Showing papers in "Cell Communication and Signaling in 2018"

A highlight on Sonic hedgehog pathway

Abstract: Hedgehog (Hh) signaling pathway plays an essential role during vertebrate embryonic development and tumorigenesis. It is already known that Sonic hedgehog (Shh) pathway is important for the evolution of radio and chemo-resistance of several types of tumors. Most of the brain tumors are resistant to chemotherapeutic drugs, consequently, they have a poor prognosis. So, a better knowledge of the Shh pathway opens an opportunity for targeted therapies against brain tumors considering a multi-factorial molecular overview. Therefore, emerging studies are being conducted in order to find new inhibitors for Shh signaling pathway, which could be safely used in clinical trials. Shh can signal through a canonical and non-canonical way, and it also has important points of interaction with other pathways during brain tumorigenesis. So, a better knowledge of Shh signaling pathway opens an avenue of possibilities for the treatment of not only for brain tumors but also for other types of cancers. In this review, we will also highlight some clinical trials that use the Shh pathway as a target for treating brain cancer.

Regulation of the master regulator FOXM1 in cancer

Abstract: FOXM1 (forkhead box protein M1) is a critical proliferation-associated transcription factor that is widely spatiotemporally expressed during the cell cycle It is closely involved with the processes of cell proliferation, self-renewal, and tumorigenesis In most human cancers, FOXM1 is overexpressed, and this indicates a poor prognosis for cancer patients FOXM1 maintains cancer hallmarks by regulating the expression of target genes at the transcriptional level Due to its potential role as molecular target in cancer therapy, FOXM1 was named the Molecule of the Year in 2010 However, the mechanism of FOXM1 dysregulation remains indistinct A comprehensive understanding of FOXM1 regulation will provide novel insight for cancer and other diseases in which FOXM1 plays a major role Here, we summarize the transcriptional regulation, post-transcriptional regulation and post-translational modifications of FOXM1, which will provide extremely important implications for novel strategies targeting FOXM1

Activation of the JAK/STAT3 and PI3K/AKT pathways are crucial for IL-6 trans-signaling-mediated pro-inflammatory response in human vascular endothelial cells

Abstract: IL-6 classic signaling is linked to anti-inflammatory functions while the trans-signaling is associated with pro-inflammatory responses. Classic signaling is induced via membrane-bound IL-6 receptor (IL-6R) whereas trans-signaling requires prior binding of IL-6 to the soluble IL-6R. In both cases, association with the signal transducing gp130 receptor is compulsory. However, differences in the downstream signaling mechanisms of IL-6 classic- versus trans-signaling remains largely elusive. In this study, we used flow cytometry, quantitative PCR, ELISA and immuno-blotting techniques to investigate IL-6 classic and trans-signaling mechanisms in Human Umbilical Vein Endothelial Cells (HUVECs). We show that both IL-6R and gp130 are expressed on the surface of human vascular endothelial cells, and that the expression is affected by pro-inflammatory stimuli. In contrast to IL-6 classic signaling, IL-6 trans-signaling induces the release of the pro-inflammatory chemokine Monocyte Chemoattractant Protein-1 (MCP-1) from human vascular endothelial cells. In addition, we reveal that the classic signaling induces activation of the JAK/STAT3 pathway while trans-signaling also activates the PI3K/AKT and the MEK/ERK pathways. Furthermore, we demonstrate that MCP-1 induction by IL-6 trans-signaling requires simultaneous activation of the JAK/STAT3 and PI3K/AKT pathways. Collectively, our study reports molecular differences in IL-6 classic- and trans-signaling in human vascular endothelial cells; and elucidates the pathways which mediate MCP-1 induction by IL-6 trans-signaling.

Copper signalling: causes and consequences.

Abstract: Copper-containing enzymes perform fundamental functions by activating dioxygen (O2) and therefore allowing chemical energy-transfer for aerobic metabolism. The copper-dependence of O2 transport, metabolism and production of signalling molecules are supported by molecular systems that regulate and preserve tightly-bound static and weakly-bound dynamic cellular copper pools. Disruption of the reducing intracellular environment, characterized by glutathione shortage and ambient Cu(II) abundance drives oxidative stress and interferes with the bidirectional, copper-dependent communication between neurons and astrocytes, eventually leading to various brain disease forms. A deeper understanding of of the regulatory effects of copper on neuro-glia coupling via polyamine metabolism may reveal novel copper signalling functions and new directions for therapeutic intervention in brain disorders associated with aberrant copper metabolism.

M2 macrophages promote myofibroblast differentiation of LR-MSCs and are associated with pulmonary fibrogenesis

Abstract: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the histopathological pattern of usual interstitial pneumonia and is associated with a high mortality rate. Recently, lung resident mesenchymal stem cells (LR-MSCs) have been identified as an important contributor to myofibroblast activation in pulmonary fibrosis. Macrophages are also believed to play a critical role in pulmonary fibrosis. However, the underlying connections between LR-MSCs and macrophages in the pathogenesis of pulmonary fibrosis are still elusive. In this study, we investigated the interaction between LR-MSCs and macrophages using a bleomycin-induced mouse pulmonary fibrosis model and a coculture system. Here, we show that blocking pulmonary macrophage infiltration attenuated bleomycin-induced pulmonary fibrosis. In addition, as determined by flow cytometry, we discovered that the recruited macrophages in fibrotic lungs of bleomycin-treated mice were mainly M2 macrophages. In particular, we found that M2, rather than M1 macrophages, promoted myofibroblast differentiation of LR-MSCs. Moreover, we demonstrated that suppression of the Wnt/β-catenin signaling pathway could attenuate myofibroblast differentiation of LR-MSCs induced by M2 macrophages and bleomycin-induced pulmonary fibrosis. Tissue samples from IPF patients confirmed the infiltration of M2 macrophages and activation of Wnt/β-catenin signaling pathway. In summary, this study furthered our understanding of the pulmonary fibrosis pathogenesis and highlighted M2 macrophages as a critical target for treating pulmonary fibrosis.

