Robert Q. Miao

A noncoding antisense RNA in tie-1 locus regulates tie-1 function in vivo

Recently, messenger RNAs in eukaryotes have shown to associate with antisense (AS) transcript partners that are often referred to as long noncoding RNAs (lncRNAs) whose function is largely unknown. Here, we have identified a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse, and humans. In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript and in additional locations (ear and brain). The tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1 transcript levels, resulting in specific defects in endothelial cell contact junctions in vivo and in vitro. The ratio of tie-1 versus tie-1AS lncRNA is altered in human vascular anomaly samples. These results directly implicate noncoding RNA-mediated transcriptional regulation of gene expression as a fundamental control mechanism for physiologic processes, such as vascular development.

Nogo-B receptor is essential for angiogenesis in zebrafish via Akt pathway.

Our previous work has shown that axon guidance gene family Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro. To investigate NogoB-NgBR function in vivo, we cloned the zebrafish ortholog of both genes and studied loss of function in vivo using morpholino antisense technology. Zebrafish ortholog of Nogo-B is expressed in somite while expression of zebrafish NgBR is localized in intersomitic vessel (ISV) and axial dorsal aorta during embryonic development. NgBR or Nogo-B knockdown embryos show defects in ISV sprouting in the zebrafish trunk. Mechanistically, we found that NgBR knockdown not only abolished its ligand Nogo-B-stimulated endothelial cell migration but also reduced the vascular endothelial growth factor (VEGF)-stimulated phosphorylation of Akt and vascular endothelial growth factor-induced chemotaxis and morphogenesis of human umbilical vein endothelial cells. Further, constitutively activated Akt (myristoylated [myr]Akt) or human NgBR can rescue the NgBR knockdown umbilical vein endothelial cell migration defects in vitro or NgBR morpholino-caused ISV defects in vivo. These data place Akt at the downstream of NgBR in both Nogo-B- and VEGF-coordinated sprouting of ISVs. In summary, this study identifies the in vivo functional role for Nogo-B and its receptor (NgBR) in angiogenesis in zebrafish.

Endothelial cell–specific chemotaxis receptor (ecscr) promotes angioblast migration during vasculogenesis and enhances VEGF receptor sensitivity

Endothelial cell-specific chemotaxis receptor (ECSCR) is a cell surface protein expressed by blood endothelial cells with roles in endothelial cell migration and signal transduction. We investigated the function of ecscr in the development of the zebrafish vasculature. Zebrafish ecscr is expressed in angioblasts and in axial vessels during angioblast migration and vasculogenesis. Morpholino-directed ecscr knockdown resulted in defective angioblast migration in the posterior lateral plate mesoderm, a process known to depend on vascular endothelial-derived growth factor (VEGF). In cultured cells, transfected ECSCR localized to actin-rich membrane protrusions, colocalizing with kinase insert domain protein receptor (KDR)/VEGF receptor 2 in these regions. ECSCR-silenced cells show reduced VEGF-induced phosphorylation of KDR but not of FMS-like tyrosine kinase 1 (FLT1)/VEGF receptor 1. Finally, chemical inhibition of VEGF receptor activity in zebrafish resulted in angioblast deficiencies that partially overlap with those seen in ecscr morphants. We propose that ecscr promotes migration of zebrafish angioblasts by enhancing endothelial kdr sensitivity to VEGF.

