ratio, which regulates the effects of metabolic, dietary, and microbial factors on acute and chronic CNS inflammation, as a potential marker in multiple sclerosis (MS).MethodsTGF-α, VEGF-B, and AHR agonistic activity were determined in serum of 252 patients with relapsing-remitting (RR) MS, primary and secondary progressive MS, as well as during active disease (clinically isolated syndrome [CIS] and RRMS relapse).ResultsThe TGF-α/VEGF-B ratio and AHR agonistic activity were decreased in all MS subgroups with a stable disease course as compared to controls. During active CNS inflammation in CIS and RRMS relapse, the TGF-α/VEGF-B ratio and AHR agonistic activity were increased. Conversely, in patients with minimal clinical impairment despite long-standing disease, the TGF-α/VEGF-B ratio and AHR agonistic activity were unaltered. Finally, the TGF-α/VEGF-B ratio and AHR agonistic activity correlated with neurologic impairment and time to conversion from CIS to MS.ConclusionsThe AHR-dependent TGF-α/VEGF-B ratio is altered in a subtype, severity, and disease activity–specific manner and correlates with time to conversion from CIS to MS. It may thus represent a novel marker and serve as additive guideline for immunomodulatory strategies in MS.Classification of EvidenceThis study provides Class III evidence that serum levels of AHR, TGF-α, and VEGF-B distinguish subtypes of MS and predict the severity and disease activity of MS.

The anticancer effects of arazyme, a bacterial metalloprotease, have been revealed in previous studies. Because of the overexpression of epidermal growth factor receptor (EGFR) in tumor cells, targeting this receptor is one of the approaches to cancer therapy. In the present study, we designed fusion protein by using a non-mitogenic binding sequence of TGFα, arazyme, and a suitable linker. The I-TASSER and Robetta web servers were employed to predict the territory structures of the Arazyme-linker-TGFαL3, and TGFαL3-linker-Arazyme. Then, models were validated by using PROCHECK, ERRAT, ProSA, and MolProbity web servers. After docking to EGFR, Arazyme-linker-TGFαL3 showed a higher binding affinity and was selected to be optimized through 100 ns Molecular dynamic (MD) simulation. Next, the stability of ligand-bound receptor was examined utilizing MD simulation for 100 ns. Furthermore, the binding free energy calculation and free energy decomposition were carried out employing MM-PBSA and MM-GBSA methods, respectively. The root mean square deviation and fluctuation (RMSD, RMSF), the radius of gyration, H-bond, and binding free energy analysis revealed the stability of the complex during simulation time. Finally, linear and conformational epitopes of B cells, as well as MHC class I and MHC class II were predicted by using different web servers. Meanwhile, the potential B cell and T cell epitopes were identified throughout arazyme protein sequence. Collectively, this study suggests a novel chimera protein candidate prevent cancer cells potentially by inducing an immune response and inhibiting cell proliferation. Communicated by Ramaswamy H. Sarma.

A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with nonsmall cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and crossphosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF beta-chain-Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling.

A small library of phorbol 12,13-diesters bearing low lipophilicity ester chains was prepared as potential neurogenic agents in the adult brain. They were also used in a targeted UHPLC-HRMS screening of the latex of Euphorbia resinifera. Two new 12-deoxy-16-hydroxyphorbol 13,16-diesters were isolated, and their structures were deduced using two-dimensional NMR spectroscopy and NOE experiments. The ability of natural and synthetic compounds to stimulate transforming growth factor alpha (TFG alpha) release, to increase neural progenitor cell proliferation, and to stimulate neurogenesis was evaluated. All compounds that facilitated TGF alpha release promoted neural progenitor cell proliferation. The presence of two acyloxy moieties on the tigliane skeleton led to higher levels of activity, which decreased when a free hydroxyl group was at C-12. Remarkably, the compound bearing isobutyryloxy groups was the most potent on the TGF alpha assay and at inducing neural progenitor cell proliferation in vitro, also leading to enhanced neurogenesis in vivo when administered intranasally to mice., This work was supported by the Spanish Ministerio de Ciencia, Innovacion y Universidades (grant nos RTI2018-099908-BC21 and RTI2018-099908-B-C22 MICINN/FEDER granted to CC and RHG, respectively) and Consejeria de Economia, Conocimiento, Empresas y Universidades Junta de Andalucia (grant no FEDER-ANDALUCIA sol2018-00106647-tra). We thank the "Servicio de experimentacion y produccion animal (SEPA) de la Universidad de Cadiz" as well as the "Servicios Centrales de apoyo a la investigacion en Ciencias de la Salud (SCICS) de la Universidad de Cadiz" and "Servicios centrales de Ciencia Cadiz".

