Inhibition Of Akt Kinase Activity Research Articles

Down-Regulation of AKT Proteins Slows the Growth of Mutant-KRAS Pancreatic Tumors.

Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinomas (PDACs) harbor activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilized proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. The PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, the inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. The concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions, and the IGF-1 growth stimulation effect was AKT-dependent. The RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth, and the pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer.

Depletion of Akt1 and Akt2 Impairs the Repair of Radiation-Induced DNA Double Strand Breaks via Homologous Recombination.

Homologous recombination repair (HRR), non-homologous end-joining (NHEJ) and alternative NHEJ are major pathways that are utilized by cells for processing DNA double strand breaks (DNA-DSBs); their function plays an important role in the radiation resistance of tumor cells. Conflicting data exist regarding the role of Akt in homologous recombination (HR), i.e., the regulation of Rad51 as a major protein of this pathway. This study was designed to investigate the specific involvement of Akt isoforms in HRR. HCT116 colon cancer cells with stable AKT-knock-out and siRNA-mediated AKT-knockdown phenotypes were used to investigate the role of Akt1 and Akt2 isoforms in HR. The results clearly demonstrated that HCT116 AKT1-KO and AKT2-KO cells have a significantly reduced Rad51 foci formation 6 h post irradiation versus parental cells. Depletion of Akt1 and Akt2 protein levels as well as inhibition of Akt kinase activity resulted in an increased number of residual-γH2AX in CENP-F positive cells mainly representing the S and G2 phase cells. Furthermore, inhibition of NHEJ and HR using DNA-PK and Rad51 antagonists resulted in stronger radiosensitivity of AKT1 and AKT2 knockout cells versus wild type cells. These data collectively show that both Akt1 and Akt2 are involved in DSBs repair through HRR.

Synthesis and Bioevaluation of Quaternary Centered 3-hydroxy-3 (alkynyl)indolin-2-one Derivatives as Potential Cytotoxic Agents and Akt Kinase Inhibitors.

Alkynes are fundamental building blocks in synthetic chemistry with high pharmaceutical applications. Among the bioactive acetylenic molecules, propargylic alcohol is most important as almost all the marketed drugs contains quaternary centered propargylic alcohol functionality. In this study we have synthesised and evaluated 3-hydroxy-3-ethynylindolin-2-one derivatives for in vitro cytotoxic activity. An expeditious method for direct alkynylation of isatins (ketones) has been developed using tetrabutylammonium fluoride (TBAF) as a catalyst in THF solvent at room temperature under metal-free conditions. Furthermore, this method is an economically viable process that also compliments green aspects like being a ligand/metal free process under ambient conditions. This reaction tolerated a wide range of substrates with good to excellent yields (80-94%). The results showed that the synthesized compounds (4m, 4n and 4p) has the ability to inhibit Akt kinase activity with IC50 values ranging from 7.7 to 9.8 µM. All the 3-hydroxy-3-ethynylindolin-2-one derivatives were subjected for in vitro cytotoxic activity on five different cancer cell lines. Further, the synthesized compounds (4m, 4n and 4p) were evaluated for their ability to inhibit Akt kinase activity and exhibited good inhibition with IC50 values ranging from 7.7 to 9.8 µM..

Upregulation of kinesin family member 4A enhanced cell proliferation via activation of Akt signaling and predicted a poor prognosis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer-related death worldwide, and the molecular pathogenesis and development of HCC are largely unknown. In the present study, we found that KIF4A expression was upregulated in HCC (678 samples, P = 2.03E-8) based on a meta-analysis of Oncomine database. We further confirmed that both KIF4A mRNA and protein expressions were overexpressed in human HCC tumour tissues as well as cancer cell lines. Higher KIF4A expression was correlated with poorer overall survival (P < 0.0001) and disease-free survival (P < 0.0337) in HCC patients. We constructed in vitro KIF4A overexpression and depletion HCC cell models. KIF4A overexpression significantly enhanced cellular proliferation and clonogenic abilities, whereas KIF4A depletion caused a dramatic increase of cells with abnormal chromosome segregation and subsequently resulted in augmentation of apoptosis in HCC cells. In addition, we demonstrated that KIF4A depletion was related to inhibition of Akt kinase activity and induction of intrinsic apoptosis signaling pathway. Taken together, KIF4A may act as a prognostic biomarker and potential therapeutic target in human HCC.