Nrf2 activation drive macrophages polarization and cancer cell epithelial-mesenchymal transition during interaction

Abstract: The M2 phenotype of tumor-associated macrophages (TAM) inhibits the anti-tumor inflammation, increases angiogenesis and promotes tumor progression. The transcription factor Nuclear Factor (erythroid-derived 2)-Like 2 (Nrf2) not only modulates the angiogenesis but also plays the anti-inflammatory role through inhibiting pro-inflammatory cytokines expression; however, the role of Nrf2 in the cancer cell and macrophages interaction is not clear. Hepatocellular carcinoma cells (Hep G2 and Huh 7) and pancreatic cancer cells (SUIT2 and Panc-1) were co-cultured with monocytes cells (THP-1) or peripheral blood monocytes derived macrophages, then the phenotype changes of macrophages and epithelial-mesenchymal transition of cancer cells were detected. Also, the role of Nrf2 in cancer cells and macrophages interaction were investigated. In this study, we found that cancer cells could induce an M2-like macrophage characterized by up-regulation of CD163 and Arg1, and down-regulation of IL-1b and IL-6 through Nrf2 activation. Also, Nrf2 activation of macrophages promoted VEGF expression. The Nrf2 activation of macrophages correlated with the reactive oxygen species induced by cancer cells derived lactate. Cancer cells educated macrophages could activate Nrf2 of the cancer cells, in turn, to increase cancer cells epithelial-mesenchymal transition (EMT) through paracrine VEGF. These findings suggested that Nrf2 played the important role in the cancer cells and macrophages interaction. Macrophage Nrf2 activation by cancer cell-derived lactate skews macrophages polarization towards an M2-like phenotype and educated macrophages activate Nrf2 of the cancer cells to promote EMT of cancer cells. This study provides a new understanding of the role of Nrf2 in the cancer cell and TAM interaction and suggests a potential therapeutic target.

Cold shock proteins: from cellular mechanisms to pathophysiology and disease.

Abstract: Cold shock proteins are multifunctional RNA/DNA binding proteins, characterized by the presence of one or more cold shock domains. In humans, the best characterized members of this family are denoted Y-box binding proteins, such as Y-box binding protein-1 (YB-1). Biological activities range from the regulation of transcription, splicing and translation, to the orchestration of exosomal RNA content. Indeed, the secretion of YB-1 from cells via exosomes has opened the door to further potent activities. Evidence links a skewed cold shock protein expression pattern with cancer and inflammatory diseases. In this review the evidence for a causative involvement of cold shock proteins in disease development and progression is summarized. Furthermore, the potential application of cold shock proteins for diagnostics and as targets for therapy is elucidated.

YAP/TAZ regulates TGF-β/Smad3 signaling by induction of Smad7 via AP-1 in human skin dermal fibroblasts.

Abstract: Transcription factors YAP and TAZ function as the primary mediators of the Hippo pathway. Yet, crosstalk of YAP and TAZ with other signaling pathways remains relatively unexplored. We have explored the impact of YAP and TAZ levels on the TGF-β/Smad signaling pathway in human skin dermal fibroblasts. YAP and TAZ levels in dermal fibroblasts were reduced in dermal fibroblasts by siRNA-mediated knockdown. The effects of YAP and TAZ reduction on TGF-β/Smad signaling were examined by quantitative real-time PCR, Western analysis, and immunostaining. Luciferase reporter assays and electrophoretic mobility shift assays were conducted to investigate the transcription factor DNA-binding and transcriptional activities. Knockdown of both YAP and TAZ (YAP/TAZ), but not either separately, impaired TGF-β1-induced Smad3 phosphorylation and Smad3 transcriptional activity, thereby inhibiting the expression of TGF-β target genes. This reduction by reduced levels of YAP/TAZ results from induction of inhibitory Smad7, which inhibits Smad3 phosphorylation and activity by TGF-β1. Conversely, prevention of Smad7 induction restores Smad3 phosphorylation and Smad3 transcriptional activity in fibroblasts that have reduced YAP/TAZ. In agreement with these findings, inhibition of YAP/TAZ transcriptional activity, similar to the reduction of YAP/TAZ levels, also significantly induced Smad7 and impaired TGF-β/Smad signaling. Further investigations revealed that reduced levels of YAP/TAZ led to induction of activator protein-1 (AP-1) activity, Activated AP-1 bound to DNA sequences in the Smad7 gene promoter, and deletion of these AP-1 binding sequences substantially reduced Smad7 promoter reporter activity. YAP/TAZ functions in concert with transcription factor AP-1 and Smad7 to regulate TGF-β signaling, in human dermal fibroblasts. Reduction of YAP/TAZ levels leads to activation of AP-1 activity, which induces Smad7. Smad7 suppresses the TGF-β pathway.

Neutrophil extracellular traps induce aggregation of washed human platelets independently of extracellular DNA and histones

Abstract: The release of neutrophil extracellular traps (NETs), a mesh of DNA, histones and neutrophil proteases from neutrophils, was first demonstrated as a host defence against pathogens. Recently it became clear that NETs are also released in pathological conditions. NETs released in the blood can activate thrombosis and initiate a cascade of platelet responses. However, it is not well understood if these responses are mediated through direct or indirect interactions. We investigated whether cell-free NETs can induce aggregation of washed human platelets in vitro and the contribution of NET-derived extracellular DNA and histones to platelet activation response. Isolated human neutrophils were stimulated with PMA to produce robust and consistent NETs. Cell-free NETs were isolated and characterised by examining DNA-histone complexes and quantification of neutrophil elastase with ELISA. NETs were incubated with washed human platelets to assess several platelet activation responses. Using pharmacological inhibitors, we explored the role of different NET components, as well as main platelet receptors, and downstream signalling pathways involved in NET-induced platelet aggregation. Cell-free NETs directly induced dose-dependent platelet aggregation, dense granule secretion and procoagulant phosphatidyl serine exposure on platelets. Surprisingly, we found that inhibition of NET-derived DNA and histones did not affect NET-induced platelet aggregation or activation. We further identified the molecular pathways involved in NET-activated platelets. The most potent single modulator of NET-induced platelet responses included NET-bound cathepsin G, platelet Syk kinase, and P2Y12 and αIIbβ3 receptors. In vitro-generated NETs can directly induce marked aggregation of washed human platelets. Pre-treatment of NETs with DNase or heparin did not reduce NET-induced activation or aggregation of human washed platelets. We further identified the molecular pathways activated in platelets in response to NETs. Taken together, we conclude that targeting certain platelet activation pathways, rather than the NET scaffold, has a more profound reduction on NET-induced platelet aggregation.