Identification of interferon-γ as a new molecular target of liver X receptor

LXR (liver X receptor) is a ligand-activated transcription factor and plays an important role in regulation of lipid homoeostasis and inflammation. Several studies indicate that LXR inhibits IFN-γ (interferon γ)-induced biological responses; however, the influence of LXR on IFN-γ expression has not been fully elucidated. In the present study, we investigated the effects of LXR activation on IFN-γ expression at different levels. At the molecular level, we surprisingly observed that LXR ligand (T0901317) induced macrophage and T-cell IFN-γ protein expression which was associated with increased mRNA and secreted protein levels in culture medium. In contrast, selective inhibition of LXRα and/or LXRβ expression by siRNA reduced IFN-γ expression. Promoter analysis defined the multiple LXREs (LXR-responsive elements) in the proximal region of the IFN-γ promoter. EMSAs and ChIP indicated that LXR activation enhanced the binding of LXR protein to these LXREs. Inxa0vivo, T0901317 increased wild-type mouse serum IFN-γ levels and IFN-γ expression in the lung and lymph nodes. Functionally, we observed that administration of T0901317 to wild-type mice increased rates of survival and being tumour-free, and inhibited tumour growth when the animals were inoculated with LLC1 carcinoma. In contrast, these protective effects were substantially attenuated in IFN-γ-knockout (IFN-γ-/-) mice, suggesting that the induction of IFN-γ production plays a critical role in T0901317-inhibited tumour growth. Taken together, the results of the present study show that IFN-γ is another molecular target of LXR activation, and it suggests a new mechanism by which LXR inhibits tumour growth.

Endothelial Cell-specific Chemotaxis Receptor (ECSCR) Enhances Vascular Endothelial Growth Factor (VEGF) Receptor-2/Kinase Insert Domain Receptor (KDR) Activation and Promotes Proteolysis of Internalized KDR

Background: ECSCR is required for full KDR activation, but the mechanism is unknown. Results: ECSCR and KDR show both basal and VEGF-stimulated association. Conclusion: ECSCR modulates KDR activation and proteolysis of internalized KDR. Significance: ECSCR expression in vascular anomalies such as infantile hemangioma has implications for VEGF signaling in those tissues. In contrast, ECSCR levels are not increased in lung squamous cell carcinoma. The endothelial cell-specific chemotaxis receptor (ECSCR) is a cell-surface protein selectively expressed by endothelial cells (ECs), with roles in EC migration, apoptosis and proliferation. Our previous study (Verma, A., Bhattacharya, R., Remadevi, I., Li, K., Pramanik, K., Samant, G. V., Horswill, M., Chun, C. Z., Zhao, B., Wang, E., Miao, R. Q., Mukhopadhyay, D., Ramchandran, R., and Wilkinson, G. A. (2010) Blood 115, 4614–4622) showed that loss of ECSCR in primary ECs reduced tyrosine phosphorylation of vascular endothelial growth factor (VEGF) receptor 2/kinase insert domain receptor (KDR) but not VEGF receptor 1/FLT1. Here, we show that ECSCR biochemically associates with KDR but not FLT1 and that the predicted ECSCR cytoplasmic and transmembrane regions can each confer association with KDR. Stimulation with VEGF165 rapidly and transiently increases ECSCR-KDR complex formation, a process blocked by the KDR tyrosine kinase inhibitor compound SU5416 or inhibitors of endosomal acidification. Triple labeling experiments show VEGF-stimulated KDR+/ECSCR+ intracellular co-localization. Silencing of ECSCR disrupts VEGF-induced KDR activation and AKT and ERK phosphorylation and impairs VEGF-stimulated KDR degradation. In zebrafish, ecscr interacts with kdrl during intersomitic vessel sprouting. Human placenta and infantile hemangioma samples highly express ECSCR protein, suggesting a role for ECSCR-KDR interaction in these tissues.

Sucrose non-fermenting related kinase enzyme is essential for cardiac metabolism.

ABSTRACT In this study, we have identified a novel member of the AMPK family, namely Sucrose non-fermenting related kinase (Snrk), that is responsible for maintaining cardiac metabolism in mammals. SNRK is expressed in the heart, and brain, and in cell types such as endothelial cells, smooth muscle cells and cardiomyocytes (CMs). Snrk knockout (KO) mice display enlarged hearts, and die at postnatal day 0. Microarray analysis of embryonic day 17.5 Snrk hearts, and blood profile of neonates display defect in lipid metabolic pathways. SNRK knockdown CMs showed altered phospho-acetyl-coA carboxylase and phospho-AMPK levels similar to global and endothelial conditional KO mouse. Finally, adult cardiac conditional KO mouse displays severe cardiac functional defects and lethality. Our results suggest that Snrk is essential for maintaining cardiac metabolic homeostasis, and shows an autonomous role for SNRK during mammalian development.

