Positive Feedback Manner Research Articles

Acid sphingomyelinase-dependent autophagic degradation of GPX4 is critical for the execution of ferroptosis

Ferroptosis is a type of regulated cell death characterized by ROS accumulation and devastating lipid peroxidation (LPO). The role of acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, in the induction of apoptosis has been studied; however, to date its role in ferroptosis is unclear. In this study, we report that ASM plays a hitherto unanticipated role in promoting ferroptosis. Mechanistically, Erastin (Era) treatment results in the activation of ASM and generation of ceramide, which are required for the Era-induced reactive oxygen species (ROS) generation and LPO. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or removal of intracellular ROS, significantly reduced Era-induced ASM activation, suggesting that NADPH oxidase-derived ROS regulated ASM-initiated redox signaling in a positive feedback manner. Moreover, ASM-mediated activation of autophagy plays a critical role in ferroptosis inducers (FINs)-induced glutathione peroxidase 4 (GPX4) degradation and ferroptosis activation. Genetic or pharmacological inhibition of ASM diminishes Era-induced features of autophagy, GPX4 degradation, LPO, and subsequent ferroptosis. Importantly, genetic activation of ASM increases ferroptosis in cancer cells induced by various FINs. Collectively, these findings reveal that ASM plays a novel role in ferroptosis that could be exploited to improve pathological conditions that link to ferroptosis.

CBIO-20. MOLECULAR MECHANISMS OF OLIG2 TRANSCRIPTION FACTOR IN GLIOBLASTOMA

Abstract Oligodendrocyte lineage transcription factor 2 (OLIG2) promotes proliferation of normal neural stem/progenitor cells and glioma cells. However, the mechanisms underlying the regulation of OLIG2 remain largely unknown. Here, we show that a comprehensive analysis of the critical gene regulatory networks involving OLIG2 in glioma initiating cell (GIC) lines. In vitro differentiation studies showed that proneural GIC lines possess the potential to differentiate into astrocytic, neuronal, and oligodendrocytic lineages, whereas mesenchymal GICs exhibited limited potential for neural lineage differentiation following retinoic acid induction. We also showed that CDK2-mediated OLIG2 phosphorylation stabilizes OLIG2 protein from proteasomal degradation. Phosphorylated OLIG2 binds to the E-Box regions of p27 promoter and represses p27 transcription, which in turn activates CDK2 in positive feedback manner. CDK2-mediated OLIG2 phosphorylation promotes cell cycle progression, cell proliferation, and tumorigenesis. OLIG2 inhibition disrupted cell cycle control mechanism by decreasing CDK2 and elevating apoptosis-related molecules. Inhibition of CDK2 activity disrupted OLIG2-CDK2 interactions and attenuated OLIG2 protein stability. In addition, OLIG2-high glioma initiating cells are highly sensitive to CDK2 inhibitor treatment, indicating that OLIG2 can be a biomarker for personalized treatment for glioblastoma patients with CDK2 inhibitors. In conclusion, we have identified OLIG2-CDK2 interactions in glioma stem cells that can be targeted by CDK2 inhibitors and this may allow the selection of patients with high likelihood of responding to this therapy.

Abstract 5826: Molecular mechanism of OLIG2-CDK2 interaction in glioma cells

Abstract Oligodendrocyte transcription factor 2 (Olig2) promotes proliferation of normal neural stem/progenitor cells and glioma cells. However, the mechanisms underlying the regulation of Olig2 remain largely unknown. Here we identified Olig2 as a critical phosphorylation target for cyclin-dependent kinase 2 (CDK2). CDK2-mediated Olig2 phosphorylation stabilizes Olig2 protein from proteasomal degradation. Phosphorylated Olig2 binds to the E-Box regions of p27 promoter and represses p27 transcription, which in turn activates CDK2 in positive feedback manner. CDK2-mediated Olig2 phosphorylation promotes cell cycle progression, cell proliferation, and tumorigenesis. Olig2 inhibition disrupted cell cycle control mechanism by decreasing CDK2 and elevating apoptosis-related molecules. Inhibition of CDK2 activity disrupted Olig2-CDK2 interactions and attenuated Olig2 protein stability. In addition, Olig2-high glioma initiating cells are highly sensitive to CDK2 inhibitor treatment, indicating that Olig2 can be a biomarker for personalized treatment for glioblastoma patients with CDK2 inhibitors. Further investigation on these mechanisms may lead to novel targeted therapy on GBMs with high Olig2 expression. Citation Format: Norihiko Saito, Nozomi Hirai, Sho Sato, Yu Hiramoto, Satoshi Fujita, Haruo Nakayama, Morito Hayashi, Takatoshi Sakurai, Satoshi Iwabuchi. Molecular mechanism of OLIG2-CDK2 interaction in glioma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5826.

