Autocrine Feedback Loop Research Articles

Abstract 2484: Non-canonical Hedgehog/Gli1 signaling drives lung adenocarcinoma stem cells survival and its targeting inhibits CSC-derived tumors

Abstract Introduction: Lung Adenocarcinoma (AC) is the most frequent lung cancer histological subtype and is a leading cause of cancer-related death worldwide. Hedgehog/Gli (Hh/Gli) signaling pathway regulates lung development and its aberrant activation contributes to tumor pathogenesis and play a role in cancer stem cells (CSC) control. We investigated oncogenic Hh/Gli signaling in AC-CSC. Methods: human AC-CSC were derived from primary tumors. For in vitro studies AC-CSC were maintained in serum-free medium supplemented with EGF/bFGF. For in vivo experiments, immunocompromised mice were injected with AC-CSC. Gli1 inhibitor GANT61 was used both in vitro and in vivo (IP 40 mg/kg twice/we) treatments. Results: Using mRNA datasets and tissue microarray of Non-small Cell Lung Cancer (NCSLC) samples we found that in AC Gli1 is expressed independently of its canonical activator Smoothened (Smo) expression level. Similarly, CSC were Gli1 positive regardless of Smo expression levels. Indeed, we found that SMO regulatory region is highly methylated in AC cells where Smo mRNA and protein are low. Looking for Smo-independent regulation of GLI activity we found that NRP2/Erk and IGF1R/Erk axes regulate Gli1 in AC-CSC. Suppression of Gli1 with GANT61 in CSC resulted in impairment of cell growth and stemness features. We performed in-vivo experiments: CSC-derived xenograft from GANT61 treated mice showed impaired tumor growth, reduced proliferation and increased apoptosis. We next highlighted a tumor-stroma crosstalk. A positive autocrine oncogenic feedback loop sustains Gli1 in AC-CSC since we found that the CSC expressed NRP2 and its ligands VEGF-A and VEGF-C. A paracrine positive loop also sustain Hh/Gli signaling in AC where Cancer-Associated Fibroblasts (CAFs) harbor Hh/Gli1 canonical pathway that regulates the expression of VEGF-A, VEGF-C and IGF2 ligands of NRP2 and IGF1R respectively. Conclusion: Our findings suggest a non-canonical activation of Hh/Gli pathway in AC. In this context, GLI1 transcription factor is effectors of NRP2/Erk and/or IGF1R/Erk signaling inputs. Our results have implications for the understanding of AC development and sustain the inclusion of target therapy acting downstream of Smo level in the design of AC treatment. Citation Format: Agnese Po, Marianna Silvano, Evelina Miele, Adriana Eramo, Matilde Todaro, Carlo Capalbo, Valentina Salvati, Giovanni Sette, Danilo Cucchi, Zein M. Besharat, Gianluca Canettieri, Lucia Di Marcotullio, Isabella Screpanti, Giorgio Stassi, Ruggero De Maria, Ann Zeuner, Enrico De Smaele, Elisabetta Ferretti. Non-canonical Hedgehog/Gli1 signaling drives lung adenocarcinoma stem cells survival and its targeting inhibits CSC-derived tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2484.

The inflammatory/cancer-related IL-6/STAT3/NF-κB positive feedback loop includes AUF1 and maintains the active state of breast myofibroblasts.

The IL-6/STAT3/NF-κB positive feedback loop links inflammation to cancer and maintains cells at a transformed state. Similarly, cancer-associated myofibroblats remains active even in absence of cancer cells. However, the molecular basis of this sustained active state remains elusive. We have shown here that breast cancer cells and IL-6 persistently activate breast stromal fibroblasts through the stimulation of the positive IL-6/STAT3/NF-κB feedback loop. Transient neutralization of IL-6 in culture inhibited this signaling circuit and reverted myofibrobalsts to a normalized state, suggesting the implication of the IL-6 autocrine feedback loop as well. Importantly, the IL-6/STAT3/NF-κB pro-inflammatory circuit was also active in cancer-associated fibroblasts isolated from breast cancer patients. Transient inhibition of STAT3 by specific siRNA in active fibroblasts persistently reduced the level of the RNA binding protein AUF1, blocked the loop and normalized these cells. Moreover, we present clear evidence that AUF1 is also part of this positive feedback loop. Interestingly, treatment of breast myofibroblasts with caffeine, which has been previously shown to persistently inhibit active breast stromal fibroblasts, blocked the positive feedback loop through potent and sustained inhibition of STAT3, AKT, lin28B and AUF1. These results indicate that the IL-6/STAT3/NF-κB positive feedback loop includes AUF1 and is responsible for the sustained active status of cancer-associated fibroblasts. We have also shown that normalizing myofibroblasts, which could be of great therapeutic value, is possible through the inhibition of this procarcinogenic circuit.