Introduction: Epidermal growth factor (EGF) receptors (EGFRs: ERBB1-4) are activated by a family of ligands (e.g. EGF, Hb-EGF, EREG, TGFa), signaling through ERK1/2 and Akt to promote cell division and cancer. Antibody-based inhibition of ERBB2 in breast cancer can cause heart failure, but the role of other receptors and EGFR ligands in the heart, and potential cardiotoxicity of generic EGFR inhibitors is unclear. Hypothesis: We hypothesize that EGFR ligands play an important role in cardiac adaptation to hypertension, acting through EGFRs to promote adaptive remodelling. Methods & Results: EGF ligand/receptor mRNA expression was assessed in human failing hearts and normal controls (n=12/8). EGFRs were expressed at similar levels, but ligand expression differed with significant up- or downregulation of EGF/Hb-EGF vs EREG/TGFa, respectively, in failing hearts (pin vivo , C57Bl/6J mice (n=6) were treated with 0.8 mg/kg/d angiotensin II (AngII; 7d) ± 0.45 mg/kg/d afatinib. AngII promoted cardiac hypertrophy with increased left ventricular (LV) wall thickness (WT) and decreased LV internal diameter (ID; assessed by echocardiography). Afatinib enhanced AngII-induced hypertrophy with significantly increased WT:ID ratios (1.30-fold and 1.54-fold in diastole and systole, respectively; pNppb mRNA expression and cardiomyocyte cross-sectional area (208.80±9.78 vs 161.10±3.87μm 2 ; pCol1a1 mRNA expression was enhanced by afatinib, along with interstitial and perivascular fibrosis (3.21±0.38 vs 5.61±0.46, 0.98±0.06 vs 1.45±0.18 % area; p Conclusion: EGFR signaling is modulated in human heart failure, promotes cardiomyocyte hypertrophy and is required for cardiac adaptation to hypertension. Since EGFR inhibition in hypertension prevents adaptive cardiomyocyte hypertrophy whilst promoting fibrosis, EGFR inhibitors are likely to cause cardiac dysfunction and be cardiotoxic in hypertensive patients.

Background: Chronic leg ulcerations are associated with Haemoglobin disorders, Type2 Diabetes Mellitus, and long-term venous insufficiency, where poor perfusion and altered metabolism develop into a chronic inflammation that impairs wound closure. Skin equivalent organotypic cultures can be engineered in vitro to study skin biology and wound closure by modelling the specific cellular components of the skin. This study aimed to develop a novel bioactive platelet-rich plasma (PRP) leukocyte depleted scaffold to facilitate the study of common clinical skin wounds in patients with poor chronic skin perfusion and low leukocyte infiltration. A scratch assay was performed on the skin model to mimic two skin wound conditions, an untreated condition and a condition treated with recombinant tumour necrotic factor (rTNF) to imitate the stimulation of an inflammatory state. Gene expression of IL8 and TGFA was analysed in both conditions. Statistical analysis was done through ANOVA and paired student t-test. P, Results: A skin model that consisted of a leukocyte-depleted, platelet-rich plasma scaffold was setup with embedded fibroblasts as dermal equivalents and seeded keratinocytes as multi-layered epidermis. Gene expression levels of IL8 and TGFA were significantly different between the control and scratched conditions (p, peer-reviewed

Objectives To study the expression of growth factors in the regulation of tissue repair after peritoneal damage tissue response to peritoneal damage. Methods Experimental study in 35 male Wistar rats determining the evolution over time of the tissue response to aseptic peritoneal damage. A standardized bowel and peritoneal lesions were created in the right lower quadrant by laparotomy. Then, tissular expression of growth factors was evaluated by multiplex polymerase chain reaction at seven timepoints between 6 h and 30 days, postoperatively. Results Tissular responses of granulocyte-stimulating factors (Csf2, Csf3), connective tissue growth factor (Ctgf), epidermal growth factors and receptor (Egf, Egfr), fibroblast growth factors (Fgf2, 7 and 10), heparin binding EGF-like growth factor (Hbegf), hepatocyte growth factor (Hgf), insulin-like growth factor-1 (Igf1), mitogenic transforming growth factors (Tgfa, Tgfb1, Tgfbr3), and vascular endothelial growth factor A (Vegfa) were biphasic with a first expression peak at day 3, followed by a more pronounced peak at day 14. Conclusions We observed a long-lasting, widespread response of tissular growth factors for at least two weeks after peritoneal damage. To be clinically effective, the prophylaxis of postoperative adhesions might be needed for an extended period of time.

Background Pancreatic adenocarcinoma (PAAD) is a pancreatic disease with a high mortality rate in the world. This present research intends to identify the function of lncRNA LINC00857/miR-340-5p/Transforming growth factor alpha (TGFA) in the progression of PAAD. Methods Bioinformatics analysis was used to explore the differentially expressed lncRNA/miRNA/mRNA and analyze the relationship between lncRNA/miRNA/mRNA expression and prognosis of PAAD by enquiring TCGA, GEO and GTEX. KEGG pathway analysis and GO enrichment analysis were implemented to annotate the crucial genes regulated by LINC00857. The biological behaviors of PAAD cells were detected by CCK-8, colony formation and transwell assays. Interactive associations between LINC00857 and miR-340-5p, as well as miR-340-5p and TGFA were analyzed by dual luciferase assay. Results By enquiring TCGA database, we got that LINC00857 was highly expressed in patients with PAAD and positively associated with worse prognosis in PAAD patients. Moreover, LINC00857 upregulation promoted the proliferation and clone formation abilities of PAAD cells. Afterwards, the downstream miRNA and mRNA targets of LINC00857 were picked up to construct a ceRNA network. Further study revealed that TGFA expression was positively regulated by LINC00857 and negatively regulated by miR-340-5p. Besides that, the inhibitory effect of miR-340-5p on PAAD cells growth and movement can be blocked by LINC00857 upregulation. While, the malignant behavior of PAAD cells induced by TGFA overexpression can be eliminated by LINC00857 knockdown. Conclusions Upregulation of LINC00857 improved growth, invasion and migration abilities of PAAD cells by modulation of miR-340-5p/TGFA, affording potential targets and biomarkers for the clinical diagnosis and treatment.