Stretching our understanding of baroreflex control in humans: evidence of a positive feedback pulmonary baroreflex.

Stretching our understanding of baroreflex control in humans: evidence of a positive feedback pulmonary baroreflex.

CBMT-04. MOLECULAR MECHANISM OF OLIG2-CDK2 INTERACTION IN GLIOMA CELLS

Abstract Oligodendrocyte transcription factor 2 (OLIG2) promotes proliferation of normal neural stem/progenitor cells and glioma cells. However, the mechanisms underlying the regulation of OLIG2 remain largely unknown. Here we identified OLIG2 as a critical phosphorylation target for cyclin-dependent kinase 2 (CDK2). CDK2-mediated OLIG2 phosphorylation stabilizes OLIG2 protein from proteasomal degradation. Phosphorylated OLIG2 binds to the E-Box regions of p27 promoter and represses p27 transcription, which in turn activates CDK2 in positive feedback manner. CDK2-mediated OLIG2 phosphorylation promotes cell cycle progression, cell proliferation, and tumorigenesis. OLIG2 inhibition disrupted cell cycle control mechanism by decreasing CDK2 and elevating apoptosis-related molecules. Inhibition of CDK2 activity disrupted OLIG2-CDK2 interactions and attenuated OLIG2 protein stability. In addition, OLIG2-high glioma initiating cells are highly sensitive to CDK2 inhibitor treatment, indicating that OLIG2 can be a biomarker for personalized treatment for glioblastoma patients with CDK2 inhibitors. Further investigation on these mechanisms may lead to novel targeted therapy on GBMs with high OLIG2 expression.

Developmental Rewiring between Cerebellar Climbing Fibers and Purkinje Cells Begins with Positive Feedback Synapse Addition

SUMMARYDuring postnatal development, cerebellar climbing fibers alter their innervation strengths onto supernumerary Purkinje cell targets, generating a one-to-few connectivity pattern in adulthood. To get insight about the processes responsible for this remapping, we reconstructed serial electron microscopy datasets from mice during the first postnatal week. Between days 3 and 7, individual climbing fibers selectively add many synapses onto a subset of Purkinje targets in a positive-feedback manner, without pruning synapses from other targets. Active zone sizes of synapses associated with powerful versus weak inputs are indistinguishable. Changes in synapse number are thus the predominant form of early developmental plasticity. Finally, the numbers of climbing fibers and Purkinje cells in a local region nearly match. Initial over-innervation of Purkinje cells by climbing fibers is therefore economical: the number of axons entering a region is enough to assure that each ultimately retains a postsynaptic target and that none branched there in vain.

Hydrogen peroxide inducible clone-5 sustains NADPH oxidase-dependent reactive oxygen species-c-jun N-terminal kinase signaling in hepatocellular carcinoma

Target therapy aiming at critical molecules within the metastatic signal pathways is essential for prevention of hepatocellular carcinoma (HCC) progression. Hic-5 (hydrogen peroxide inducible clone-5) which belongs to the paxillin superfamily, can be stimulated by a lot of metastatic factors, such as transforming growth factor (TGF-β), hepatocyte growth factor (HGF), and reactive oxygen species (ROS). Previous studies implicated Hic-5 cross-talks with the ROS-c-jun N-terminal kinase (JNK) signal cascade in a positive feedback manner. In this report, we addressed this issue in a comprehensive manner. By RNA interference and ectopic Hic-5 expression, we demonstrated Hic-5 was essential for activation of NADPH oxidase and ROS generation leading to activation of downstream JNK and c-jun transcription factor. This was initiated by interaction of Hic-5 with the regulator and adaptor of NADPH oxidase, Rac1 and Traf4, respectively, which may further phosphorylate the nonreceptor tyrosine kinase Pyk2 at Tyr881. On the other hand, promoter activity assay coupled with deletion mapping and site directed mutagenesis strategies demonstrated the distal c-jun and AP4 putative binding regions (943–1126 bp upstream of translational start site) were required for transcriptional activation of Hic-5. Thus Hic-5 was both downstream and upstream of NADPH oxidase-ROS-JNK-c-jun cascade. This signal circuit was essential for regulating the expression of epithelial mesenchymal transition (EMT) factors, such as Snail, Zeb1, E-cadherin, and matrix metalloproteinase 9, involved in HCC cell migration and metastasis. Due to the limited expression of Hic-5 in normal tissue, it can be a promising therapeutic target for preventing HCC metastasis.

