[Keywords: Akt; IKK; NF-B; Raptor; mTOR] Supplemental material is available at http://www.genesdev.org.

Accumulated studies have shown that activation of the Akt pathway plays a pivotal role in malignant transformation and chemoresistance by inducing cell survival, growth, migration, and angiogenesis. Therefore, Akt is believed to be a critical target for cancer intervention. Here, we report the discovery of a small molecule Akt pathway inhibitor, Akt/ protein kinase B signaling inhibitor-2 (API-2), by screening the National Cancer Institute Diversity Set. API-2 suppressed the kinase activity and phosphorylation level of Akt. The inhibition of Akt kinase resulted in suppression of cell growth and induction of apoptosis in human cancer cells that harbor constitutively activated Akt due to overexpression of Akt or other genetic alterations such as PTEN mutation. API-2 is highly selective for Akt and does not inhibit the activation of phosphatidylinositol 3-kinase, phosphoinositide-dependent kinase-1, protein kinase C, serumand glucocorticoid-inducible kinase, protein kinase A, signal transducer and activators of transcription 3, extracellular signal-regulated kinase-1/2, or c-Jun NH 2 -terminal kinase. Furthermore, API-2 potently inhibited tumor growth in nude mice of human cancer cells in which Akt is aberrantly expressed/activated but not of those cancer cells in which it is not. These findings provide strong evidence for pharmacologically targeting Akt for anticancer drug discovery.

Natural Killer (NK) cells and CD8 + cytotoxic T cells are two types of immune cells that can kill target cells through similar cytotoxic mechanisms. With the remarkable success of chimeric antigen receptor (CAR)-engineered T (CAR-T) cells for treating haematological malignancies, there is a rapid growing interest in developing CAR-engineered NK (CAR-NK) cells for cancer therapy. Compared to CAR-T cells, CAR-NK cells could offer some significant advantages, including: (1) better safety, such as a lack or minimal cytokine release syndrome and neurotoxicity in autologous setting and graft-versus-host disease in allogenic setting, (2) multiple mechanisms for activating cytotoxic activity, and (3) high feasibility for ‘off-the-shelf’ manufacturing. CAR-NK cells could be engineered to target diverse antigens, enhance proliferation and persistence in vivo, increase infiltration into solid tumours, overcome resistant tumour microenvironment, and ultimately achieve an effective anti-tumour response. In this review, we focus on recent progress in genetic engineering and clinical application of CAR-NK cells, and discuss current challenges and future promise of CAR-NK cells as a novel cellular immunotherapy in cancer.

Akt negatively regulates apoptotic pathways at a premitochondrial level through phosphorylation and modulation of proteins such as Bad, Forkhead proteins, and GSK-3␤. Akt has also been shown to protect cell death at a post-mitochondrial level, although its downstream targets have not been well documented. Here, we demonstrate that Akt, including AKT1 and AKT2, interacts with and phosphorylates X-linked inhibitor of apoptosis protein (XIAP) at residue serine-87 in vitro and in vivo. Phosphorylation of XIAP by Akt protects XIAP from ubiquitination and degradation in response to cisplatin. Moreover, autoubiquitination of XIAP is also inhibited by Akt. Consistent with this, an XIAP mutant introduced into cells which mimics the Akt-phosphorylated form (i.e. XIAP-S87D) displays reduced ubiquitination and degradation as compared with wild type XIAP. The greater stability of XIAP-S87D in cells translated to increased cell survival after cisplatin treatment. Conversely, a mutant that could not be phosphorylated by Akt (XIAP-S87A) was more rapidly degraded and showed increased cisplatin-induced apoptosis. Furthermore, suppression of XIAP by either siRNA or adenovirus of antisense of XIAP induced programmed cell death and inhibited Akt-stimulated cell survival in ovarian cancer cells. These data identify XIAP as a new downstream target of Akt and a potentially important mediator of the effect of Akt on cell survival.Akt, also named protein kinase B (PKB) 1 or RAC kinase, is a family of phosphatidylinositol 3-OH-kinase-regulated serine/ threonine kinase (1-3). Three isoforms of Akt have been identified: Akt1/PKB␣, Akt2/PKB␤, and Akt3/PKB␥, all of which are activated by growth factors in a phosphatidylinositol 3-OHkinase-dependent manner (4 -6). Accumulated evidence shows that Akt and its downstream targets constitute a major cell survival pathway. Akt promotes cell survival and suppresses apoptotic death in a number of cell types induced by a variety of stimuli, including growth factor withdrawal, cell cycle discordance, and loss of cell adhesion (7). Several downstream targets containing the Akt phosphorylation consensus sequence (R-X-R-X-X-S/T) have been identified which shed light on the mechanisms by which Akt promotes cell survival and blocks apoptosis. The first anti-apoptotic Akt target identified was the pro-apoptotic protein BAD. BAD is a pro-death member of the Bcl-2 family that initiates apoptosis by binding to Bcl-x L on the outer mitochondrial membrane, causing the release of cytochrome c into the cytosol. Akt phosphorylates BAD on Ser 136 , promoting the association of BAD with 14-3-3 proteins in the cytosol and inactivating its proapoptotic function (7). The execution of cellular apoptosis also involves changes in the transcriptional program (7). Akt decreases the transcription of a subset of death genes by phosphorylation of the Forkhead family of transcription factors, which causes their nuclear exclusion and inactivation (7). Akt also phosphorylates and activates the cyclic AMP-response element-binding pr...