BACKGROUND: Thyroid hormones are key regulators of essential cellular processes including proliferation, differentiation, and finally apoptosis. AIM: The aim of study was to detect changes in the expression of apoptosis and cell cycle pathway-focused genes in patients with different forms of thyroid pathology. PATIENTS AND METHODS: 36 patients with thyroid pathology were enrolled in the study. We used the pathway-specific real-time PCR array (Neurotrophins and Receptors RT2 Profiler PCR Array, QIAGEN, Germany) to identify and verify apoptosis and cell cycle pathway-focused genes expression in patients with postoperative hypothyroidism, hypothyroidism as a result of autoimmune thyroiditis (AIT) and AIT with elevated serum an anti-thyroglobulin (anti-Tg) and anti-thyroid peroxidase (anti-TPO) antibodies. RESULTS: It was shown that patients with elevated serum anti-Tg and anti-TPO antibodies and with hypothyroidism resulting from AIT had a significantly lower expression of FAS, TGFB, TP53, TGFA, while the expression of CD40 was increased. The mRNA levels of BCL2 and BAX decreased in the patients with elevated serum anti-Tg and anti-TPO antibodies and increased in the patients with hypothyroidism resulting from AIT and postoperative hypothyroidism. The patients with hypothyroidism resulting from AIT and postoperative hypothyroidism had significantly lower expression of HSPB1. NF1 expression did not change in all groups of patients. CONCLUSION: The results of this study demonstrate that AIT and hypothyroidism affect the mRNA-level expression of apoptosis and cell cycle pathway-focused genes in gene specific manner and that these changes to gene expression can be responsible for the apoptosis signs and symptoms associated with thyroid pathology.

Human papillomaviruses (HPV) are causative agents of various tumours such as cervical cancer. HPV binding to the cell surface of keratinocytes leads to virus endocytosis at tetraspanin enriched microdomains. Complex interactions of the capsid proteins with host proteins as well as ADAM17-dependent ERK1/2 signal transduction enable the entry platform assembly of the oncogenic HPV type 16. Here, we studied the importance of tetraspanin CD9, also known as TSPAN29, in HPV16 infection of different epithelial cells. We found that both overexpression and loss of the tetraspanin decreased infection rates in cells with low endogenous CD9 levels, while reduction of CD9 expression in keratinocytes that exhibit high-CD9 protein amounts, led to an increase of infection. Therefore, we concluded that low-CD9 supports infection. Moreover, we found that changes in CD9 amounts affect the shedding of the ADAM17 substrate transforming growth factor alpha (TGFα) and the downstream phosphorylation of ERK. These effects correlate with those on infection rates suggesting that a specific CD9 optimum promotes ADAM17 activity, ERK signalling and virus infection. Together, our findings implicate that CD9 regulates HPV16 infection through the modulation of ADAM17 sheddase activity., German Research Foundation (Deutsche Forschungsgemeinschaft, DFG FL 696/3-1 for LF and DFG Project Number 125440785-SFB877 (A4) for KR). Johannes Strunk gratefully acknowledges financial support from the Max Planck Graduate Center with the University of Mainz

Enhanced expression of NADPH oxidase (NOX) and the subsequent production of reactive oxygen species (ROS) are associated with lung cancer. In the present study, fifty 6-amino-2,4,5-trimethylpyridin-3-ol derivatives were screened for anticancer activity by targeting NOX2-derived ROS. The compounds suppressed ROS production and decreased cancer cell viability (R2 = 0.79). Among the derivatives, the compound coded BJ-1207, which contained a 4-(hydroxydiphenylmethyl)piperidine moiety, exhibited the most effective anticancer activity against A549 lung cancer cell line and eight other cancer cell lines, including H1299, MCF-7, MDA-MB-231, HT-29, SW620, Mia PaCa-2, PANC-1, and U937. BJ-1207 also showed significantly lower inhibitory effects on kinase insert domain receptor (KDR) and c-KIT tyrosine kinase but higher inhibitory activity on NOX than those of sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor. In addition, BJ-1207-induced inhibition of RTK-downstream signaling pathways, such as ROS production, and expression of target genes, such as stem cell factor and transforming growth factor-α, were similar to those induced by sunitinib. In the xenograft chick tumor model, BJ-1207 inhibited lung tumor growth to a similar or much greater extent than that of sunitinib or cisplatin, respectively. Overall, the present study showed that BJ-1207, a vitamin B6-derived 2,4,5-trimethylpyridin-3-ol compound with azacyclonol moiety at C (6)-position of the pyridine ring, inhibited NOX activity and that it is a promising lead compound for developing anticancer drugs against lung cancer.