Angiotensin II promotes ovarian cancer spheroid formation and metastasis by upregulation of lipid desaturation and suppression of endoplasmic reticulum stress

BackgroundAngiotensin II (ANGII) and its receptor (AGTR1) have been proposed as significant contributors to metastasis in multiple cancers. Further, high AGTR1 levels are associated with poor epithelial ovarian cancer (EOC) outcomes. However, the mechanistic basis for these effects is unknown. Recent studies have suggested that ovarian cancer metastasis is highly dependent on the formation of multicellular spheroids (MCS). To understand the associations between the ANGII/AGTR1 pathway and cancer outcomes, we evaluated the effects of ANGII on MCS formation by ovarian cancer cells and used a proteomic approach to analyze the mechanistic basis.MethodsWe used the data from the GENT database and immunohistochemistry staining to assess the AGTR1 expression in epithelial ovarian cancer (EOC) patients and to assess its role in cancer progression. Colony formation assay, 3D culture assay, and transwell assays were used to analyze the effect of ANGII on the MCS formation and cell migration. The signaling pathways of AGTR1 and transactivation of epidermal growth factor receptor (EGFR) transactivation were investigated by the western blotting analysis. Xenograft models were used to determine the role of AGTR1 in ovarian cancer metastasis. ANGII release from ovarian cancer cells and ANGII levels in the EOC ascites fluid were measured by immunoassay. A shotgun proteomic approach was used to explore the detail molecular mechanism. Modulation of lipid desaturation and endoplasmic reticulum stress were verified by the in vitro and in vivo functional assays.ResultsAGTR1 expression was negatively correlated with EOC prognosis. AGTR1activation significantly enhanced the MCS formation and cell migration. ANGII triggered both of the classical AGTR1 pathway and the EGFR transactivation. ANGII administration increased peritoneal metastasis. In addition, ovarian cancer cells secreted ANGII and enhanced cancer metastasis in a positive feedback manner. Based on the proteomic data, lipid desaturation was activated by induction of stearoyl-CoA desaturase-1 (SCD1), which suggests that inhibition of SCD1 may significantly reduce MCS formation by increasing endoplasmic reticulum stress.ConclusionsANGII promotes MCS formation and peritoneal metastasis of EOC cells. AGTR1 activation increases the lipid desaturation via SCD1 upregulation, which ultimately reduces endoplasmic reticulum stress in MCS. This mechanism explained the association between high levels of AGTR1 and poor clinical outcomes in EOC patients.

HAT1 signaling confers to assembly and epigenetic regulation of HBV cccDNA minichromosome.

Rationale: Hepatitis B virus (HBV) is a leading cause of liver diseases. HBV covalently closed circular DNA (cccDNA) is a critical obstacle of complete elimination by anti-HBV therapy. HBV cccDNA accumulates in nucleus as a chromatin-like cccDNA minichromosome assembled by histones and non-histones. However, the underlying mechanism of modulation of cccDNA minichromosome in hepatocytes is poorly understood.Methods: A human liver-chimeric mouse model was established. The cccDNA-ChIP, Southern blot analysis, confocal assays, RIP assays and RNA pull-down assays, et al. were performed to assess the mechanism of assembly and epigenetic regulation of cccDNA minichromosome in human liver-chimeric mouse model, human primary hepatocytes (PHH), dHepaRG, HepG2-NTCP cell lines and clinical liver tissues.Results: Importantly, the expression levels of HAT1, CAF-1 and lncRNA HULC were significantly elevated in the liver from HBV-infected human liver-chimeric mice. Strikingly, the depletion of HAT1 reduced HBV replication and cccDNA accumulation, and impaired the assembly of histone H3/H4 and the deposition of HBx and p300 onto cccDNA to form cccDNA minichromosome in the cells. Mechanically, chromatin assembly factor-1 (CAF-1) was involved in the events. Interestingly, HAT1 modified the acetylation of histone H3K27/H4K5/H4K12 on cccDNA minichromosome. Moreover, lncRNA HULC-scaffold HAT1/HULC/HBc complex was responsible for the modification on cccDNA minichromosome. Additionally, HBV activated HAT1 through HBx-co-activated transcriptional factor Sp1 in a positive feedback manner.Conclusion: HAT1 signaling contributes to assembly and epigenetic regulation of HBV cccDNA minichromosome.