MAPK1E322K mutation increases head and neck squamous cell carcinoma sensitivity to erlotinib through enhanced secretion of amphiregulin.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have not been effective in unselected head and neck squamous cell carcinoma (HNSCC) populations. We previously reported an exceptional response to a brief course of erlotinib in a patient with advanced HNSCC whose tumor harbored a MAPK1E322K somatic mutation. MAPK1E322Kwas associated with increased p-EGFR, increased EGFR downstream signaling and increased sensitivity to erlotinib. In this study, we investigated the mechanism of MAPK1E322K-mediated EGFR activation in the context of erlotinib sensitivity. We demonstrated increased AREG secretion in HNSCC cell lines harboring endogenous or exogenous MAPK1E322K compared to wild type MAPK1. We found inhibition or knockdown of MAPK1 with siRNA resulted in reduced secretion of AREG and decreased sensitivity to erlotinib in the setting of MAPK1E322K. MAPK1E322K was associated with increased AREG secretion leading to an autocrine feedback loop involving AREG, EGFR and downstream signaling. Knockdown of AREG in HNSCC cells harboring MAPK1E322K abrogated EGFR signaling and decreased sensitivity to erlotinib in vitro and in vivo. These cumulative findings implicate increased AREG secretion and EGFR activation as contributing to increased erlotinib sensitivity in MAPK1E322K HNSCC.

Cetuximab Inhibits T790M-Mediated Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor in a Lung Adenocarcinoma Patient-Derived Xenograft Mouse Model

BackgroundThe epidermal growth factor receptor (EGFR) kinase domain T790M (amino acid substitution at position 790 in EGFR from threonine [T] to methionine [M]) mutation in non–small-cell lung cancer (NSCLC) results in resistance to EGFR tyrosine kinase inhibitors (TKIs). We used a patient-derived tumor xenograft (PDX) model containing an EGFR exon 19 deletion/T790M mutation to assess response to the EGFR-directed antibody cetuximab. Changes in the EGFR signaling pathway and ligand expression after treatment were investigated. MethodsPDX were randomized into control and treatment arms. Pharmacodynamic studies were performed at 2 and 24 hours and at 4 days after a single administration of cetuximab, erlotinib, or dacomitinib. Changes in the EGFR signaling pathway were assessed using Western blot analysis, and baseline mRNA expression of EGFR ligands using microarray analysis. Relative changes after treatment were assessed using quantitative polymerase chain reaction. ResultsThe xenograft showed a dramatic response to cetuximab. A complete reduction of total EGFR and phosphorylated EGFR occurred after cetuximab treatment. The PDX had increased baseline levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF) compared with other PDX models with or without EGFR mutations. Amphiregulin was significantly reduced 2 hours after treatment with cetuximab. Compared with control mice, cetuximab- and EGFR–TKI-treated mice had significantly reduced HB-EGF gene expression at 2 hours, however, by day 4 the level of HB-EGF expression was higher. The effect of cetuximab compared with EGFR TKI on HB-EGF gene expression levels differed significantly at 2 and 24 hours but not at 4 days. ConclusionWe showed a dramatic tumor response with cetuximab in an exon 19 deletion/T790M EGFR mutant lung adenocarcinoma PDX model, which suggests a role for the autocrine feedback loop in the mutant EGFR signaling pathway. Further investigation using cetuximab in NSCLC with T790M mutation is warranted.

S1P2/G12/13 Signaling Negatively Regulates Macrophage Activation and Indirectly Shapes the Atheroprotective B1-Cell Population.