AimsOur aim was to examine longitudinal associations of triacylglyceride fatty acid (TGFA) composition with insulin sensitivity (IS) and beta-cell function.MethodsAdults at-risk for T2D (n=477) had glucose and insulin measured from a glucose challenge at 3 time points over 6 years. The outcome variables Matsuda index (ISI), HOMA2-%S, Insulinogenic Index over HOMA-IR (IGI/IR), and Insulin Secretion-Sensitivity Index-2 (ISSI-2) were computed from the glucose challenge. Gas chromatography quantified TGFA composition from the baseline. We used adjusted generalized estimating equations (GEE) models and partial least squares (PLS) regression for the analysis.ResultsIn adjusted GEE models, four TGFA (14:0, 16:0, 14:1n-7, 16:1n-7 as mol%) had strong negative associations with IS while others (e.g. 18:1n-7, 18:1n-9, 20:2n-6, 20:5n-3) had strong positive associations. Few associations were seen for beta-cell function, except for 16:0, 18:1n-7, and 20:2n-6. PLS analysis indicated four TGFA (14:0, 16:0, 14:1n-7, 16:1n-7) that clustered together and strongly related with lower IS. These four TGFA also correlated highly (r>0.4) with clinically measured TG.ConclusionsWe found that higher proportions of a cluster of four TGFA strongly related with lower IS as well as hypertriglyceridemia, suggesting only a few fatty acids within the TGFA composition may primarily explain lipids’ role in glucose dysregulation.

Objective Colorectal cancer (CRC) is the 3rd most common cancer worldwide. Recently, long non-coding RNAs (lncRNAs) were found to be critical modulators in the CRC progression. The aim of this study is to investigate the potential roles of lncRNA P73 antisense RNA 1T (TP73-AS1) in CRC development and progression. Methods Quantitative real-time PCR (qRT-PCR) was performed to determine relevant gene expression levels; western blot was performed to determine protein expression levels; CCK-8, colony formation, wound healing and Transwell invasion assays were used to determined CRC cell proliferation, migration and invasion; in vivo tumor growth was assessed in xenograft mice model. Results TP73-AS1 was up-regulated in both CRC tissues and CRC cell lines. Overexpression of TP73-AS1 was associated with metastasis and advanced clinical stages in CRC patients. Overexpression of TP73-AS1 promoted CRC cell growth, proliferation, migration and invasion in vitro; and knockdown of TP73-AS1 significantly inhibited CRC cell growth, proliferation, migration and invasion in vitro as well as tumor growth in vivo. Bioinformatics analysis and luciferase reporter assay indicated that TP73-AS1 could bind directly with miR-194, and TP73-AS1 negatively regulated the expression of miR-194 in CRC cells. Further study indicated that miR-194 negatively regulated the downstream target of transforming growth factor alpha (TGFα) via targeting its 3' untranslated region, and TP73-AS1 positively regulated the expression of TGFα in CRC cells. Moreover, overexpression of miR-194 suppressed CRC cell proliferation and invasion, and attenuated the effects of TP73-AS1 overexpression on CRC cell proliferation and invasion. Silence of TGFα inhibited CRC cell proliferation and invasion, and also reversed the effects of TP73-AS1 overexpression on CRC cell proliferation and invasion. Conclusions this study demonstrated that TP73-AS1 regulated CRC progression by acting as a competitive endogenous RNA to sponge miR-194 to modulate the expression of TGFα.

Background Cleft lip with or without cleft palate (CL/P) is one of the most common congenital defects, which etiology involves both genetic and environmental factors. Previous studies have shown that miR-199a-5p may mediate the occurrence of CL/P. However, the key target genes regulated by miR-199a-5p are not clear. In this study, we employed a systematic bioinformatics analysis of target genes regulated by miR-199a-5p which may be involved in CL/P. Methods The miRBase, Human miRNA tissue atlas, miRecords, miRpathDB, miRWalk, miRTarBase, DIANA-TarBase (v7.0), Literature search, DAVID software, Cytoscape plugin ClueGO + Cluepedia app, MalaCards, TargetScanhuman7.1, Venny 2.1, STRING and GEO databases were comprehensive employed to identify the key genes regulated by miR-199a-5p associated with CL/P. Results Total 429 experimentally validated target genes were obtained from five miRNAs related databases. Expressions of miR-199a-5p and its experimentally validated target genes were elevated in bone, brain and skin. KEGG pathway analysis revealed that the target genes were enriched in focal adhesion, microRNAs in cancer and hippo signaling pathway. Biological process categorization revealed that significant portions of the target genes were grouped as transcription, DNA-templated. Total eight intersection genes were identified by using MalaCards and TargetScanhuman7.1. The target gene transforming growth factor alpha (TGFA) of miR-199a-5p involved in CL/P is screened and verified. Conclusion MiR-199a-5p may mediate CL/P by regulating key target gene TGFA. The study may contribute to a better understanding of the etiology of CL/P.

Triple-negative breast cancer (TNBC) is aggressive and typically has a poor prognosis. Chemokines have chemoattractant potential for cancer metastasis. Here, we investigated the chemokine signatures in BC subtypes and the underlying mechanisms that enhance proinflammatory chemokines in TNBC. Analysis from microarray dataset revealed that basal-like BC subtype including TNBC expressed dominantly proinflammatory chemokines, such as CXCL1 and 8, compared to non-TNBC. Chemokine PCR array confirmed the dominant chemokines in TNBC cells. To identify a driving factor for proinflammatory chemokines in TNBC cells, we determined the expression and signaling profiles of epidermal growth factor receptor (EGFR) family members. TNBC cells expressed higher levels of EGFR and phosphorylated Akt/Erk than non-TNBC cells. In addition, EGF further enhanced the proinflammatory chemokines in TNBC cells, including CXCL2. Knockdown of Akt reduced the CXCL2 promoter activity, while overexpression of Akt enhanced it. MK2206, an Akt inhibitor, reduced the CXCL2 promoter activity, while inhibition and knockdown of Erk did not reduce its activity. We found that transforming growth factor alpha (TGFα) could serve as a main ligand for EGFR to drive EGFR-mediated Akt activation in TNBC cells. MK2206 decreased TGFα promoter activity, while overexpression of Akt increased it. MK2206 also reduced TGFα release from TNBC cells. Moreover, MK2206 downregulated CXCL2 mRNA expression, while TGFα upregulated it. Taken together, the TGFα-EGFR-Akt signaling axis can play a role in enhancing proinflammatory chemokine expression in TNBC, subsequently contributing to the inflammatory burden that ultimately lead to cancer progression and a higher mortality rate among TNBC patients.