Indian Hedgehog links obesity to development of hepatocellular carcinoma.

Obesity increases the risk of hepatocellular carcinoma (HCC), but precise identification and characterization of druggable oncogenic pathways that contribute to the progression of NAFLD to HCC, and hence to the increased incidence and aggressiveness of HCC in obese individuals is lacking. In this regard, we demonstrate that the Indian Hedgehog (Ihh) signaling pathway is upregulated in the fatty livers of mice consuming a high fat diet, and furthermore sustained in HCC tumors specifically within the context of a NAFLD microenvironment. Using a diet-induced mouse model of HCC wherein only obese mice develop HCC, targeted ablation of hepatocyte-secreted Ihh results in a decreased tumor burden and lower grade tumors. Ihh activation regulates the transdifferentiation of ciliated stellate cells and proliferation of Epcam+ ductal cells to promote fibrosis. Mechanistically, increased expression of hitherto uncharacterized effectors of Hh pathway, namely Myc and Tgf-β2 is critical to the observed physiology. This pro-tumorigenic response is driven by increased expression of Wnt5a to effect a poorly-differentiated and invasive tumor phenotype. Wnt5a secreted from activated stellate cells act on Ror2-expressing hepatocytes. We further demonstrate that Wnt5a expression is also elevated in poorly-differentiated HCC cells, suggesting that these ligands are also able to function in an autocrine positive feedback manner to sustain poorly-differentiated tumors. Taken together, our study provides a mechanistic understanding for how Ihh signaling promotes HCC tumorigenesis specifically in obese mice. We propose that therapeutic targeting of the Hh pathway offers benefit for patients with dietary / NAFLD-driven steatotic HCC.

OxInflammation: From Subclinical Condition to Pathological Biomarker.

Inflammation is a complex systemic response evolved to cope with cellular injury, either due to infectious agents or, in general, with sporadic events challenging tissue integrity and function. Researchers involved in different fields have the tendency to look at the inflammatory response with different angles, according to their specific interest. Established its complexity, one of the most evident features of the inflammatory response is the generation of a pro-oxidative environment due to the production of high fluxes of pro-oxidant species. This production begins locally, close to the sites of tissue damage or infection, but eventually becomes a chronic challenge for the organism, if the inflammatory response is not properly controlled. In this review, we focus on this specific aspect of chronic, low-level sub-clinical inflammatory response. We propose the term “OxInflammation” as a novel operative term describing a permanent pro-oxidative feature that interact, in a positive feed-back manner, to a not yet clinically detectable inflammatory process, leading in a long run (chronically) to a systemic/local damage, as a consequence of the cross talk between inflammatory, and oxidative stress mediators. Therefore, it could be useful to analyze inflammatory markers in pathologies where there is an alteration of the redox homeostasis, although an inflammatory status is not clinically evident.

OP-41 - Nitroxide radical-containing nanoparticles impairs the tumorigenic potential of triple negative breast cancer

Persistent elevated intracellular reactive oxygen species (ROS), requisite for cancerous phenotypes, has been known to affect cancers in a positive feedback manner, by playing critical roles in survival, proliferation and metastasis processes. Hence, scavenging of ROS to abrogate cell survival signaling pathways may therefore offer a potential therapeutic regime to mitigate the progression of various cancers. We therefore synthesized ROS scavenging nitroxide radical-containing nanoparticles (RNPs); pH insensitive RNPO and pH sensitive RNPN, to impede the proliferative and metastatic characteristics of the triple negative breast cancer cell line, MDA-MB-231, both in vitro and in vivo. RNPs significantly inhibited proliferation, colony forming potential, migration and invasion of MDA-MB-231 cells, accompanied by decrease in ROS level. Furthermore, anti-tumor and anti-metastatic potential of RNPs was observed in an MDA-MB-231 xenograft mouse model via downregulation of ROS-sensitive transcriptional factor, NF-κB and migration regulator, MMP-2 with negligible adverse effect compared to low molecular weight TEMPOL and paclitaxel. Conclusively, our findings provide insights into the use of nanoparticle over low molecular weight antioxidants in the redox-based cancer therapeutics.