Casein kinase 1α: biological mechanisms and theranostic potential

Abstract: Casein kinase 1α (CK1α) is a multifunctional protein belonging to the CK1 protein family that is conserved in eukaryotes from yeast to humans. It regulates signaling pathways related to membrane trafficking, cell cycle progression, chromosome segregation, apoptosis, autophagy, cell metabolism, and differentiation in development, circadian rhythm, and the immune response as well as neurodegeneration and cancer. Given its involvement in diverse cellular, physiological, and pathological processes, CK1α is a promising therapeutic target. In this review, we summarize what is known of the biological functions of CK1α, and provide an overview of existing challenges and potential opportunities for advancing theranostics.

Adipocyte-derived IL-6 and leptin promote breast Cancer metastasis via upregulation of Lysyl Hydroxylase-2 expression.

Abstract: Adipocytes make up the major component of breast tissue, accounting for 90% of stromal tissue. Thus, the crosstalk between adipocytes and breast cancer cells may play a critical role in cancer progression. Adipocyte-breast cancer interactions have been considered important for the promotion of breast cancer metastasis. However, the specific mechanisms underlying these interactions are unclear. In this study, we investigated the mechanisms of adipocyte-mediated breast cancer metastasis. Breast cancer cells were cocultured with mature adipocytes for migration and 3D matrix invasion assays. Next, lentivirus-mediated loss-of-function experiments were used to explore the function of lysyl hydroxylase (PLOD2) in breast cancer migration and adipocyte-dependent migration of breast cancer cells. The role of PLOD2 in breast cancer metastasis was further confirmed using orthotopic mammary fat pad xenografts in vivo. Clinical samples were used to confirm that PLOD2 expression is increased in tumor tissue and is associated with poor prognosis of breast cancer patients. Cells were treated with cytokines and pharmacological inhibitors in order to verify which adipokines were responsible for activation of PLOD2 expression and which signaling pathways were activated in vitro. Gene expression profiling and Western blotting analyses revealed that PLOD2 was upregulated in breast cancer cells following coculture with adipocytes; this process was accompanied by enhanced breast cancer cell migration and invasion. Loss-of-function studies indicated that PLOD2 knockdown suppressed cell migration and disrupted the formation of actin stress fibers in breast cancer cells and abrogated the migration induced by following coculture with adipocytes. Moreover, experiments performed in orthotopic mammary fat pad xenografts showed that PLOD2 knockdown could reduce metastasis to the lung and liver. Further, high PLOD2 expression correlated with poor prognosis of breast cancer patients. Mechanistically, adipocyte-derived interleukin-6 (IL-6) and leptin may facilitate PLOD2 upregulation in breast cancer cells and promote breast cancer metastasis in tail vein metastasis assays. Further investigation revealed that adipocyte-derived IL-6 and leptin promoted PLOD2 expression through activation of the JAK/STAT3 and PI3K/AKT signaling pathways. Our study reveals that adipocyte-derived IL-6 and leptin promote PLOD2 expression by activating the JAK/STAT3 and PI3K/AKT signaling pathways, thus promoting breast cancer metastasis.

lncRNA KRAL reverses 5-fluorouracil resistance in hepatocellular carcinoma cells by acting as a ceRNA against miR-141.

Abstract: 5-Fluorouracil (5-FU) has been widely applied to treat various types of cancers, including hepatocellular carcinoma (HCC). However, primary or acquired 5-FU resistance prevents the clinical application of this drug in cancer therapy. Herein, our study is the first to demonstrate that lower expression of KRAL, a long non-coding RNA (lncRNA), mediates 5-FU resistance in HCC via the miR-141/Keap1 axis. Cell proliferation assays, western blot analysis, qRT-PCR, the dual-luciferase reporter assay and RNA immunoprecipitation were performed to investigate the mechanisms by which KRAL mediates 5-fluorouracil resistance in HCC cell lines. The quantitative analysis indicated that KRAL and Keap1 were significantly decreased and that Nrf2 was increased in HepG2/5-FU and SMMC-7721/5-FU cells compared with the corresponding expression levels in the respective parental cells. Overexpression of KRAL increased Keap1 expression, and inactivating the Nrf2-dependent antioxidant pathway could reverse the resistance of HepG2/5-FU and SMMC-7721/5-FU cells to 5-FU. Moreover, KRAL functioned as a competitive endogenous RNA (ceRNA) by effectively binding to the common miR-141 and then restoring Keap1 expression. These findings demonstrated that KRAL is an important regulator of Keap1; furthermore, the ceRNA network involving KRAL may serve as a treatment strategy against 5-FU resistance in hepatocellular carcinoma cells. KRAL/miR-141/Keap1 axis mediates 5-fluorouracil resistance in HCC cell lines.

Four-octyl itaconate activates Keap1-Nrf2 signaling to protect neuronal cells from hydrogen peroxide

Abstract: Four-octyl itaconate (OI), the itaconate’s cell-permeable derivative, can activate Nrf2 signaling via alkylation of Keap1 at its cysteine residues. The current study tested the potential neuroprotective function of OI in hydrogen peroxide (H2O2)-treated neuronal cells. SH-SY5Y neuronal cells and epigenetically de-repressed (by TSA treatment) primary murine neurons were treated with OI and/or H2O2. Nrf2 pathway genes were examined by Western blotting assay and real-time quantitative PCR analysis. Neuronal cell death was tested by the LDH and trypan blue staining assays. Apoptosis was tested by TUNEL and Annexin V assays. In SH-SY5Y neuronal cells and primary murine neurons, OI activated Nrf2 signaling, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation, as well as expression of Nrf2-regulated genes (HO1, NQO1 and GCLC) and ninjurin2 (Ninj2). Functional studies showed that OI attenuated H2O2-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage as well as neuronal cell death and apoptosis. shRNA-mediated knockdown, or CRISPR/Cas9-induced knockout of Nrf2 almost abolished OI-induced neuroprotection against H2O2. Keap1 is the primary target of OI. Keap1 knockout by CRISPR/Cas9 method mimicked and abolished OI-induced actions in SH-SY5Y cells. Introduction of a Cys151S mutant Keap1 in SH-SY5Y cells reversed OI-induced Nrf2 activation and anti-H2O2 neuroprotection. OI activates Keap1-Nrf2 signaling to protect SH-SY5Y cells and epigenetically de-repressed primary neurons from H2O2 in vitro.