Background: A series of epidemiological studies were conducted to investigate the association between transforming growth factor alpha ( TGFA) polymorphism and nonsyndromic cleft lip and/or palate (CL/P) risk, but the findings remain conflicting. The present meta-analysis summarizes the association between the TGFA Taq I polymorphisms and nonsyndromic CL/P risk. Methods: We searched PubMed, EMBASE, Web of Science, and Chinese Biomedical Literature databases from their inception to May 1, 2015. Fixed-effects or random-effects models were used to calculate the pooled odds ratio for two genetic comparisons (heterozygous mutation versus wild type, homozygous mutation versus wild type). All of the statistical tests were conducted by STATA 10.0 (StataCorp, College Station, TX). Results: A total of 26 case-control studies were identified for this meta-analysis. There was evidence of a significant association between the TGFA Taq I polymorphism and nonsyndromic CL/P risk in the overall population. The TGFA polymorphism was associated with nonsyndromic CL/P susceptibility in Asian populations under any of genetic models. However, in subgroup analysis, we did not find a significant association of TGFA gene polymorphisms with a reduced cancer risk in White and other populations and (recessive model, odds ratio = 2.37, 95% confidence intervals = 0.92-6.07; odds ratio = 3.45, 95% confidence intervals = 1.07-11.09, respectively). Conclusion: Our study indicates that the TGFA gene polymorphism might be associated with nonsyndromic CL/P susceptibility. However, these findings still need to be confirmed by single, large, well-designed prospective studies.

Degenerative disk disease of the spine is a major cause of back pain and disability. Optimization of regenerative medical therapies for degenerative disk disease requires a deep mechanistic understanding of the factors controlling the structural integrity of spinal tissues. In this investigation, we sought to identify candidate regulatory genes controlling extracellular matrix synthesis in spinal tissues. To achieve this goal we performed high throughput next generation RNA sequencing on 39 annulus fibrosus and 21 nucleus pulposus human tissue samples. Specimens were collected from patients undergoing surgical discectomy for the treatment of degenerative disk disease. Our studies identified associations between extracellular matrix genes, growth factors, and other important regulatory molecules. The fibrous matrix characteristic of annulus fibrosus was associated with expression of the growth factors platelet derived growth factor beta (PDGFB), vascular endothelial growth factor C (VEGFC), and fibroblast growth factor 9 (FGF9). Additionally we observed high expression of multiple signaling proteins involved in the NOTCH and WNT signaling cascades. Nucleus pulposus extracellular matrix related genes were associated with the expression of numerous diffusible growth factors largely associated with the transforming growth signaling cascade, including transforming factor alpha (TGFA), inhibin alpha (INHA), inhibin beta A (INHBA), bone morphogenetic proteins (BMP2, BMP6), and others. Clinical significance this investigation provides important data on extracellular matrix gene regulatory networks in disk tissues. This information can be used to optimize pharmacologic, stem cell, and tissue engineering strategies for regeneration of the intervertebral disk and the treatment of back pain. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1356-1369, 2018.

Bone metastasis (BM) is the advanced complication of breast cancer, while bone marrow-derived mesenchymal stem cells (BMSCs) in the microenvironment unclearly contribute to cancer metastasis. This study investigated potential roles of transforming growth factor- (TGF-) α in the interaction between breast cancer and BMSCs in BM. Clinical cases of breast cancer with bone metastasis (BMBC), breast cancer without bone metastasis (Non-BM-BC), and benign fibroadenoma (Benign) were enlisted in a retrospective study. TGF-α was found obviously overexpressed in BM lesion of BMBC compared to primary lesion of both BMBC and Non-BM-BC (P<0.01), and TGF-α was higher in primary lesion of both BMBC and Non-BM-BC (P<0.01) than Benign group. Interestingly, TGF-α in nontumor tissues of both BMBC and Non-BM-BC was at a higher level than Benign group (P<0.01), and numbers of macrophages in nontumor tissues of both BMBC and Non-BM-BC (P<0.01) were higher than Benign group. Furthermore, in cultured human BMSCs, TGF-α stimulated production of procancer cytokines including IL-6, VEGF, FGF10, FGF17, and TGF-β1 in a dose-dependent manner. Thus, TGF-α in BC could potentially be an important signal of carcinogenesis and metastasis. Macrophages in the nontumor tissue of BC may not be protective but could promote cancer metastasis.