Overexpression of secretory phospholipase A2-IIa supports cancer stem cell phenotype via HER/ERBB-elicited signaling in lung and prostate cancer cells.

Resistance to conventional chemotherapies remains a significant clinical challenge in treatment of cancer. The cancer stem cells (CSCs) have properties necessary for tumor initiation, resistance to therapy, and progression. HER/ERBB‑elicited signaling supports CSC properties. Our previous studies revealed that secretory phospholipaseA2 groupIIa (sPLA2‑IIa) is overexpressed in both prostate and lung cancer cells, leading to an aberrant high level in the interstitial fluid, i.e.,tumor microenvironment and blood. HER/ERBB-PI3K-Akt-NF-κB signaling stimulates sPLA2‑IIa overexpression, and in turn, sPLA2‑IIa activates EGFR family receptors and HER/ERBB-elicited signaling and stimulates sPLA2‑IIa overexpression in a positive feedback manner. The present study determined the molecular mechanisms of sPLA2‑IIa in stimulating HER/ERBB-elicited signaling and supporting CSC properties. We found that sPLA2‑IIa binds both EGFR and HER3 demonstrated by co-immunoprecipitation experiments and also indirectly interacts with HER2, suggesting that sPLA2‑IIa functions as a ligand for both EGFR and HER3. Furthermore, both side population CSCs from non-small cell lung cancer (NSCLC) A549 and H1975 cells and ALDH1‑high CSCs from castration-resistant prostate cancer (CRPC) 22Rv1 cells overexpress sPLA2‑IIa and produce tumors when inoculated into subcutis of nude mice. Given an aberrant high level of sPLA2‑IIa in the tumor microenvironment that should be much higher than that in the blood, our findings support the notion that sPLA2‑IIa functions as a ligand for EGFR family receptors and supports CSC properties via HER/ERBB-elicited signaling, which may contribute to resistance to therapy and cancer progression.

Induction of triglyceride accumulation and mitochondrial maintenance in muscle cells by lactate.

Muscle exercise induces intramuscular triglyceride (TG) accumulation and promotes mitochondrial maintenance in myotubes. However, the mechanism underlying exercise effects remains unknown. In this study, lactic acid was tested as a signaling molecule in C2C12 myotubes to understand the mechanism. Intracellular TG storage was induced in the cells by sodium lactate. The lactate activity was observed with an inhibition of the cAMP-PKA pathway as indicated by a reduction in the phosphorylation status of CREB (pCREB). Induction of pCREB signal by forskolin was blocked by pretreatment of cells with lactate. The impact of lactate on mitochondrial function was examined with a focus on the activities of two enzymes, MCAT (malonylCoA:ACP transferase) and PDH (pyruvate dehydrogenase). The enzyme activities were induced in the cells by lactate. Expression of the lactate receptor (GPR81) and lactate transporters (MCT1/4) were induced as well by lactate. The lactate activities were observed at concentrations between 4–64 mM, and were not dependent on the increase in intracellular pyruvate. Pyruvate treatment did not generate the same effects in the cells. Those results suggest that lactate may induce intramuscular TG storage and mitochondrial maintenance in myotubes through inhibition of the cAMP pathway by activation of GPR81 in a positive feedback manner.

Abstract PR01: Disruption of DDR2-collagen interactions in tumor and stroma with a novel small molecule inhibitor blunts cancer metastasis