Emerging roles and regulation of MiT/TFE transcriptional factors.

Abstract: The MiT/TFE transcription factors play a pivotal role in the regulation of autophagy and lysosomal biogenesis. The subcellular localization and activity of MiT/TFE proteins are primarily regulated through phosphorylation. And the phosphorylated protein is retained in the cytoplasm and subsequently translocates to the nucleus upon dephosphorylation, where it stimulates the expression of hundreds of genes, leading to lysosomal biogenesis and autophagy induction. The transcription factor-mediated lysosome-to-nucleus signaling can be directly controlled by several signaling molecules involved in the mTORC1, PKC, and AKT pathways. MiT/TFE family members have attracted much attention owing to their intracellular clearance of pathogenic factors in numerous diseases. Recently, multiple studies have also revealed the MiT/TFE proteins as master regulators of cellular metabolic reprogramming, converging on autophagic and lysosomal function and playing a critical role in cancer, suggesting that novel therapeutic strategies could be based on the modulation of MiT/TFE family member activity. Here, we present an overview of the latest research on MiT/TFE transcriptional factors and their potential mechanisms in cancer.

HMGB2 is associated with malignancy and regulates Warburg effect by targeting LDHB and FBP1 in breast cancer.

Abstract: High-mobility group box 2 (HMGB2) is implicated in tumorigenesis in various cancers. However, the clinical significance of HMGB2 signaling in human breast cancer progression remains unknown. We investigated HMGB2 expression in 185 cases of primary breast cancer and matched normal breast tissue specimens, and explored the underlying mechanisms of altered HMGB2 expression as well as the impact of this altered expression on breast cancer growth and on aerobic glycolysis using in vitro and animal models of breast cancer. HMGB2 was more highly expressed in tumor-cell nuclei of breast cancer cells than in the adjacent normal breast tissues (P < 0.05). Higher HMGB2 expression correlated with larger tumor size (P = 0.003) and advanced tumor stage (P = 0.033). A Cox proportional hazards model revealed that HMGB2 expression was an independent prognostic factor for breast cancer after radical resection (P < 0.05). Experimentally, knockdown of HMGB2 expression by stable transfected shRNA significantly decreased the growth and glycolysis of breast cancer cells both in vitro and in mouse models. Mechanically, promotion of breast cancer progression by HMGB2 directly and significantly correlated with activation of LDHB expression and inactivation of FBP1 expression. These results disclose a novel role for HMGB2 in reprogramming the metabolic process in breast cancer cells by targeting LDHB and FBP1 and provide potential prognostic predictors for breast cancer patients.

Enhanced metastatic capacity of breast cancer cells after interaction and hybrid formation with mesenchymal stroma/stem cells (MSC)

Abstract: Fusion of breast cancer cells with tumor-associated populations of the microenvironment including mesenchymal stroma/stem-like cells (MSC) represents a rare event in cell communication whereby the metastatic capacity of those hybrid cells remains unclear. Functional changes were investigated in vitro and in vivo following spontaneous fusion and hybrid cell formation between primary human MSC and human MDA-MB-231 breast cancer cells. Thus, lentiviral eGFP-labeled MSC and breast cancer cells labeled with mcherry resulted in dual-fluorescing hybrid cells after co-culture. Double FACS sorting and single cell cloning revealed two different aneuploid male hybrid populations (MDA-hyb1 and MDA-hyb2) with different STR profiles, pronounced telomerase activities, and enhanced proliferative capacities as compared to the parental cells. Microarray-based mRNA profiling demonstrated marked regulation of genes involved in epithelial-mesenchymal transition and increased expression of metastasis-associated genes including S100A4. In vivo studies following subcutaneous injection of the breast cancer and the two hybrid populations substantiated the in vitro findings by a significantly elevated tumor growth of the hybrid cells. Moreover, both hybrid populations developed various distant organ metastases in a much shorter period of time than the parental breast cancer cells. Together, these data demonstrate spontaneous development of new tumor cell populations exhibiting different parental properties after close interaction and subsequent fusion of MSC with breast cancer cells. This formation of tumor hybrids contributes to continuously increasing tumor heterogeneity and elevated metastatic capacities.

Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

Abstract: Macrophages are one of the most important players in the tumor microenvironment. The polarization status of tumor associated macrophages into a pro-inflammatory type M1 or anti-inflammatory type M2 may influence cancer progression and patient survival. Extracellular vesicles (EVs) are membrane-bound vesicles containing different biomolecules that are involved in cell to cell signal transfer. Accumulating evidence suggests that cancer-derived EVs are taken up by macrophages and modulate their phenotype and cytokine profile. However, the interactions of cancer-derived EVs with monocytes and macrophages at various differentiation and polarization states are poorly understood. In the current study, we have analyzed the uptake and functional effects of primary (SW480) and metastatic (SW620) isogenic colorectal cancer (CRC) cell line-derived EVs on monocytes (M), inactive macrophages (M0) and M1 and M2 polarized macrophages. THP-1 monocytes were differentiated into M0 macrophages by addition of phorbol-12-myristate-13-acetate. Then M0 macrophages were further polarized into M1 and M2 macrophages in the presence of LPS, IFN- γ, IL-4, and IL-13 respectively. Internalization of SW480 and SW620-derived EVs was analyzed by flow cytometry and fluorescence microscopy. Changes in monocyte and macrophage immunophenotype and secretory profile upon EV exposure were analyzed by flow cytometry, quantitative PCR and Luminex assays. THP-1 monocytes and M0 macrophages efficiently take up SW480 and SW620-derived EVs, and our results indicate that dynamin-dependent endocytic pathways may be implicated. Interestingly, SW480 and SW620-derived EVs increased CD14 expression in M0 macrophages whereas SW480-derived EVs decreased HLA-DR expression in M1 and M2 polarized macrophages. Moreover, SW480-derived EVs significantly increased CXCL10 expression in monocytes and M0 macrophages. In contrast, SW620-derived EVs induced secretion of IL-6, CXCL10, IL-23 and IL-10 in M0 macrophages. However, addition of CRC cell line-derived EVs together with LPS, IFN- γ (M1) and IL-4, IL-13 (M2) stimuli during macrophage polarization had no additional effect on cytokine expression in M1 and M2 macrophages. Our results suggest that CRC cell line-derived EVs are internalized and reprogram the immunophenotype and secretory profile in monocytes and inactive macrophages inducing mixed M1 and M2 cytokine response. Although CRC EVs decreased HLA-DR expression in M1, M2 polarized macrophages, their effect on the secretory profile of M1 and M2 polarized macrophages was negligible.