Relevance. The modern approach to prognostication of the course of renal carcinoma requires identification of the biological markers, which determine the key properties of the tumor in particular cases. The purpose of this study is to clarify the nature of expression of the molecules connected with the phenotype of the cancer stem cell (CD133, N-cadherin), TGF-α growth factor for tubular epithelium, and carboanhydrase IX (CA-IX) - one of the key molecules connected with VHL by the molecular way of carcinogenesis in cases of renal cancer. Materials and methods. We studied 61 observations of renal cell carcinoma (RCC). The immunohystochemical examination was based on a panel of antibodies, which included antibodies to TGF-α (Аbcam), N-cadgerin (M3613 clone; Dako), CD133 (Prominin-1, Biorbyt), carboanhydrase IX (CAIX; NCL-L-CAIX, Leica Biosystems). The degree of the markers' expression was evaluated by a semiquantitative method depending on the content of the positive-reaction cells in the tumor. The differences between the compared groups were identified through the use of the Mann-Whitney U test and the chi-squared test. Results. Expression of TGF-α was found in 36 observations (59%) of RCC. TGF-α was most frequently present in the papillary (100%) and the clear-cell (55.9%) versions, and only rarely in chromophobic carcinoma (23.1%). A connection was found between the expression level of this marker and the neoplastic differentiation degree (p < 0.05). Expression of N-cadgerin was met in 38 observations (62.3%), CD133 occurred in 37 observations of renal carcinoma (60.6%). N-cadgerin and CD133 were found in the clear-cell and the papillary versions of RCC. There was a connection between the expression of N-cadgerin in the tumor and its invasive potential (p < 0.05). A higher occurrence rate of CD133 expression was recorded in the patients with metastatic forms of RCC, and also in cases of sarcomatoid transformation of the tumor (p < 0.05). Expression of carboanhydrase IX (CA-IX) occurred in 35 observations (57.4%). It was found in the clear-cell version of RCC. In cases of symptomatic RCC, expression of CA-IX was more frequently met (p < 0.05). There was a consistent association between the expression of CA-IX and the tumor size, stage and invasive potential. (For citation: Pasechnik DG, Kogan MI, Akhokhov ZM. The features of expression and prognostic significance of the peptide molecules connected with the key properties of renal cell carcinoma. Urologicheskie vedomosti. 2017;7(4):5-16. doi: 10.17816/uroved745-16).

The complex and heterogeneous nature of neuroblastoma poses a great challenge to the scientific community for developing an effective therapeutic drug. Neuroblastoma tumors show relapse and resistance to available drugs and it is proving to be an obstacle for efficient treatment options. The tumor accounts for about 7% of the malignancies worldwide and is seen mostly in children around one year. Our present study aims to understand various mechanisms underlying Neuroblastoma. The gene interaction network of 92 Neuroblastoma related genes along with a total of 339 functional interactions are used for the gene interaction network construction and analysis. The clustering analysis resulted in four densely interconnected gene clusters. We further performed functional enrichment analysis and found important pathways associated with neuroblastoma in PI3K/AKT pathway, MAPK pathway, Ras pathway, Rap1 pathway, Cytokine- cytokine interaction, Neurotrophin signaling, EGFR tyrosine kinase inhibitor resistance, and VEGF signaling pathways. The predominant genes involved in the enriched pathways are ANGPT2, FGF2, NGFR, NTRK1, NTRK2, PDGFC, TGFA, TP53, VEGFB, VEGFC, BDNF, NRAS, and VEGFA. The topological parameter analysis has shown the genes with more direct interactions are considered as the hub genes and are important in the network. The genes TP53, VEGFA, BDNF, MYCN, and FGF2 with the maximum number of functional interactors, can be used as potential therapeutic targets for Neuroblastoma. The results obtained in our study will be helpful for researchers in better understanding the molecular level associations during the Neuroblastoma progression.

Hepatocytes are responsive to mitogenic effects of several ligands acting via EGFR. Studying primary cultures of rat hepatocytes, we found that, as compared to EGF, HB-EGF had a markedly higher affinity of the EGFR, while AR and TGFα had lower affinity. HB-EGF was also more potent compared to the other growth factors regarding phosphorylation of EGFR, Shc, ERK1/2 and Akt. All ligands induced phosphorylation of ErbB2, indicating receptor heterodimerization. TGFα, despite having much lower receptor affinity, was about equally potent and efficacious as HB-EGF as a stimulator of DNA synthesis. In contrast, EGF had relatively high affinity but markedly lower efficacy in stimulation of DNA synthesis. The results suggest that amplifying and/or inhibitory mechanisms may modulate the mitogenic responses downstream of the initial signalling steps, and that this may affect the effects of the EGFR ligands differentially.

Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signaling pathways and promotes tumorigenesis in melanoma and breast cancer cells. However, the contribution of STAP-2 to the behavior of other types of cancer cells is unclear. Here, we show that STAP-2 promotes tumorigenesis of prostate cancer cells through up-regulation of EGF receptor (EGFR) signaling. Tumor growth of a prostate cancer cell line, DU145, was strongly decreased by STAP-2 knockdown. EGF-induced gene expression and phosphorylation of AKT, ERK, and STAT3 were significantly decreased in STAP-2-knockdown DU145 cells. Mechanistically, we found that STAP-2 interacted with EGFR and enhanced its stability by inhibiting c-CBL-mediated EGFR ubiquitination. Our results indicate that STAP-2 promotes prostate cancer progression via facilitating EGFR activation.