Monocyte/macrophage recruitment and activation at vascular predilection sites plays a central role in the pathogenesis of atherosclerosis. Heterotrimeric G proteins of the G12/13 family have been implicated in the control of migration and inflammatory gene expression, but their function in myeloid cells, especially during atherogenesis, is unknown. Mice with myeloid-specific deficiency for G12/13 show reduced atherosclerosis with a clear shift to anti-inflammatory gene expression in aortal macrophages. These changes are because of neither altered monocyte/macrophage migration nor reduced activation of inflammatory gene expression; on the contrary, G12/13-deficient macrophages show an increased nuclear factor-κB-dependent gene expression in the resting state. Chronically increased inflammatory gene expression in resident peritoneal macrophages results in myeloid-specific G12/13-deficient mice in an altered peritoneal micromilieu with secondary expansion of peritoneal B1 cells. Titers of B1-derived atheroprotective antibodies are increased, and adoptive transfer of peritoneal cells from mutant mice conveys atheroprotection to wild-type mice. With respect to the mechanism of G12/13-mediated transcriptional control, we identify an autocrine feedback loop that suppresses nuclear factor-κB-dependent gene expression through a signaling cascade involving sphingosine 1-phosphate receptor subtype 2, G12/13, and RhoA. Together, these data show that selective inhibition of G12/13 signaling in macrophages can augment atheroprotective B-cell populations and ameliorate atherosclerosis.

Regulation of De Novo Adipocyte Differentiation Through Cross Talk Between Adipocytes and Preadipocytes.

There are many known adipokines differentially secreted from the different adipose depots; however, their paracrine and autocrine effects on de novo adipocyte formation are not fully understood. By developing a coculture method of preadipocytes with primary subcutaneous and visceral adipocytes or tissue explants, we could show that the total secretome inhibited preadipocyte differentiation. Using a proteomics approach with fractionated secretome samples, we were able to identify a spectrum of factors that either positively or negatively affected adipocyte formation. Among the secreted factors, Slc27a1, Vim, Cp, and Ecm1 promoted adipocyte differentiation, whereas Got2, Cpq, interleukin-1 receptor-like 1/ST2-IL-33, Sparc, and Lgals3bp decreased adipocyte differentiation. In human subcutaneous adipocytes of lean subjects, obese subjects, and obese subjects with type 2 diabetes, Vim and Slc27a1 expression was negatively correlated with adipocyte size and BMI and positively correlated with insulin sensitivity, while Sparc and Got2 showed the opposite trend. Furthermore, we demonstrate that Slc27a1 was increased upon weight loss in morbidly obese patients, while Sparc expression was reduced. Taken together, our findings identify adipokines that regulate adipocyte differentiation through positive or negative paracrine and autocrine feedback loop mechanisms, which could potentially affect whole-body energy metabolism.

Transcriptional and Functional Characterization of the G Protein-Coupled Receptor Repertoire of Gastric Somatostatin Cells.

In the stomach, somatostatin (SST) acts as a general paracrine negative regulator of exocrine secretion of gastric acid and pepsinogen and endocrine secretion of gastrin, ghrelin, and histamine. Using reporter mice expressing red fluorescent protein (RFP) under control of the SST promotor, we have characterized the G protein-coupled receptors expressed in gastric Sst-RFP-positive cells and probed their effects on SST secretion in primary cell cultures. Surprisingly, besides SST, amylin and PYY were also highly enriched in the SST cells. Several receptors found to regulate SST secretion were highly expressed and/or enriched. 1) The metabolite receptors calcium-sensing receptor and free fatty acid receptor 4 (GPR120) functioned as positive and negative regulators, respectively. 2) Among the neurotransmitter receptors, adrenergic receptors α1a, α2a, α2b, and β1 were all highly expressed, with norepinephrine and isoproterenol acting as positive regulators. The muscarinic receptor M3 acted as a positive regulator, whereas M4 was conceivably a negative regulator. 3) Of the hormone receptors, the GLP-1 and GIP receptors, CCKb (stimulated by both CCK and gastrin) and surprisingly the melanocortin MC1 receptor were all positive regulators. 4) The neuropeptide receptors for calcitonin gene-related peptide, adrenomedullin, and vasoactive intestinal peptide acted as positive regulators, no effect was observed using galanin and nociceptin although transcripts for the corresponding receptors appeared highly expressed. 5) The SST receptors 1 and 2 functioned in an autocrine negative feedback loop. Thus, the article provides a comprehensive map of receptors through which SST secretion is regulated by hormones, neurotransmitters, neuropeptides and metabolites that act directly on the SST cells in the gastric mucosa.