Abstract Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase that utilizes the extracellular matrix protein collagen as its ligand. Recently, DDR2 was shown to be critical in facilitating breast cancer metastasis. In tumor cells DDR2 expression, which is absent in normal breast epithelium, is induced during Epithelial Mesenchymal transition (EMT), and serves to stabilize protein levels of the EMT inducing transcription factor, Snail1. DDR2 expression is present in a majority of human invasive ductal breast carcinomas, and expression is predominately localized to the tumor-stroma boundary. In these tumors DDR2 is acting in a positive feedback manner to maintain Snail1 levels and activity at the leading, invasive edge of the tumors, where cells have undergone EMT and come into contact with extracellular matrix (ECM) collagen I. This allows continued invasion through the ECM and contributes to breast cancer metastasis. Interestingly, in genetic models of breast cancer in mice, selectively eliminating DDR2 in either the tumor (MMTV-PyMT; DDR2 fl/fl; K14-Cre) or the fibroblasts within the stroma (MMTV-PyMT; DDR2 fl/fl; FSP-Cre) leads to a dramatic inhibition of tumor metastasis. This indicates that in addition to maintaining EMT in the tumor cells, DDR2 functions within the stromal compartment to facilitate metastasis as well. In culture, the matrix synthesized and remodeled by cancer associated fibroblasts (CAFs) is organized very differently than that of normal fibroblasts. When DDR2 is depleted from CAFS, the matrix more closely resembles that produced by normal fibroblasts, with less organization of matrix proteins. Therefore, it appears that DDR2 engagement by collagen provides a means for both tumor and stromal cells to enhance metastatic potential. As such, DDR2 is a novel RTK target for the treatment of breast cancer metastasis, and we have developed potent and selective small molecule inhibitors of DDR2. These compounds provide a means of blunting breast cancer metastasis by targeting the tumor and stromal cells, thereby disrupting DDR2 signaling in both compartments. The developed inhibitors have nanomolar cellular potency at blocking DDR2 activation. Unlike traditional tyrosine kinase inhibitors (TKIs) that target the intracellular kinase domain, the inhibitors that we have developed act on the extracellular domain of DDR2 and disrupt DDR2 signaling by accelerating the disassembly of the DDR2-collagen complex. Therefore these inhibitors have a unique mode of action that is distinct from the canonical classes of TKIs. These compounds have the ability to attenuate DDR2 signaling both in cell culture, as well as in vivo as demonstrated by using a Snail1-clic beetle green reporter in a syngeneic transplant model. Treating CAFs with the developed inhibitors in culture reverts the organizational phenotype of the resulting matrix to that resembling the matrix produced by normal fibroblasts. Furthermore, using mouse models of late stage breast tumor metastasis, we have shown that these inhibitors reduce metastatic burden in the lungs of mice, to a level comparable to that of genetic knockdown of DDR2. Together these data support further investigation of this novel class of DDR2 inhibitors as anti-metastasis agents, potentially for use in combination with standard of care therapy to halt cancer progression and prevent relapse. Further, it will be important to study the role of these inhibitors in other cancers where DDR2 expression has been shown to promote metastasis, including, but not limited to, ovarian, head and neck, and nonsmall cell lung carcinoma. This abstract is also presented as Poster B13. Citation Format: Whitney R. Grither, Gregory D. Longmore. Disruption of DDR2-collagen interactions in tumor and stroma with a novel small molecule inhibitor blunts cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR01.

ApoA-I induces S1P release from endothelial cells through ABCA1 and SR-BI in a positive feedback manner.

Sphingosine-1-phosphate (S1P), which has emerged as a pivotal signaling mediator that participates in the regulation of multiple cellular processes, is derived from various cells, including vascular endothelial cells. S1P accumulates in lipoproteins, especially HDL, and the majority of free plasma S1P is bound to HDL. We hypothesized that HDL-associated S1P is released through mechanisms associated with the HDL maturation process. ApoA-I, a major HDL apolipoprotein, is a critical factor for nascent HDL formation and lipid trafficking via ABCA1. Moreover, apoA-I is capable of promoting bidirectional lipid movement through SR-BI. In the present study, we confirmed that apoA-I can facilitate the production and release of S1P by HUVECs. Furthermore, we demonstrated that ERK1/2 and SphK activation induced by apoA-I is involved in the release of S1P from HUVECs. Inhibitor and siRNA experiments showed that ABCA1 and SR-BI are required for S1P release and ERK1/2 phosphorylation induced by apoA-I. However, the effects triggered by apoA-I were not suppressed by inhibiting ABCA1/JAK2 or the SR-BI/Src pathway. S1P released due to apoA-I activation can stimulate the (ERK1/2)/SphK1 pathway through S1PR (S1P receptor) 1/3. These results indicated that apoA-I not only promotes S1P release through ABCA1 and SR-BI but also indirectly activates the (ERK1/2)/SphK1 pathway by releasing S1P to trigger their receptors. In conclusion, we suggest that release of S1P induced by apoA-I from endothelial cells through ABCA1 and SR-BI is a self-positive-feedback process: apoA-I-(ABCA1 and SR-BI)-(S1P release)-S1PR-ERK1/2-SphK1-(S1P production)-(more S1P release induced by apoA-I).