TNFAIP8 promotes the proliferation and cisplatin chemoresistance of non-small cell lung cancer through MDM2/p53 pathway.

Abstract: The highly refractory nature of non-small cell lung cancer (NSCLC) to chemotherapeutic drugs is an important factor resulting in its poor prognosis. Recent studies have revealed that tumour necrosis factor alpha-induced protein 8 (TNFAIP8) is involved in various biological and pathological processes of cells, but their underlying mechanisms in processes ranging from cancer development to drug resistance have not been fully elucidated. TNFAIP8 expression in clinical NSCLC samples was examined through immunohistochemistry (IHC). After adjusting for patients’ characteristics with propensity score matching, Kaplan-Meier analysis and Cox regression analysis were performed for comparison of patients’ survival according to the TNFAIP8 level. Lentiviral transfection with TNFAIP8-specific shRNAs was used to establish stable TNFAIP8 knockdown (TNFAIP8 KD) NCI-H460, A549 and cis-diamminedichloroplatinum II resistant A549 (A549/cDDP) cell lines. Cell proliferation and viability were assessed by CCK-8 assay. Cell cycle was examined by flow cytometry. Multiple pathways regulated by TNFAIP8 KD were revealed by microarray analysis. We found that high TNFAIP8 expression was associated with advanced pT stage, advanced pTNM stage, lymph node metastasis and unfavourable survival in NSCLC patients. TNFAIP8 shRNAs reduced in vitro cancer cell proliferation and in vivo tumor growth. Additionally, TNFAIP8 KD increased the sensitivity of NSCLC cells to cisplatin in vitro and in vivo. Conversely, up-regulation of TNFAIP8 promoted the proliferation and drug resistance to cisplatin of NSCLC cells. TNFAIP8 influences cancer progression pathways involving the MDM2/p53 pathway. Indeed, we observed that TNFAIP8 KD mediated the MDM2 downregulation and the p53 ubiquitination, thereby decreasing the degradation of p53 protein. shRNA p53 reversed TNFAIP8 shRNA-mediated regulation of cell proliferation, cell cycle, cisplatin sensitivity, and expression levels of RAD51, a DNA repair gene. Our work uncovers a hitherto unappreciated role of TNFAIP8 in NSCLC proliferation and cisplatin chemoresistance that is mediated through the MDM2/p53 pathway. These findings might offer potential therapeutic targets for reversing cisplatin resistance in NSCLC patients with high TNFAIP8 expression.

AMPK-dependent and independent actions of P2X7 in regulation of mitochondrial and lysosomal functions in microglia.

Abstract: P2X7 is ubiquitously expressed in myeloid cells and regulates the pathophysiology of inflammatory diseases. Since mitochondrial function in microglia is highly associated with microglial functions in controlling neuronal plasticity and brain homeostasis, we interested to explore the roles of P2X7 in mitochondrial and lysosomal functions as well as mitophagy in microglia. P2X7−/− bone marrow-derived macrophages (BMDM), primary microglia and BV-2 immortalized microglial cells were used to detect the particular protein expression by immunoblotting. Mitochondrial reactive oxygen species (mitoROS), intracellular calcium, mitochondrial mass and lysosomal integrity were examined by flow cytometry. Mitochondrial oxygen consumption rate (OCR) was recorded using Seahorse XF flux analyzer. Confocal microscopic images were performed to indicate the mitochondrial dynamics and mitophagy after P2X7 activation. In primary microglia, BV-2 microglial cells and BMDM, P2X7 agonist BzATP triggered AMPK activation and LC3II accumulation through reactive oxygen species (ROS) and CaMKKII pathways, and these effects were abolished by P2X7 antagonist A438079 and P2X7 deficiency. Moreover, we detected the dramatic decreases of mitochondrial OCR and mass following P2X7 activation. AMPK inhibition by compound C or AMPK silencing reversed the P2X7 actions in reduction of mitochondrial mass, induction of mitochondrial fission and mitophagy, but not in uncoupling of mitochondrial respiration. Interestingly, we found that P2X7 activation induced nuclear translocation of TFEB via an AMPK-dependent pathway and led to lysosomal biogenesis. Mimicking the actions of BzATP, nigericin also induced ROS-dependent AMPK activation, mitophagy, mitochondrial fission and respiratory inhibition. Longer exposure of BzATP induced cell death, and this effect was accompanied by the lysosomal instability and was inhibited by autophagy and cathepsin B inhibitors. Altogether ROS- and CaMKK-dependent AMPK activation is involved in P2X7-mediated mitophagy, mitochondrial dynamics and lysosomal biogenesis in microglial cells, which is followed by cytotoxicity partially resulting from mitophagy and cathepsin B activation.

Functional implications of Rab27 GTPases in Cancer.

Abstract: The Rab27 family of small GTPases promotes the progression of breast cancer, melanoma, and other human cancers. In this review, we discuss the role of Rab27 GTPases in cancer progression and the potential applications of these targets in cancer treatment. Elevated expression of Rab27 GTPases is associated with poor prognosis and cancer metastasis. Moreover, these GTPases govern a variety of oncogenic functions, including cell proliferation, cell motility, and chemosensitivity. In addition, small GTPases promote tumor growth and metastasis by enhancing exosome secretion, which alters intracellular microRNA levels, signaling molecule expression, and the tumor microenvironment. Rab27 GTPases may have applications as prognostic markers and therapeutic targets in cancer treatment.