Inhibition of tumor growth by U0126 is associated with induction of interferon‐γ production

Several MEK1/2 inhibitors have been in clinical trial evaluation for cancer treatment. Interferon‐γ (IFN‐γ) is a cytokine with multiple biological functions including antitumor activity. Expression of IFN‐γ can be induced by liver X receptor (LXR), a ligand‐activated transcription factor. However, it remains unknown if the anti‐cancer action of MEK1/2 inhibitors is completed, at least in part, by activating IFN‐γ expression. In this study, we determined that U0126, a MEK1/2 inhibitor, increased tumor‐free and survival rates and decreased growth of inoculated Lewis lung carcinomas in wild type mice. However, the protective effects were substantially attenuated in IFN‐γ deficient (IFN‐γ−/−) mice. At cellular and molecular levels, MEK1/2 inhibitors increased IFN‐γ protein and mRNA expression and activated natural IFN‐γ promoter but not the IFN‐γ promoters with mutations of the LXR responsive elements (LXREs). MEK1/2 inhibitors also enhanced formation of the LXRE‐nuclear protein complexes by inducing LXR expression and nuclear translocation. Similarly, MEK1/2 siRNA inhibited phosphorylation of ERK1/2 by MEK1/2 while activated IFN‐γ expression. In contrast, inhibition of LXR expression by siRNA blocked MEK1/2 inhibitors‐induced IFN‐γ expression. U0126 also inhibited chemicals‐induced pulmonary carcinomas, which was associated with increased IFN‐γ expression in the lung. Taken together, our study suggests that MEK1/2 inhibitors induce IFN‐γ production in an LXR‐dependent manner and the induction of IFN‐γ expression can partially contribute to the anti‐tumorigenic properties of U0126.

Abstract A31: Roles of Nogo-B receptor (NgBR) in regulating Ras translocation and modulating breast cancer cell sensitivity to chemotherapeutic drug and irradiation

Ras, a critical oncogene, is one of small GTPase family members. It is active when bound to GTP and inactive when bound to GDP. The activation process needs membrane translocation of Ras because its activators are recruited by membrane receptors, such as EGF receptors. In addition, appropriate localization of activated Ras also is critical for interacting with the downstream effectors. Therefore, regulation of Ras membrane translocation is very important for Ras function. Nogo-B receptor (NgBR) is a type I receptor with a single transmembrane domain. Our previous work has shown that axon guidance gene family Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro and blood vessel formation in Zebrafish. Here, we demonstrate that NgBR can bind and recruit farnesylated Ras to plasma membrane, which results in the activation of PI3K-Akt and Raf-MEK-ERK signaling pathways in human breast cancer cells. Direct interaction between NgBR and farnesylated Ras has been demonstrated by co-immunoprecipitation of MBP-NgBR from incubation of purified recombinant farnesylated Ras and MBP-NgBR protein. Knockdown of NgBR in MDA-MB-231 human breast cancer cells remarkably reduces the amount of membrane-associated Ras. These results suggest that NgBR is a potential modulator for Ras translocation. Knockdown of NgBR expression in MDA-MB-231 breast cancer cells decreases cancer cell colony formation, and abolishes EGF-stimulated Ras activation and phosphorylation of Akt in MDA-MB-231 cells. In addition, overexpression of NgBR caused the transformation of NIH-3T3 cells via increasing Ras plasma membrane translocation and enhancing EGF-stimulated Ras signaling. Knockdown of NgBR decreases the survival and colony formation of MDA-MB-231 cells after irradiation and increases the cisplatin-induced apoptosis and Caspase-3 activity of MDA-MB-231 Cells. These results suggest that NgBR can increase MDA-MB-231 breast cancer cell sensitivity to chemotherapeutic drug and irradiation. Consequently, our preliminary results further demonstrated that inducible knockdown of NgBR in tumor xenograft of MDA-MB-231 cells in nude mice significantly retards tumor growth. Thus, these data demonstrate that NgBR is a unique receptor that recruits farnesylated Ras to the plasma membrane via its hydrophobic cytoplasmic domain and then activates both PI3K-Akt and Raf-MEK-ERK pathways in human breast cancer cells. Collectively, it is the first study to demonstrate that NgBR is a potential therapeutic target for inhibiting Ras-dependent growth of breast cancer and decreasing the sensitivity of breast cancer cells to chemotherapeutic drug and irradiation.