Exosomes miR-126a released from MDSC induced by DOX treatment promotes lung metastasis.

Acquired resistance to chemotherapy remains a major stumbling block in cancer treatment. Chronic inflammation plays a crucial role in induction of chemo resistance, and results in part from the induction and expansion of inflammatory cells that include myeloid derived suppressor cells (MDSC) and IL-13+Th2 cells. The mechanisms that lead to induction of activated MDSCs and IL-13+Th2 cells have not yet been identified. Here we demonstrated that doxorubicin treatment of 4T1 breast tumor bearing mice led to the induction of IL-13R+miR-126a+MDSC (DOX-MDSC). DOX-MDSC promote breast tumor lung metastasis through MDSC miR-126a+exosomal mediated induction of IL-13+Th2 cells and tumor angiogenesis. The induction of DOX-MDSC is regulated in a paracrine manner. DOX treatment not only increases IL-33 released from breast tumor cells, which is crucial for the induction of IL-13+Th2 cells, but it also participates in the induction of IL-13 receptors and miR-126a expressed on/in the MDSCs. IL-13 released from IL-13+Th2 cells then promotes the production of DOX-MDSC and MDSC miR-126a+exosomes via MDSC IL-13R. MDSC miR-126a+exosomes further induce IL13+Th2 cells in a positive feed-back loop manner. We also showed that MDSC miR-126a rescues doxorubicin induced MDSC death in a S100A8/A9 dependent manner and promotes tumor angiogenesis. Our findings provide insight into the MDSC exosomal mediated chemo resistance mechanism, which will be useful for the design of inhibitors targeting the blocking of induction of miR-126a+MDSC.

A Remote Role for Renalase

A Remote Role for Renalase

Allele-specific regulation of mutantHuntingtinby Wig1, a downstream target of p53

p53 has been implicated in the pathophysiology of Huntington's disease (HD). Nonetheless, the molecular mechanism of how p53 may play a unique role in the pathology remains elusive. To address this question at the molecular and cellular biology levels, we initially screened differentially expressed molecules specifically dependent on p53 in a HD animal model. Among the candidate molecules, wild-type p53-induced gene 1 (Wig1) is markedly upregulated in the cerebral cortex of HD patients. Wig1 preferentially upregulates the level of mutant Huntingtin (Htt) compared with wild-type Htt. This allele-specific characteristic of Wig1 is likely to be explained by higher affinity binding to mutant Htt transcripts than normal counterpart for the stabilization. Knockdown of Wig1 level significantly ameliorates mutant Htt-elicited cytotoxicity and aggregate formation. Together, we propose that Wig1, a key p53 downstream molecule in HD condition, play an important role in stabilizing mutant Htt mRNA and thereby accelerating HD pathology in the mHtt-p53-Wig1 positive feedback manner.

PP47 - Natural Nrf2 activators protect cells from oxidative damage inflicted by angiotensin II or aldosterone

The blood pressure regulating hormones angiotensin II (AngII) and aldosterone (Ald) cause oxidative stress and oxidative DNA damage in kidney cells in vitro and in vivo. Strategies to prevent oxidative damage with antioxidants were ineffective in humans. Fortifying the intrinsic cellular antioxidative defense is a new approach to protect cells from oxidative attack. In kidney cells the effect of the three natural Nrf2 activators curcumin, methysticin and sulforaphane (Sulf) on Nrf2 activation and their impact on Ald-induced DNA damage was studied. Sulforaphane was additionally tested for its potential to prevent AngII-induced DNA damage and NF-kB activation. All Nrf2 activators were able to protect from Ald-induced DNA damage. Sulf and curcumin led to a significant activation of Nrf2, detected as translocation to the nucleus. These two substances were also able to increase the expression of Nrf2 in a positive feedback manner. Sulf also protected from damage inflicted by AngII. Besides increasing the expression of the antioxidative protein HO-1, Sulf also prevented activation of the pro-inflammatory transcription factor NF-kB. In conclusion, all Nrf2 activators efficiently protected from Ald- and AngII-induced oxidative DNA damage. The role of the anti-inflammatory effect of Sulf in the prevention of genomic damage needs further investigation.