WNK pathways in cancer signaling networks

Abstract: The with no lysine [K] (WNK) pathway consists of the structurally unique WNK kinases, their downstream target kinases, oxidative stress responsive (OSR)1 and SPS/Ste20-related proline-alanine-rich kinase (SPAK), and a multitude of OSR1/SPAK substrates including cation chloride cotransporters. While the best known functions of the WNK pathway is regulation of ion transport across cell membranes, WNK pathway components have been implicated in numerous human diseases. The goal of our review is to draw attention to how this pathway and its components exert influence on the progression of cancer, specifically by detailing WNK signaling intersections with major cell communication networks and processes. Here we describe how WNKs and associated proteins interact with and influence PI3K-AKT, TGF-β, and NF-κB signaling, as well as its unanticipated role in the regulation of angiogenesis.

Interleukin-15 facilitates muscle regeneration through modulation of fibro/adipogenic progenitors.

Abstract: Chronic muscle injury is characteristics of fatty infiltration and fibrosis. Recently, fibro/adipogenic progenitors (FAPs) were found to be indispensable for muscular regeneration while were also responsible for fibrosis and fatty infiltration in muscle injury. Many myokines have been proven to regulate the adipose or cell proliferation. Because the fate of FAPs is largely dependent on microenvironment and the regulation of myokines on FAPs is still unclear. We screened the potential myokines and found Interleukin-15 (IL-15) may regulate the fatty infiltration in muscle injury. In this study, we investigated how IL-15 regulated FAPs in muscle injury and the effect on muscle regeneration. Cell proliferation assay, western blots, qRT-PCR, immunohistochemistry, flow cytometric analysis were performed to investigate the effect of IL-15 on proliferation and adipogensis of FAPs. Acute muscle injury was induced by injection of glycerol or cardiotoxin to analyze how IL-15 effected on FAPs in vivo and its function on fatty infiltration or muscle regeneration. We identified that the expression of IL-15 in injured muscle was negatively associated with fatty infiltration. IL-15 can stimulate the proliferation of FAPs and prevent the adipogenesis of FAPs in vitro and in vivo. The growth of FAPs caused by IL-15 was mediated through JAK-STAT pathway. In addition, desert hedgehog pathway may participate in IL-15 inhibiting adipogenesis of FAPs. Our study showed IL-15 can cause the fibrosis after muscle damage and promote the myofiber regeneration. Finally, the expression of IL-15 was positively associated with severity of fibrosis and number of FAPs in patients with chronic rotator cuff tear. These findings supported the potential role of IL-15 as a modulator on fate of FAPs in injured muscle and as a novel therapy for chronic muscle injury.

Notch signaling triggered via the ligand DLL4 impedes M2 macrophage differentiation and promotes their apoptosis.

Abstract: Notch signaling controls many cellular processes, including cell fate determination, cell differentiation, proliferation and apoptosis. In mammals, four Notch receptors (Notch 1–4) can interact with five distinct ligands [Jagged1, Jagged2, Delta-like 1 (DLL1), DLL3, and DLL4]. We previously reported that Notch activation is modulated in endothelial cells and monocytes during inflammation and showed that inflammation upregulates DLL4 on endothelial cells. DLL4 promotes differentiation of blood monocytes into proinflammatory M1 macrophages. Here, we further investigated the ability of DLL4 to interfere with the polarization of blood monocytes into immunosuppressive M2 macrophages. Human blood monocytes were differentiated in vitro into M0 macrophages and then polarized into M1 or M2 macrophages with LPS/IFNγ and IL-4, respectively. Polarization steps were performed in the presence of immobilized recombinant DLL4. Immune phenotype and apoptosis of macrophage subsets were analyzed and quantified by flow cytometry. Regulatory effects of DLL4 on gene expression, cell signaling and apoptotic pathways were investigated by QPCR and western blots. The phenotype of M2 macrophages was subject to specific alterations in the presence of recombinant DLL4. DLL4 inhibits the upregulation of IL-4 induced M2 markers such as CD11b, CD206, and CD200R. Survival of macrophages upon M2 polarization was also strongly reduced in the presence of DLL4. DLL4 induces a caspase3/7-dependent apoptosis during M2 but not M1 macrophage polarization. The Notch ligand DLL1 has no apoptotic effect. Both DLL4 signaling via Notch1 as well as DLL4-mediated apoptosis are Notch-dependent. Fully differentiated M2 macrophages became resistant to DLL4 action. Mechanistically, DLL4 selectively upregulates gene expression in macrophages upon M2 polarization, thereby affecting the Notch pattern (Notch1, 3, Jag1), activity (HES1), and transcription (IRF5, STAT1). The pro-apoptotic effectors Bax and Bak and the BH3-only proteins Bid and Bim seem to convey DLL4 apoptotic signal. Interplay between the DLL4/Notch and IL-4/IL-4R signaling pathways impairs M2 differentiation. Thus, DLL4 may drive a Notch-dependent selection process not only by promoting M1 macrophage differentiation but also by preventing M2 macrophage differentiation through inhibition of M2-specific gene expression and apoptotic cell death.