Functional interplay between liver X receptor and AMP‐activated protein kinase α inhibits atherosclerosis in apolipoprotein E‐deficient mice − a new anti‐atherogenic strategy

The liver X receptor (LXR) agonist T317 reduces atherosclerosis but induces fatty liver. Metformin activates energy metabolism by activating AMPKα. In this study, we determined if interactions between metformin and T317 could inhibit atherosclerosis without activation of hepatic lipogenesis.

Abstract B26: Roles of NgBR in promoting Ras translocation and Ras signaling in human breast cancer cells

NgBR is a type I receptor with a single transmembrane domain. Our previous work has shown that axon guidance gene family Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro and blood vessel formation in Zebrafish. Here, we demonstrate that NgBR binds and translocates farnesylated Ras to plasma membranes, which results in the activation of PI3K-Akt and Raf-MEK-ERK signaling pathways in human breast cancer cells. Comparing NgBR to other sequences in the NCBI database reveals that regions of the cytoplasmic domain have a high degree of similarity (49%) to the cis-lsoprenyl Disphosphate Synthases (cis-IPPS) family of lipid modifying enzymes. However, NgBR lacks key residues required for catalytic activity, but contains a conserved hydrophobic tunnel for shuttling prenyl lipids and/or prenylated proteins. Farnesylation of Ras is essential for Ras translocation and activation. Direct interaction between NgBR and farnesylated Ras has been demonstrated by co-immunoprecipitation of MBP-NgBR from incubation of purified recombinant MBP-NgBR and farnesylated Ras protein. Knockdown of NgBR in MDA-MB-231 human breast cancer cells remarkably reduces the amount of membrane-associated Ras. These results suggest that NgBR is a potential modulator for Ras translocation. Immunohistochemistry analysis of human breast tumor tissue arrays revealed that NgBR is highly expressed in human breast cancer tissues. We further used real-time PCR to determine the copy number of NgBR transcripts in human breast tumor tissues. Accompanying pathology reports were used to categorize the 84 breast tumor tissues into four different grades from I to IV. Real-time PCR results show that NgBR expression is significantly higher in Grade II and Grade III-IV breast tumors when compared with normal breast samples (12 samples) and Grade I breast tumor samples (21 samples). 62% of Grade II (34 samples) and 55% of Grade III-IV (29 samples) breast tumors had increased NgBR transcripts (>50% higher) when compared with normal breast tissues. These data suggest that NgBR expression is strongly associated with advanced breast cancers. Interestingly, NgBR is highly expressed in several breast cancer cells such as MDA-MB-231 cells as compared to immortalized mammary epithelial cells MCF-10A. Knockdown of NgBR expression in MDA-MB-231 breast cancer cells decreases cancer cell colony formation, and abolishes EGF-stimulated Ras activation and phosphorylation of Akt in MDA-MB-231 cells. In addition, overexpression of NgBR caused the transformation of NIH-3T3 cells via enhancing Ras plasma membrane translocation and EGF-stimulated Ras signaling. Consequently, our preliminary results further demonstrated that inducible knockdown of NgBR in tumor xenograft of MDA-MB-231 cells in nude mice significantly retards tumor growth. Thus, these data demonstrate that NgBR is a unique receptor that recruits farnesylated Ras to the plasma membrane via its hydrophobic cytoplasmic domain and then activates both PI3K-Akt and Raf-MEK-ERK pathways in human breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B26.