P0302 : Constitutive gp130 activation accelerates transformation of proliferating human hepatocytes via increased levels of oxidative stress

Background and Aims: Transforming growth factor (TGF)-b is a ubiquitously expressed cytokine with fundamental roles in various aspects of cell physiology. In carcinogenesis, TGF-b signaling plays a dual role. While it suppresses the proliferation of epithelial cells and adenoma cells at early stages by inducing growth arrest and apoptosis, it triggers epithelial to mesenchymal transition (EMT) and gain of metastatic abilities at later stages of carcinoma development. The molecular mechanisms underlying this ’TGF-bswitch’ are only beginning to be unravelled. To mimic the pathophysiological situation as closely as possible, we exposed the cells to TGFb long-term. Through this approach we aimed to identify those cooperating factors and signaling pathways that cause HCC cells to interpret the TGF-b signal in a tumor progressive way. Methods: In vitro comparison of migratory behaviour of various HCC cell lines treated long-term (>10 days) with TGF-b. Analysis of regulatory networks and target genes underlying the TGF-b treatment. Results: HCC cell lines that have undergone EMT secrete TGF-b and show elevated levels of Smad2/3 phosphorylation indicating an autocrine regulatory feedback loop. Inhibition of TGF-b by LY2109 abrogates autocrine stimulation and diminishes the migratory potential of mesenchymal HCC cells. Silencing of either TGF-bR1 or Smad4 indicated the importance of canonical TGF-b/Smad signaling HCC cell migration. Short-term treatment of cells with TGF-b could not improve migratory abilities. Interestingly, long-term TGF-b treatment revealed crucial differences between mesenchymal HCC cell lines. While HLF cells showed an increase in migration when treated with TGF-b for more than 10 days, SNU449 displayed a dramatic reduction in migration. However, both cell lines displayed no modulation in Smad phosphorylation, indicating a change in the utilization of TGF-b signaling in long-term treated SNU449 cells. Conclusions: EMT-transformed HCC cells establish an autocrine TGF-b loop which stimulates migration. However, TGF-b cannot add up to the autocrine loop but causes a different, even opposing reaction, over time. Interpretation of long-term TGF-b signaling, which mimics the patient’s situation more closely, depends on duration and intensity and is controlled by co-acting factors and signaling pathways. P0301 Withdrawn

Exploring the IL-21–STAT3 Axis as Therapeutic Target for Sézary Syndrome

Sézary syndrome is an aggressive cutaneous T-cell lymphoma. The malignant cells (Sézary cells) are present in skin, lymph nodes, and blood, and express constitutively activated signal transducer and activator of transcription (STAT)3. STAT3 can be activated by IL-21 in vitro and the IL-21 gene itself is a STAT3 target gene, thereby creating an autocrine positive feedback loop that might serve as a therapeutic target. Sézary cells underwent apoptosis when incubated with Stattic, a selective STAT3 inhibitor. STAT3 activation in Sézary cells did not affect expression of the supposed anti-apoptotic STAT3 target genes BCL2, BCL-xL, and SURVIVIN, whereas expression of (proto)oncogenes miR-21, TWIST1, MYC, and PIM1 was significantly increased. CD3/CD28-mediated activation of Sézary cells induced IL-21 expression, accompanied by STAT3 activation and increased proliferation. Blocking IL-21 in CD3/CD28-activated cells had no effects, whereas Stattic abrogated IL-21 expression and cell proliferation. Thus, specific inhibition of STAT3 is highly efficient in the induction of apoptosis of Sézary cells, likely mediated via the regulation of (proto)oncogenes. In contrast, blocking IL-21 alone seems insufficient to affect STAT3 activation, cell proliferation, or apoptosis. These data provide further insights into the pathogenic role of STAT3 in Sézary syndrome and strengthen the notion that STAT3 represents a promising therapeutic target in this disease.

Abstract LB-236: Preclinical efficacy of targeting FGF autocrine signaling in mesothelioma with the FGF ligand trap, FP-1039/GSK3052230