BACKGROUND: The aim of this study is to evaluate the association of TGFA TaqI polymorphism with nonsyndromic cleft lip and/or palate (NSCLP) in an Iranian population. METHODS: In this case-control study, 113 children with NSCLP and 209 controls were included. Genotyping of the TaqI polymorphism was performed by polymerase chain reaction and restriction fragment length polymorphism methods. A p-value of

Objective: The aim of the study is to evaluate the effects of silencing a disintegrin and metalloproteinase 17 (ADAM17) gene expression by lentivirus‐mediated RNA interference (RNAi) in the gefitinib‐resistant lung adenocarcinoma cells, and then to explore whether the recombinant lentivirus mediated ADAM17 RNAi reversed the acquired resistance of lung adenocarcinoma to gefitinib in vitro. Methods: The gefitinib‐resistant RPC‐9 cells were established and the mutations of EGFR were detected by gene sequencing. The ADAM17 shRNA expression vectors were constructed and packaged to recombinant lentivirus. The cell proliferation viability was detected by MTT, and cellular apotosis was analyzed by flow cytometry assay. The expression levels of ADAM17, EGFR and the phosphorylated EGFR were respectively detected by reverse transcription polymerase chain reaction and western blot. TGF‐α production in the supernatant was detected by enzyme‐linked immunosorbent assay. Results: The gefitinib‐resistant RPC‐9 cells in which mutated EGFR (exon 20) carried 790T > T/M mutation were established. When the concentrations of gefitinib were less than 10μmol/L, there were no significant changes in the apoptosis and cellular proliferation of RPC‐9 with the dose‐escalation of gefitinib. The cell proliferation viability of RPC‐9 was significantly decreased by lentivirus mediated ADAM17 RNAi (P 0.05). Gefitinib had no significant effects on TGF alpha production in the supernatants (P > 0.05). Gefitinib did not inhibit EGFR expression in gefitinib‐sensitive PC‐9 and gefitinib‐resistant RPC‐9 cells (P > 0.05). The phosphorylation of EGFR in gefitinib‐sensitive PC‐9 cells was significantly inhibited by gefitinib (P 0.05). Lentivirus mediated ADAM17 RNAi significantly inhibited the mRNA and protein expression of ADAM17 in gefitinib‐resistant RPC‐9 cells (P

The epidermal growth factor receptor (EGFR) is a member of the erbB family of receptors and is overexpressed in many tumor types. A repebody is a newly designed nonantibody protein scaffold for tumor targeting that contains leucine-rich repeat modules. In this study, 3 64Cu-labeled anti-EGFR repebodies with different chelators were synthesized, and their biologic characteristics were assessed in cultured cells and tumor-bearing mice. Methods: Repebodies were synthesized with the chelators 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-N,N',N,″-triacetic acid trihydrochloride ([p-SCN-Bn]-NOTA), 2,2',2″-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA-N-hydroxysuccinimide ester), or 1-(p-isothiocyanatobenzyl)diethylenetriamine pentaacetic acid trihydrochloride ([p-SCN-Bn]-DTPA) in 1.0 M NaHCO3 buffer (pH 9.2) for 24 h. Purified NOTA-, DOTA-, and DTPA-conjugated repebody were radiolabeled with 64Cu in 0.1 M NH4OAc buffer (pH 5.5). To compare the EGFR-binding affinities of the repebodies, cellular uptake studies were performed with the human non-small cell lung cancer cell line H1650 (high expression of EGFR) and the human colon adenocarcinoma cell line SW620 (low expression of EGFR). Biodistribution and small-animal PET imaging studies were performed using H1650 tumor-bearing mice. Results: Radiochemical yields of the 64Cu-labeled repebodies were approximately 70%-80%. Cellular uptake of the NOTA-, DOTA-, and DTPA-repebodies was over 4-fold higher in H1650 cells than in SW620 cells at 1 h. The 3 repebodies had accumulated specifically in H1650 tumor-bearing nude mice by 1 h after intravenous injection and were retained for over 24 h, as measured by the percentage injected dose per gram of tissue (%ID/g). Tumor uptake of all repebodies increased from 1 to 6 h (at 1 h, 6.28, 8.46, and 6.91 %ID/g for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 9.4, 8.28, and 10.1 %ID/g, respectively). H1650 tumors were clearly visible after injection of each repebody, with high tumor-to-background ratios (at 1 h, 3.43, 4.89, and 2.38 for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 3.05, 4.36, and 2.08; at 24 h, 3.81, 4.58, and 2.86). Conclusion: The 3 64Cu-repebody complexes demonstrated specific and rapid uptake in EGFR-expressing tumors within 1 h and may have potential as novel EGFR imaging agents for PET.

Transforming growth factor (TGF)-β family proteins control cell physiology, proliferation, and growth, and direct cell differentiation, thus playing key roles in normal development and disease. The mechanisms of how TGF-β family ligands interact with heteromeric complexes of cell surface receptors to then activate Smad signaling that directs changes in gene expression are often seen as established. Even though TGF-β-induced Smad signaling may be seen as a linear signaling pathway with predictable outcomes, this pathway provides cells with a versatile means to induce different cellular responses. Fundamental questions remain as to how, at the molecular level, TGF-β and TGF-β family proteins activate the receptor complexes and induce a context-dependent diversity of cell responses. Among the areas of progress, we summarize new insights into how cells control TGF-β responsiveness by controlling the TGF-β receptors, and into the key roles and versatility of Smads in directing cell differentiation and cell fate selection.