Mast cells and mast cell tryptase enhance migration of human lung fibroblasts through protease-activated receptor 2

Abstract: Mast cells may activate fibroblasts and contribute to remodeling processes in the lung. However, the mechanism behind these actions needs to be further investigated. Fibroblasts are major regulators of on-going remodeling processes. Protease activated receptor 2 (PAR2) expressed by fibroblasts may be activated by serine proteases, such as the mast cell mediator tryptase. The objective in this study was to investigate the effects of mast cells and specifically mast cell tryptase on fibroblast migration and the role of PAR2 activation. Human lung fibroblasts (HFL-1) were cultured together with human peripheral blood-derived mast cells or LAD2 mast cells and stimulated with either conditioned medium from LAD2 cells or tryptase. Analyses of immunological stimulation of mast cells by IgE/anti IgE in the co-culture system were also performed. The importance of PAR2 activation by mast cells and mast cell tryptase for the migratory effects of fibroblasts was investigated by pre-treatment with the PAR2 antagonist P2pal-18S. The expression of PAR2 was analyzed on fibroblasts and mast cells. The migratory capacity of HFL-1 cells was enhanced by blood-derived mast cells (p < 0.02), LAD2 cells (p < 0.001), conditioned medium (p < 0.05) and tryptase (p < 0.006). P2pal-18S decreased the induced migration caused by mast cells (p < 0.001) and tryptase (p < 0.001) and the expression of PAR2 was verified in HFL-1 cells. Mast cells immunologically stimulated with IgE/Anti IgE had no further effects on fibroblast migration. Mast cells and the mast cell mediator tryptase may have crucial roles in inducing lung fibroblast migration via PAR-2 activation, which may contribute to remodeling processes in chronic lung diseases.

LncRNA HOTAIR promotes cell migration and invasion by regulating MKL1 via inhibition miR206 expression in HeLa cells

Abstract: Long non-coding RNAs (lncRNAs) have emerged as a new and crucial layer of gene regulation in recent years and regulate various biological processes such as carcinogenesis and metastasis. LncRNA HOTAIR, an oncogenic lncRNA, is involved in human tumorigenesis and dysregulated in cervical cancer. Megakaryoblastic leukemia 1 (MKL1), as a transcription coactivity factor, involved in cancer metastasis and cell differentiation. However, the precise mechanism of biological roles of HOTAIR and MKL1 in cancer cells remain unclear. The expression levels of HOTAIR and MKL1 were measured by quantitative PCR (qPCR), immunoblotting, in situ hybridization (ISH) and immunohistochemistry (IHC). Wound-healing and transwell assays were used to examine the invasive abilities of HeLa cells. Luciferase reporter assays and CHIP were used to determine how MKL1 regulates HOTAIR. Tissue microarray and immunohistochemical staining were used to assess the correlation between HOTAIR and MKL1 in Cervical cancer tissues in vivo. In this study, we have identified that MKL1 had a role in the induction of migration and invasion in cervical cancer cells. Moreover, the expression level of MKL1, as the targeting gene of miR206, was decreased after HOTAIR inhibition in HeLa cells. Agreement with it, Highly level of MKL1 correlation with HOTAIR is validated in cervical cancer tissues. Importantly, HOTAIR is observed to participate in the silencing of miR206 expression. Interestingly, HOTAIR inhibition could also accelerate the expression of MKL1 in cytoplasm. What is more, MKL1 can activate the transcription of HOTAIR through binding the CArG box in the promoter of HOTAIR. These elucidates that the phenotypic effects of migration and invasion observed after HOTAIR inhibition, at least in part, through the regulation of MKL1 via inhibition of miR206 expression in HeLa cells. These data indicate the existence of a positive feedback loop between HOTAIR and MKL1. Together, these findings suggest that MKL1 is an important player in the functions of HOTAIR in the migration and invasion of cancer cells.

Utilization of adipocyte-derived lipids and enhanced intracellular trafficking of fatty acids contribute to breast cancer progression

Abstract: To determine whether adipocyte-derived lipids could be transferred into breast cancer cells and investigate the underlying mechanisms of subsequent lipolysis and fatty acid trafficking in breast cancer cells. A Transwell co-culture system was used in which human breast cancer cells were cultured in the absence or presence of differentiated murine 3 T3-L1 adipocytes. Migration/invasion and proliferation abilities were compared between breast cancer cells that were cultivated alone and those co-cultivated with mature adipocytes. The ability of lipolysis in breast cancer cells were measured, as well as the expression of the rate-limiting lipase ATGL and fatty acid transporter FABP5. ATGL and FABP5 were then ablated to investigate their impact on the aggressiveness of breast cancer cells that were surrounded by adipocytes. Further, immunohistochemistry was performed to detect differential expression of ATGL and FABP5 in breast cancer tissue sections. The migration and invasion abilities of cancer cells were significantly enhanced after co-culture with adipocytes, accompanied by elevated lipolysis and expression of ATGL and FABP5. Abrogation of ATGL and FABP5 sharply attenuated the malignancy of co-cultivated breast cancer cells. However, this phenomenon was not observed if a lipid emulsion was added to the culture medium to substitute for adipocytes. Furthermore, epithelial-mesenchymal transaction was induced in co-cultivated breast cancer cells. That may partially due to the stimulation of PPARβ/δ and MAPK, which was resulted from upregulation of FABP5. As evidenced by immunohistochemistry, ATGL and FABP5 also had higher expression levels at the invasive front of the breast tumor, in where the adipocytes abound, compared to the central area in tissue specimens. Lipid originating from tumor-surrounding adipocytes could be transferred into breast cancer cells. Adipocyte-cancer cell crosstalk rather than lipids alone induced upregulation of lipases and fatty acid transport protein in cancer cells to utilize stored lipids for tumor progression. The increased expression of the key lipase ATGL and intracellular fatty acid trafficking protein FABP5 played crucial roles in this process via fueling or signaling.

Reawakening of dormant estrogen-dependent human breast cancer cells by bone marrow stroma secretory senescence.