Abstract Fibroblast growth factor (FGF) ligand-dependent signaling plays an important role in cancer development and tumor maintenance through autocrine production of FGFs directly from cancer cells and/or through paracrine production of FGFs from the local stroma. FGF-FGFR autocrine feedback loops have been characterized in several tumor types and may also play a role in drug resistance upon exposure to chemotherapy or targeted agents. FP-1039/GSK3052230 is a ligand trap that sequesters multiple FGFs and inhibits FGF receptor signaling. We previously demonstrated that increased FGF2 mRNA levels correlated with response to GSK3052230. To further extend this hypothesis, we investigated the preclinical efficacy of this drug in models of mesothelioma, a tumor type shown to express high levels of FGF2 mRNA in cell lines and in primary tumor specimens. GSK3052230 inhibited MAPK signaling as evidenced by decreased phospho-ERK levels in both NCI-H226 and MSTO-211H cells. When both cell lines were grown as tumor xenografts in mice, GSK3052230 inhibited tumor growth in a dose-dependent manner (NCI-H226: 16 - 78% TGI; MSTO-211H: 20 - 50% TGI). However, in contrast to what was observed in cultured cells, GSK3052230 had minimal effects on phospho-ERK levels in NCI-H226 tumors. Similarly, two genes downstream of ERK known to be regulated by FGF signaling, DUSP6 and ETV4, displayed modestly reduced mRNA levels upon GSK3052230 treatment. These data demonstrate the limitation of FGF pathway downstream signaling as a pharmacodynamic (PD) readout for this drug in this study despite observing significant tumor growth inhibition. Because FGFs also play a key role in angiogenesis, we explored the effects of GSK3052230 on tumor vessel formation in NCI-H226 xenografts as a potential approach to measure PD. Dose-dependent and statistically-significant reductions in tumor vessel density were observed in GSK3052230-treated tumors compared to vehicle-treated tumors using MECA-32 endothelial cell immunohistochemical staining. These data support the clinical evaluation of this drug in mesothelioma patients. Citation Format: M. Phillip De Young, Christian Sherk, Maureen Bleam, Mary Barnette, Gopi Ganji, Bao Hoang, James Tunstead, David Bellovin, Gerrit Los, Elisabeth Minthorn, Rakesh Kumar. Preclinical efficacy of targeting FGF autocrine signaling in mesothelioma with the FGF ligand trap, FP-1039/GSK3052230. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-236. doi:10.1158/1538-7445.AM2014-LB-236

Autocrine FGF feedback can establish distinct states of Nanog expression in pluripotent stem cells: a computational analysis.

BackgroundThe maintenance of stem cell pluripotency is controlled by a core cluster of transcription factors, NANOG, OCT4 and SOX2 – genes that jointly regulate each other’s expression. The expression of some of these genes, especially of Nanog, is heterogeneous in a population of undifferentiated stem cells in culture. Transient changes in expression levels, as well as heterogeneity of the population is not restricted to this core regulator, but involve a large number of other genes that include growth factors, transcription factors or signal transduction proteins.ResultsAs the molecular mechanisms behind NANOG expression heterogeneity is not yet understood, we explore by computational modeling the core transcriptional regulatory circuit and its input from autocrine FGF signals that act through the MAP kinase cascade. We argue that instead of negative feedbacks within the core NANOG-OCT4-SOX2 transcriptional regulatory circuit, autocrine signaling loops such as the Esrrb - FGF - ERK feedback considered here are likely to generate distinct sub-states within the “ON” state of the core Nanog switch. Thus, the experimentally observed fluctuations in Nanog transcription levels are best explained as noise-induced transitions between negative feedback-generated sub-states. We also demonstrate that ERK phosphorilation is altered and being anti-correlated with fluctuating Nanog expression – in accord with model simulations. Our modeling approach assigns an empirically testable function to the transcriptional regulators Klf4 and Esrrb, and predict differential regulation of FGF family members.ConclusionsWe argue that slow fluctuations in Nanog expression likely reflect individual cell-specific changes in parameters of an autocrine feedback loop, such as changes in ligand capture efficiency, receptor numbers or the presence of crosstalks within the MAPK signal transduction pathway. We proposed a model that operates with binding affinities of multiple transcriptional regulators of pluripotency, and the activity of an autocrine signaling pathway. The resulting model produces varied expression levels of several components of pluripotency regulation, largely consistent with empirical observations reported previously and in this present work.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-014-0112-4) contains supplementary material, which is available to authorized users.

Vasopressin regulation of epithelial colonic proliferation and permeability is mediated by pericryptal platelet-derived growth factor A.