Transforming growth factor-β (TGF-β), serine proteinases such as trypsin, and proteinase-activated receptor 2 (PAR2) promote tumor development by stimulating invasion and metastasis. Previously, we found that in cancer cells derived from pancreatic ductal adenocarcinoma (PDAC) PAR2 protein is necessary for TGF-β1-dependent cell motility. Here, we show in the same cells that, conversely, the type I TGF-β receptor activin receptor-like kinase 5 is dispensable for trypsin and PAR2 activating peptide (PAR2-AP)-induced migration. To reveal whether Gq-calcium signaling is a prerequisite for PAR2 to enhance TGF-β signaling, we investigated the effects of PAR2-APs, PAR2 mutation and PAR2 inhibitors on TGF-β1-induced migration, reporter gene activity, and Smad activation. Stimulation of cells with PAR2-AP alone failed to enhance basal or TGF-β1-induced C-terminal phosphorylation of Smad3, Smad-dependent activity of a luciferase reporter gene, and cell migration. Consistently, in complementary loss of function studies, abrogation of the PAR2-Gq-calcium signaling arm failed to suppress TGF-β1-induced cell migration, reporter gene activity, and Smad3 activation. Together, our findings suggest that the calcium-regulating motif is not required for PAR2 to synergize with TGF-β1 to promote cell motility. Additional experiments in PDAC cells revealed that PAR2 and TGF-β1 synergy may involve TGF-β1 induction of enzymes that cause autocrine cleavage/activation of PAR2, possibly through a biased signaling function. Our results suggest that although reducing PAR2 protein expression may potentially block TGF-β's prooncogenic function, inhibiting PAR2-Gq-calcium signaling alone would not be sufficient to achieve this effect.

Objectives: Angiogenic factors are proteins that can potentially be related to certain foetal chromosomal abnormalities. The goal of this study was to determine the concentrations of 60 angiogenic factors in the amniotic fluid of women carrying foetuses with Down syndrome (DS). Methods: After analysis of the karyotyping results, for the purpose of this study, we chose 12 women with foetal DS. For the control group, we selected 12 healthy patients with uncomplicated pregnancies (15-18 weeks of gestation) who delivered healthy newborns at term. To assess the concentrations of proteins in the amniotic fluid, we used a protein macroarray, which enabled the simultaneous determination of 60 angiogenic factors per sample. Results: In the amniotic fluid of women with foetal DS compared to patients with healthy foetuses, we reported significant decreases in the concentrations of 14 angiogenic factors, including leptin, angiopoietin 1 (ANG-1), angiostatin, epidermal growth factor (EGF), interleukin 1-beta (IL-1b), interleukin 4 (IL-4), interleukin 12p40 (IL-12p40), monocyte chemotactic protein 2 (MCP-2), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-9 (MMP-9), platelet endothelial cell adhesion molecule 1 (PECAM-1), transforming growth factor alpha (TGF alpha), vascular endothelial growth factor 2 (VEGFR2), and vascular endothelial growth factor 3 (VEGFR3). Conclusions: Based on our findings, we hypothesise that angiogenic factors may play roles in the pathogenesis of DS. Defining the factors' potential as biochemical factors of DS requires further investigation in a larger group of patients.

Structure-based design and synthesis of pyrimidine containing reversible epidermal growth factor receptor (EGFR) inhibitors 1a-d are reported. The compounds (1a-d) inhibited the EGFR kinase activity in vitro with IC50 range 740 nm to 3 μm. mRNA expression of EGFR downstream target genes, that is twist, c-fos and aurora were found to be altered upon treatment with compounds 1a-d. The compounds 1a-d exhibited excellent anticancer activity at low micromolar level (3.2-9 μm) in lung, colon and breast cancer cell lines. Furthermore, compounds induced the alteration in mitochondrial membrane potential and reactive oxygen species level and. Selected compound 1b was found to increase sub-G1 population indicative of cell death, the mode of cell death was apoptotic as evident from Annexin V verses propidium iodide assay. Molecular modelling further helped to investigate the binding recognition pattern of the compounds in ATP binding EGFR domain similar to erlotinib and dissimilar to WZ4002.

Transforming growth factor-alpha (TGFA) has been shown to play a role in experimental chronic kidney disease associated with nephron reduction, while its role in diabetic kidney disease (DKD) is unknown. We show here that intrarenal TGFA mRNA expression, as well as urine and serum TGFA, are increased in human DKD. We used a TGFA neutralizing antibody to determine the role of TGFA in two models of renal disease, the remnant surgical reduction model and the uninephrectomized (uniNx) db/db DKD model. In addition, the contribution of TGFA to DKD progression was examined using an adeno-associated virus approach to increase circulating TGFA in experimental DKD. In vivo blockade of TGFA attenuated kidney disease progression in both nondiabetic 129S6 nephron reduction and Type 2 diabetic uniNx db/db models, whereas overexpression of TGFA in uniNx db/db model accelerated renal disease. Therapeutic activity of the TGFA antibody was enhanced with renin angiotensin system inhibition with further improvement in renal parameters. These findings suggest a pathologic contribution of TGFA in DKD and support the possibility that therapeutic administration of neutralizing antibodies could provide a novel treatment for the disease.

Transforming growth factor (TGF)-β cytokines signal via a complex network of pathways to regulate proliferation, differentiation, adhesion, migration, and other functions in many cell types. A high percentage of colorectal tumors contain mutations that disrupt TGF-β family member signaling. We review how TGF-β family member signaling is altered during development of colorectal cancer, models of study, interaction of pathways, and potential therapeutic strategies.