Abstract: Dormant estrogen receptor positive (ER+) breast cancer micrometastases in the bone marrow survive adjuvant chemotherapy and recur stochastically for more than 20 years. We hypothesized that inflammatory cytokines produced by stromal injury can re-awaken dormant breast cancer cells. We used an established in vitro dormancy model of Michigan Cancer Foundation-7 (MCF-7) breast cancer cells incubated at clonogenic density on fibronectin-coated plates to determine the effects of inflammatory cytokines on reactivation of dormant ER+ breast cancer cells. We measured induction of a mesenchymal phenotype, motility and the capacity to re-enter dormancy. We induced secretory senescence in murine stromal monolayers by oxidation, hypoxia and estrogen deprivation with hydrogen peroxide (H2O2), carbonyl-cyanide m-chlorophenylhydrazzone (CCCP) and Fulvestrant (ICI 182780), respectively, and determined the effects on growth of co-cultivated breast cancer cells. Exogenous recombinant human (rh) interleukin (IL)-6, IL-8 or transforming growth factor β1 (TGFβ1) induced regrowth of dormant MCF-7 cells on fibronectin-coated plates. Dormant cells had decreased expression of E-cadherin and estrogen receptor α (ERα) and increased expression of N-cadherin and SNAI2 (SLUG). Cytokine or TGFβ1 treatment of dormant clones induced formation of growing clones, a mesenchymal appearance, increased motility and an impaired capacity to re-enter dormancy. Stromal injury induced secretion of IL-6, IL-8, upregulated tumor necrosis factor alpha (TNFα), activated TGFβ and stimulated the growth of co-cultivated MCF-7 cells. MCF-7 cells induced secretion of IL-6 and IL-8 by stroma in co-culture. Dormant ER+ breast cancer cells have activated epithelial mesenchymal transition (EMT) gene expression programs and downregulated ERα but maintain a dormant epithelial phenotype. Stromal inflammation reactivates these cells, induces growth and a mesenchymal phenotype. Reactivated, growing cells have an impaired ability to re-enter dormancy. In turn, breast cancer cells co-cultured with stroma induce secretion of IL-6 and IL-8 by the stroma, creating a positive feedback loop.

The nuclear transportation routes of membrane-bound transcription factors

Abstract: Membrane-bound transcription factors (MTFs) are transcription factors (TFs) that are anchored in membranes in a dormant state. Activated by external or internal stimuli, MTFs are released from parent membranes and are transported to the nucleus. Existing research indicates that some plasma membrane (PM)-bound proteins and some endoplasmic reticulum (ER) membrane-bound proteins have the ability to enter the nucleus. Upon specific signal recognition cues, some PM-bound TFs undergo proteolytic cleavage to liberate the intracellular fragments that enter the nucleus to control gene transcription. However, lipid-anchored PM-bound proteins enter the nucleus in their full length for depalmitoylation. In addition, some PM-bound TFs exist as full-length proteins in cell nucleus via trafficking to the Golgi and the ER, where membrane-releasing mechanisms rely on endocytosis. In contrast, the ER membrane-bound TFs relocate to the nucleus directly or by trafficking to the Golgi. In both of these pathways, only the fragments of the ER membrane-bound TFs transit to the nucleus. Several different nuclear trafficking modes of MTFs are summarized in this review, providing an effective supplement to the mechanisms of signal transduction and gene regulation. Moreover, targeting intracellular movement pathways of disease-associated MTFs may significantly improve the survival of patients.

Overexpressed HDAC8 in cervical cancer cells shows functional redundancy of tubulin deacetylation with HDAC6

Abstract: Histone deacetylases (HDACs) are involved in epigenetic gene regulation via deacetylation of acetylated lysine residues of both histone and non-histone proteins. Among the 18 HDACs identified in humans, HDAC8, a class I HDAC, is best understood structurally and enzymatically. However, its precise subcellular location, function in normal cellular physiology, its protein partners and substrates still remain elusive. The subcellular localization of HDAC8 was studied using immunofluorescence and confocal imaging. The binding parterns were identified employing immunoprecipitation (IP) followed by MALDI-TOF analysis and confirmed using in-silico protein-protein interaction studies, HPLC-based in vitro deacetylation assay, intrinsic fluorescence spectrophotometric analysis, Circular dichroism (CD) and Surface Plasmon Resonance (SPR). Functional characterization of the binding was carried out using immunoblot and knockdown by siRNA. Using one way ANOVA statistical significance (n = 3) was determined. Here, we show that HDAC8 and its phosphorylated form (pHDAC8) localized predominantly in the cytoplasm in cancerous, HeLa, and non-cancerous (normal), HEK293T, cells, although nucleolar localization was observed in HeLa cells. The study identified Alpha tubulin as a novel interacting partner of HDAC8. Further, the results indicated binding and deacetylation of tubulin at ac-lys40 by HDAC8. Knockdown of HDAC8 by siRNA, inhibition of HDAC8 and/or HDAC6 by PCI-34051 and tubastatin respectively, cell-migration, cell morphology and cell cycle analysis clearly explained HDAC8 as tubulin deacetylase in HeLa cells and HDAC6 in HEK 293 T cells. HDAC8 shows functional redundancy with HDAC6 when overexpressed in cervical cancer cells, HeLa, and deacetylaes ac-lys40 of alpha tubulin leading to cervical cancer proliferation and progression.

Wilms’ tumor 1-associating protein plays an aggressive role in diffuse large B-cell lymphoma and forms a complex with BCL6 via Hsp90

Abstract: Wilms’ tumor 1-associating protein (WTAP) is a nuclear protein, which is ubiquitously expressed in many tissues. Furthermore, in various types of malignancies WTAP is overexpressed and plays a role as an oncogene. The function of WTAP in diffuse large B-cell lymphoma (DLBCL), however, remains unclear. Immunohistochemistry was applied to evaluate the levels of WTAP expression in DLBCL tissues and normal lymphoid tissues. Overexpression and knock-down of WTAP in DLBCL cell lines, verified on mRNA and protein level served to analyze cell proliferation and apoptosis in DLBCL cell lines by flow cytometry. Finally, co-immunoprecipitation (Co-IP), IP, and GST-pull down assessed the interaction of WTAP with Heat shock protein 90 (Hsp90) and B-cell lymphoma 6 (BCL6) as well as determined the extend of its ubiquitinylation. WTAP protein levels were consistently upregulated in DLBCL tissues. WTAP promoted DLBCL cell proliferation and improved the ability to confront apoptosis, while knockdown of WTAP in DLBCL cell lines allowed a significant higher apoptosis rate after treatment with Etoposide, an anti-tumor drug. The stable expression of WTAP was depended on Hsp90. In line, we demonstrated that WTAP could form a complex with BCL6 via Hsp90 in vivo and in vitro. WTAP is highly expressed in DLBCL, promoting growth and anti-apoptosis in DLBCL cell lines. WTAP is a client protein of Hsp90 and can appear in a complex with BCL6 and Hsp90 in DLBCL. Down-regulation of WTAP could improve the chemotherapeutic treatments in DLBCL.