Arginine vasopressin (AVP) has trophic effects on the rat distal colon, increasing the growth of pericryptal myofibroblasts and reducing the colonic crypt wall permeability. This study aimed to reproduce in vitro the effects of AVP observed in vivo using cultures of human CCD-18Co myofibroblasts and T84 colonic epithelial cells. Proliferation of myofibroblasts was quantified by bromodeoxyuridine incorporation; the expression of platelet-derived growth factorA (PDGFA), platelet-derived growth factorB, epidermal growth factor, transforming growth factor-β and vascular endothelial growth factor was measured by PCR and the expression of epithelial junction proteins by Western blot. Arginine vasopressin stimulated myofibroblast proliferation and the expression of PDGFA without affecting the expression of platelet-derived growth factorB, epidermal growth factor, transforming growth factor-β or vascular endothelial growth factor. These effects were prevented when AVP receptor inhibitors were present in the medium. Pre-incubation of CCD-18Co cells with anti-PDGF antibody or with an inhibitor of the PDGF receptor abolished the effects of AVP. When colonocytes were incubated with medium obtained from myofibroblasts incubated with AVP, both cell proliferation and the expression of epithelial junction proteins increased; however, direct incubation of colonocytes with AVP did not modify these variables. These results demonstrate that AVP stimulates myofibroblast proliferation and induces PDGFA secretion, implying that PDGFA mediates local myofibroblast proliferation by an autocrine feedback loop and regulates epithelial proliferation and permeability by a paracrine mechanism.

Prostaglandin F2α Inhibits Adipogenesis Via an Autocrine‐Mediated Interleukin‐11/Glycoprotein 130/STAT1‐Dependent Signaling Cascade

Prostaglandin F2α (PGF2α) is a potent inhibitor of adipocyte differentiation in vitro, that has also recently been implicated in the regulation of the adipogenic process in vivo, by opposing adipose tissue accretion and the subsequent development of obesity and its attendant metabolic consequences. In previous studies, we have demonstrated that PGF2α inhibits adipocyte differentiation by means of a calcium-dependent signaling pathway that is critically dependent upon the activity of the calcineurin phosphatase. In the current study, we have now extended these findings to further elucidate the mechanism by which the PGF2α/calcineurin-pathway inhibits the adipogenic process. We now report that the IL-11 cytokine, a member of the gp130 cytokine co-receptor-related family, is a downstream transcriptional target of this pathway in 3T3-L1 preadipocytes and is actively secreted in differentiating cells in response to PGF2α stimulation. Using a combined shRNA and dominant-negative receptor mutant approach, we provide evidence that IL-11/gp130-signaling is required to mediate the inhibitory effects of PGF2α on adipogenesis. Moreover, by taking advantage of a well-characterized panel of chimeric gp130 mutant receptors, we demonstrate that gp130 signaling is sufficient to inhibit adipocyte differentiation and specifically requires the activation of the STAT1 transcription factor. Conversely, we find that depleting endogenous STAT1 levels rescues adipogenesis in the presence of both IL-11/gp130 signaling and PGF2α. Collectively, our findings support a model in which PGF2α inhibits adipocyte differentiation by means of an IL-11 mediated autocrine negative feedback loop, that acts via gp130 to block adipogenesis through the essential actions of the STAT1 transcription factor.

Matricellular protein CCN1/CYR61: a new player in inflammation and leukocyte trafficking

Cystein-rich protein 61 (CYR61/CCN1) is a component of the extracellular matrix, which is produced and secreted by several cell types including endothelial cells, fibroblasts and smooth muscle cells. CCN1 has been implicated in leukocyte migration and the inflammatory process, but it is also involved in cardiovascular development and carcinogenesis. It exerts its functions through binding to multiple integrins present in many different cell types. This multiplicity in function is now known to contribute to the diverse array of cellular processes it can regulate. The expression of CCN1 is tightly regulated by cytokines and growth factors. However, CCN1 can directly modulate cell adhesion and migratory processes whilst simultaneously regulating the production of other cytokines and chemokines through paracrine and autocrine feedback loops. This complex functionality of CCN1 has highlighted the pivotal role this molecule can play in regulating the immunosurveillance process. Furthermore, CCN1 has now emerged as an important partner when targeting components of the infectious or chronic inflammatory disease processes such as atherosclerosis or rheumatoid arthritis. This review will focus on CYR61/CCN1 and its ability to control the migration of leukocytes, the production of cytokines and cell proliferation or senescence at the site of inflammation.