Recent studies have identified a specialized subset of CD31hiEMCNhi vascular endothelium that positively regulates bone formation. However, it remains unclear how CD31hiEMCNhi endothelium levels are coupled to anabolic bone formation. Mice with an osteoblast-specific deletion of Shn3, which have markedly elevated bone formation, demonstrated an increase in CD31hiEMCNhi endothelium. Transcriptomic analysis identified SLIT3 as an osteoblast-derived, SHN3-regulated proangiogenic factor. Genetic deletion of Slit3 reduced skeletal CD31hiEMCNhi endothelium, resulted in low bone mass due to impaired bone formation and partially reversed the high bone mass phenotype of Shn3−/− mice. This coupling between osteoblasts and CD31hiEMCNhi endothelium is essential for bone healing, as shown by defective fracture repair in SLIT3-mutant mice and enhanced fracture repair in SHN3-mutant mice. Finally, administration of recombinant SLIT3 both enhanced bone-fracture healing and counteracted bone loss in a mouse model of postmenopausal osteoporosis. Thus, drugs that target the SLIT3 pathway may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss.

Normal cellular functions of hamartin and tuberin, encoded by the TSC1 and TSC2 tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Here we show that insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. We further demonstrate that Akt/PKB associates with hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27 kip1 , thereby promoting CDK2 activity and cellular proliferation. Our results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling hamartin-tuberin function.Tuberous sclerosis complex (TSC) 1 is an autosomal dominant disorder and is characterized by the presence of hamartomas in many organs such as brain, skin, heart, lung, and kidney (1). It is caused by mutation of either the TSC1 or TSC2 tumor suppressor gene (2-5). TSC1 encodes a protein, hamartin, containing two coiled-coil domains that have been shown to mediate binding to hamartin (6). The TSC2 gene codes for tuberin, which contains a small region of homology to the rap1GTPase-activating protein, rap1GAP (7). These two proteins function within the same pathway(s) regulating cell cycle, cell growth, adhesion, and vesicular trafficking (4,5). However, the regulation of hamartin and tuberin in the context of physiologic role as tumor suppressor genes has not been documented.Among the various properties of these two proteins, the ability to interact and to form stable complex has been the most consistent finding. This led to the hypothesis that hamartin and tuberin function as a complex and that factors regulating their interaction are important in understanding physiologic roles. There is evidence to suggest that phosphorylation of tuberin may be a major mechanism of regulation of the hamartin-tuberin complex (8, 9). However, the kinases that are responsible for phosphorylation of this complex are currently unknown. Recent Drosophila genetic studies showed that dTsc1 and dTsc2 play an important role in the insulin/dPI3K/ dakt signal transduction pathway by demonstrating that reduced cell size and cell proliferation caused by either mutations in dINR and dakt or by overexpression of dPTEN are overridden by homozygous mutants of dTsc1 or dTsc2. This implies that dTsc1 and dTsc2 are either direct downstream targets of dakt or on a parallel pathway of the insulin cascade downstream from dakt (10 -13). Akt, also known as protein kinase B (PKB),...

Septic shock is a severe systemic response to bacterial infection. Receptor for advanced glycation end products (RAGE) plays a role in immune reactions to recognize specific molecular patterns as pathogen recognition receptors. However, the interaction between LPS, the bioactive component of bacterial cell walls, and RAGE is unclear. In this study, we found direct LPS binding to RAGE by a surface plasmon resonance assay, a plate competition assay, and flow cytometry. LPS increased TNF-α secretion from peritoneal macrophages and an NF-κB promoter-driven luciferase activity through RAGE. Blood neutrophils and monocytes expressed RAGE, and TLR2 was counterregulated in RAGE−/− mice. After LPS injection, RAGE+/+ mice showed a higher mortality, higher serum levels of IL-6, TNF-α, high mobility group box 1, and endothelin-1, and severe lung and liver pathologies compared with RAGE−/− mice without significant differences in plasma LPS level. Administration of soluble RAGE significantly reduced the LPS-induced cytokine release and tissue damage and improved the LPS-induced lethality even in RAGE−/− as well as RAGE+/+ mice. The results thus suggest that RAGE can associate with LPS and that RAGE system can regulate inflammatory responses. Soluble RAGE would be a therapeutic tool for LPS-induced septic shock.

During the past decade, Akt (also known as protein kinase B, PKB) has been extensively studied. It regulates a variety of cellular processes by mediating extracellular (mitogenic growth factor, insulin and stress) and intracellular (altered tyrosine receptor kinases, Ras and Src) signals. Activation of Akt by these signals is via its pleckstrin homology (PH) domain binding to products of phosphatidylinositol 3-kinase (PI3K). This process is negatively regulated by a dual phosphatase PTEN tumor suppressor. Today, more than 30 Akt substrates have been identified. These phosphorylation events mediate the effects of Akt on cell survival, growth, differentiation, angiogenesis, migration and metabolism. Further, PI3K/PTEN/Akt pathway is frequently altered in many human malignancies and overexpression of Akt induces malignant transformation and chemoresistance. Thus, the Akt pathway is a major target for anti-cancer drug development. This review focuses on Akt signaling mechanism in oncogenesis and chemoresistance, and ongoing translational efforts to therapeutically target Akt.