In addition, data from tumor xenografts and human cancer specimens indicate that AGO1-mediated translational desuppression of VEGF may be associated with tumor angiogenesis and poor prognosis. These findings provide evidence for an angiogenic pathway involving HRMs that target AGO1 and suggest that this pathway may be a suitable target for anti-or proangiogenesis strategies.

The retinoblastoma binding protein RBP2 (KDM5A) is a histone demethylase that promotes gastric cancer cell growth and is enriched in drug-resistant lung cancer cells. In tumor-prone mice lacking the tumor suppressor gene RB or MEN1, genetic ablation of RBP2 can suppress tumor initiation, but the pathogenic breadth and mechanistic aspects of this effect relative to human tumors have not been defined. Here, we approached this question in the context of lung cancer. RBP2 was overexpressed in human lung cancer tissues where its depletion impaired cell proliferation, motility, migration, invasion, and metastasis. RBP2 oncogenicity relied on its demethylase and DNA-binding activities. RBP2 upregulated expression of cyclins D1 and E1 while suppressing the expression of cyclin-dependent kinase inhibitor p27 (CDKN1B), each contributing to RBP2-mediated cell proliferation. Expression microarray analyses revealed that RBP2 promoted expression of integrin-b1 (ITGB1), which is implicated in lung cancer metastasis. Mechanistic investigations established that RBP2 bound directly to the p27, cyclin D1, and ITGB1 promoters and that exogenous expression of cyclin D1, cyclin E1, or ITGB1 was sufficient to rescue proliferation or migration/invasion, respectively. Taken together, our results establish an oncogenic role for RBP2 in lung tumorigenesis and progression and uncover novel RBP2 targets mediating this role. Cancer Res; 73(15); 4711-21. Ó2013 AACR.

Glucagon is important for regulating lipid metabolism in part through its inhibition of fatty acid synthesis in adipocytes. Acetyl-CoA carboxylase 1 (ACC1) is the rate-limiting enzyme for fatty acid synthesis. Glucagon has been proposed to activate cAMP-dependent protein kinase A (PKA), which phosphorylates ACC1 to attenuate the lipogenic activity of ACC1. Because AMP-activated protein kinase (AMPK) also inhibits fatty acid synthesis by phosphorylation of ACC1, we examined the involvement of AMPK and its upstream kinase in the glucagon-elicited signaling in adipocytes in vitro and in vivo. LC-MS-MS analysis suggested that ACC1 was phosphorylated only at Ser 79 , an AMPKspecific site, in glucagon-treated adipocytes. Pharmacological inhibitors and siRNA knockdown of AMPK or PKA in adipocytes demonstrate that glucagon regulates ACC1 and ACC2 activity through AMPK but not PKA. By using Ca 2ϩ /calmodulin-dependent protein kinase kinase-␤ knockout (CaMKK␤ Ϫ/Ϫ ) mice and cultured adipocytes, we further show that glucagon activates the CaMKK␤/ AMPK/ACC cascade. Additionally, fasting increases the phosphorylation of AMPK and ACC in CaMKK␤ ϩ/ϩ but not CaMKK␤mice. These results indicate that CaMKK␤/AMPK signaling is an important molecular component in regulating lipid metabolism in adipocytes responding to glucagon and could be a therapeutic target for the dysregulation of energy storage.acetyl-coenzyme A carboxylase; Ca 2ϩ /calmodulin-dependent protein kinase kinase-␤; adenosine 5=-monophosphate-activated protein kinase; lipid metabolism; fatty acid synthesis; energy mobilization ENERGY HOMEOSTASIS AT THE WHOLE BODY LEVEL is tightly controlled by a myriad of biochemical signaling in tissues mediating metabolism, i.e., adipose tissue, muscle, liver, and hypothalamus. Regulating multiple metabolic processes, AMP-activated protein kinase (AMPK) is widely recognized as a cellular energy gauge (15). Activated AMPK upregulates catabolic pathways and downregulates anabolic pathways to promote ATP generation in peripheral tissues (27,34,35). Furthermore, orexigenic and anorexigenic signals regulate hypothalamic AMPK to modulate food intake (28). Upon energy deficiency, AMPK is phosphorylated at Thr 172 in its catalytic ␣-subunit by one of two AMPK kinases (AMPKK): Ca 2ϩ / calmodulin-dependent protein kinase kinase-␤ (CaMKK␤ or CaMKK2) or tumor suppressor LKB1. Expressed abundantly in the brain (41), CaMKK␤ controls food intake by regulating hypothalamic AMPK, leading to the production of the orexigenic hormone neuropeptide Y (4). Although many studies show that LKB1 acts as an AMPKK in the periphery (8,20,26), CaMKK␤ may still be involved in AMPK activation, which is shown by experiments using cultured adipocytes (12).Among the pantheon of hormones regulating energy mobilization, glucagon is widely thought to exert its major effects on regulating glycogenolysis and gluconeogenesis in the liver (42). However, the metabolic effects of glucagon may also involve tissues other than the liver because the glucagon receptor is expressed ...

Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer.

<p>Supplementary Movie 3 � MOV file 1911K, cell moving in RBP2 KD2</p>

<p>Supplementary Table S1 - PDF file 159K, RBP2-regulated genes involved in tumorigenesis and metastasis</p>

<p>Supplementary Materials and Methods - PDF file 152K, Additional data including Western Blot, Plasmids, RT-qPCR, ChIP and DNA primers</p>

<p>Supplementary Figures S1-S7 - PDF file 476K, IHC staining of RBP2 protein in normal and cancerous human lung tissues (S1); Knockdown of RBP2 inhibits growth of lung cancer cell A549 (S2); RBP2 knockdown impairs formation of metastatic nodules in lungs of nude mice (S3); Construction of shRNA-resistant HA-tagged RBP2 plasmids (S4); The protein level of p21 is increased in gastric AGS cell line but decreased in lung cancer cells when RBP2 is depleted (S5); RBP2 knockdown effect on G1 cyclin gene expression and the efficiency of p27 depletion (S6); Successful expression of exogenous ITGB1 in RBP2-depeleted H1299 cells does not restore cell growth defect (S7)</p>