Periostin Induces Intracellular Cross-talk between Kinases and Hyaluronan in Atrioventricular Valvulogenesis

Periostin (PN), a novel fasciclin-related matricellular protein, has been implicated in cardiac development and postnatal remodeling, but the mechanism remains unknown. We examined the role of PN in mediating intracellular kinase activation for atrioventricular valve morphogenesis using well defined explant cultures, gene transfection systems, and Western blotting. The results show that valve progenitor (cushion) cells secrete PN into the extracellular matrix, where it can bind to INTEGRINs and activate INTEGRIN/focal adhesion kinase signaling pathways and downstream kinases, PI3K/AKT and ERK. Functional assays with prevalvular progenitor cells showed that activating these signaling pathways promoted adhesion, migration, and anti-apoptosis. Through activation of PI3K/ERK, PN directly enhanced collagen expression. Comparing PN-null to WT mice also revealed that expression of hyaluronan (HA) and activation of hyaluronan synthase-2 (Has2) are also enhanced upon PN/INTEGRIN/focal adhesion kinase-mediated activation of PI3K and/or ERK, an effect confirmed by the reduction of HA synthase-2 in PN-null mice. We also identified in valve progenitor cells a potential autocrine signaling feedback loop between PN and HA through PI3K and/or ERK. Finally, in a three-dimensional assay to simulate normal valve maturation in vitro, PN promoted collagen compaction in a kinase-dependent fashion. In summary, this study provides the first direct evidence that PN can act to stimulate a valvulogenic signaling pathway.

Interleukin 6 promotes endometrial cancer growth through an autocrine feedback loop involving ERK–NF-κB signaling pathway

Interleukin (IL)-6 as an inflammation factor, has been proved to promote cancer proliferation in several human cancers. However, its role in endometrial cancer has not been studied clearly. Previously, we demonstrated that IL-6 promoted endometrial cancer progression through local estrogen biosynthesis. In this study, we proved that IL-6 could directly stimulate endometrial cancer cells proliferation and an autocrine feedback loop increased its production even after the withdrawal of IL-6 from the medium. Next, we analyzed the mechanism underlying IL-6 production in the feedback loop and found that its production and IL-6-stimulated cell proliferation were effectively blocked by pharmacologic inhibitors of nuclear factor-kappa B (NF-κB) and extra-cellular signal-regulated kinase (ERK). Importantly, activation of ERK was upstream of the NF-κB pathways, revealing the hierarchy of this event. Finally, we used an orthotopic nude endometrial carcinoma model to confirm the effects of IL-6 on the tumor progression. Taken together, these data indicate that IL-6 promotes endometrial carcinoma growth through an expanded autocrine regulatory loop and implicate the ERK–NF-κB pathway as a critical mediator of IL-6 production, implying IL-6 to be an important therapeutic target in endometrial carcinoma.

Positive Feedback Loop of Autocrine BDNF from Microglia Causes Prolonged Microglia Activation

Background/Aims: Microglia, which represent the immune cells of the central nervous system (CNS), have long been a subject of study in CNS disease research. Substantial evidence indicates that microglial activation functions as a strong neuro-inflammatory response in neuropathic pain, promoting the release of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α. In addition, activated microglia release brain-derived neurotrophic factor (BDNF), which acts as a powerful cytokine. In this study, we performed a series of in vitro experiments to examine whether a positive autocrine feedback loop existed between microglia-derived BDNF and subsequent microglial activation as well as the mechanisms underlying this positive feedback loop. Methods: Because ATP is a classic inducer of microglial activation, firstly, we examined ATP-activated microglia in the present study. Secondly, we used TrkB/Fc, the BDNF sequester, to eliminate the effects of endogenous BDNF. ATP-stimulated microglia without BDNF was examined. Finally, we used exogenous BDNF to further determine whether BDNF could directly activate BV2 microglia. In all experiments, to quantify BV2 microglia activation, the protein levels of CD11b, a microglial activation marker, were measured by western blot. A Transwell migration assay was used to examine microglial migration. To assess the synthesis and release of proinflammatory cytokines, western blot was used to measure BDNF synthesis, and ELISA was used to quantify TNF-α release. Results: In our present research, we have observed that ATP dramatically activates microglia, enhancing microglial migration, increasing the synthesis of BDNF and up-regulating the release of TNF-α. Microglial activation is inhibited following the sequestration of endogenous BDNF, resulting in impaired microglial migration and decreased TNF-α release. Furthermore, exogenous BDNF can also activate microglia to subsequently enhance migration and increase TNF-α release. Conclusion: Therefore, we suggest that microglial activation increases the synthesis of BDNF and that the release of BDNF can, in turn, activate microglia. A positive autocrine BDNF feedback loop from microglia may contribute to prolonged microglial activation.

An heregulin-EGFR-HER3 autocrine signaling axis can mediate acquired lapatinib resistance in HER2+ breast cancer models.

IntroductionThe human epidermal growth factor receptor 2 (HER2) receptor tyrosine kinase (RTK) oncogene is an attractive therapeutic target for the treatment of HER2-addicted tumors. Although lapatinib, an FDA-approved small-molecule HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), represents a significant therapeutic advancement in the treatment of HER2+ breast cancers, responses to lapatinib have not been durable. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER-directed therapies is of critical importance.MethodsUsing a functional protein-pathway activation mapping strategy, along with targeted genomic knockdowns applied to a series of isogenic-matched pairs of lapatinib-sensitive and resistant cell lines, we now report an unexpected mechanism of acquired resistance to lapatinib and similar TKIs.ResultsThe signaling analysis revealed that whereas HER2 was appropriately inhibited in lapatinib-resistant cells, EGFR tyrosine phosphorylation was incompletely inhibited. Using a targeted molecular knockdown approach to interrogate the causal molecular underpinnings of EGFR-persistent activation, we found that lapatinib-resistant cells were no longer oncogene addicted to HER2-HER3-PI3K signaling, as seen in the parental lapatinib-sensitive cell lines, but instead were dependent on a heregulin (HRG)-driven HER3-EGFR-PI3K-PDK1 signaling axis. Two FDA-approved EGFR TKIs could not overcome HRG-HER3-mediated activation of EGFR, or reverse lapatinib resistance. The ability to overcome EGFR-mediated acquired therapeutic resistance to lapatinib was demonstrated through molecular knockdown of EGFR and treatment with the irreversible pan-HER TKI neratinib, which blocked HRG-dependent phosphorylation of HER3 and EGFR, resulting in apoptosis of resistant cells. In addition, whereas HRG reversed lapatinib-mediated antitumor effects in parental HER2+ breast cancer cells, neratinib was comparatively resistant to the effects of HRG in parental cells. Finally, we showed that HRG expression is an independent negative predictor of clinical outcome in HER2+ breast cancers, providing potential clinical relevance to our findings.ConclusionsMolecular analysis of acquired therapeutic resistance to lapatinib identified a new resistance mechanism based on incomplete and "leaky" inhibition of EGFR by lapatinib. The selective pressure applied by incomplete inhibition of the EGFR drug target resulted in selection of ligand-driven feedback that sustained EGFR activation in the face of constant exposure to the drug. Inadequate target inhibition driven by a ligand-mediated autocrine feedback loop may represent a broader mechanism of therapeutic resistance to HER TKIs and suggests adopting a different strategy for selecting more effective TKIs to advance into the clinic.

Asbestos and erionite prime and activate the NLRP3 inflammasome that stimulates autocrine cytokine release in human mesothelial cells

BackgroundPleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear.ResultsWe document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1β, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model.ConclusionsThese novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis.

Neuropeptide Y Gates a Stress-Induced, Long-Lasting Plasticity in the Sympathetic Nervous System

Acute stress evokes the fight-or-flight reflex, which via release of the catecholamine hormones affects the function of every major organ. Although the reflex is transient, it has lasting consequences that produce an exaggerated response when stress is reexperienced. How this change is encoded is not known. We investigated whether the reflex affects the adrenal component of the sympathetic nervous system, a major branch of the stress response. Mice were briefly exposed to the cold-water forced swim test (FST) which evoked an increase in circulating catecholamines. Although this hormonal response was transient, the FST led to a long-lasting increase in the catecholamine secretory capacity measured amperometrically from chromaffin cells and in the expression of tyrosine hydroxylase. A variety of approaches indicate that these changes are regulated postsynaptically by neuropeptide Y (NPY), an adrenal cotransmitter. Using immunohistochemistry, RT-PCR, and NPY(GFP) BAC mice, we find that NPY is synthesized by all chromaffin cells. Stress failed to increase secretory capacity in NPY knock-out mice. Genetic or pharmacological interference with NPY and Y1 (but not Y2 or Y5) receptor signaling attenuated the stress-induced change in tyrosine hydroxylase expression. These results indicate that, under basal conditions, adrenal signaling is tonically inhibited by NPY, but stress overrides this autocrine negative feedback loop. Because acute stress leads to a lasting increase in secretory capacity in vivo but does not alter sympathetic tone, these postsynaptic changes appear to be an adaptive response. We conclude that the sympathetic limb of the stress response exhibits an activity-dependent form of long-lasting plasticity.