Identification of 4-phenylquinolin-2(1H)-one as a specific allosteric inhibitor of Akt

Akt plays a major role in tumorigenesis and the development of specific Akt inhibitors as effective cancer therapeutics has been challenging. Here, we report the identification of a highly specific allosteric inhibitor of Akt through a FRET-based high-throughput screening, and characterization of its inhibitory mechanism. Out of 373,868 compounds screened, 4-phenylquinolin-2(1H)-one specifically decreased Akt phosphorylation at both T308 and S473, and inhibited Akt kinase activity (IC50 = 6 µM) and downstream signaling. 4-Phenylquinolin-2(1H)-one did not alter the activity of upstream kinases including PI3K, PDK1, and mTORC2 as well as closely related kinases that affect cell proliferation and survival such as SGK1, PKA, PKC, or ERK1/2. This compound inhibited the proliferation of cancer cells but displayed less toxicity compared to inhibitors of PI3K or mTOR. Kinase profiling efforts revealed that 4-phenylquinolin-2(1H)-one does not bind to the kinase active site of over 380 human kinases including Akt. However, 4-phenylquinolin-2(1H)-one interacted with the PH domain of Akt, apparently inducing a conformation that hinders S473 and T308 phosphorylation by mTORC2 and PDK1. In conclusion, we demonstrate that 4-phenylquinolin-2(1H)-one is an exquisitely selective Akt inhibitor with a distinctive molecular mechanism, and a promising lead compound for further optimization toward the development of novel cancer therapeutics.

MicroRNA-214 Reduces Insulin-like Growth Factor-1 (IGF-1) Receptor Expression and Downstream mTORC1 Signaling in Renal Carcinoma Cells

Elevated IGF-1/insulin-like growth factor-1 receptor (IGF-1R) autocrine/paracrine signaling in patients with renal cell carcinoma is associated with poor prognosis of the disease independent of their von Hippel-Lindau (VHL) status. Increased expression of IGF-1R in renal cancer cells correlates with their potency of tumor development and progression. The mechanism by which expression of IGF-1R is increased in renal carcinoma is not known. We report that VHL-deficient and VHL-positive renal cancer cells possess significantly decreased levels of mature, pre-, and pri-miR-214 than normal proximal tubular epithelial cells. We identified an miR-214 recognition element in the 3'UTR of IGF-1R mRNA and confirmed its responsiveness to miR-214. Overexpression of miR-214 decreased the IGF-1R protein levels, resulting in the inhibition of Akt kinase activity in both types of renal cancer cells. IGF-1 provoked phosphorylation and inactivation of PRAS40 in an Akt-dependent manner, leading to the activation of mTORC1 signal transduction to increase phosphorylation of S6 kinase and 4EBP-1. Phosphorylation-deficient mutants of PRAS40 and 4EBP-1 significantly inhibited IGF-1R-driven proliferation of renal cancer cells. Expression of miR-214 suppressed IGF-1R-induced phosphorylation of PRAS40, S6 kinase, and 4EBP-1, indicating inhibition of mTORC1 activity. Finally, miR-214 significantly blocked IGF-1R-forced renal cancer cell proliferation, which was reversed by expression of 3'UTR-less IGF-1R and constitutively active mTORC1. Together, our results identify a reciprocal regulation of IGF-1R levels and miR-214 expression in renal cancer cells independent of VHL status. Our data provide evidence for a novel mechanism for IGF-1R-driven renal cancer cell proliferation involving miR-214 and mTORC1.

Simultaneous Targeting of Bladder Tumor Growth, Survival, and Epithelial-to-Mesenchymal Transition with a Novel Therapeutic Combination of Acetazolamide (AZ) and Sulforaphane (SFN).

Current chemotherapies for advanced stage metastatic bladder cancer often result in severe side effects, and most patients become drug resistant over time. Thus, there is a need for more effective therapies with minimal side effects. The acid/base balance in tumor cells is essential for tumor cell functioning. We reasoned that simultaneous targeting of pH homeostasis and survival pathways would improve therapeutic efficacy. We evaluated the effectiveness of targeting pH homeostasis with the carbonic anhydrase inhibitor acetazolamide (AZ) in combination with the survival pathway targeting isothiocyanate sulforaphane (SFN) on the HTB-9 and RT112(H) human bladder tumor cell lines. We assessed viability, proliferation, and survival in vitro and effect on xenografts in vivo. Combination AZ + SFN treatment induced dose-dependent suppression of growth, produced a potent anti-proliferative and anti-clonogenic effect, and induced apoptosis through caspase-3 and PARP activation. The anti-proliferative effect was corroborated by significant reductions in Ki-67, pHH3, cyclin D1, and sustained induction of the cell cycle inhibitors, p21 and p27. Both active p-Akt (Ser473) and p-S6 were significantly downregulated in the AZ + SFN combination treated cells with a concomitant inhibition of Akt kinase activity. The inhibitory effects of the AZ + SFN combination treatment showed similar efficacy as the dual PI3K/mTOR pathway inhibitor NVP-BEZ235, albeit at an expected higher dose. In terms of the effect on the metastatic potential of these bladder cancers, we found downregulated expression of carbonic anhydrase 9 (CA9) concomitant with reductions in both E-cadherin, N-cadherin, and vimentin proteins mitigating the epithelial-to-mesenchymal transition (EMT), suggesting negation of this program. We suggest that reductions in these components could be linked with downregulation of the survival mediated Akt pathway and suggested an active role of the Akt pathway in bladder cancer. Altogether, our in vitro and pre-clinical model data support the potential use of an AZ + SFN combination for the treatment of bladder cancer.

High glucose enhances microRNA-26a to activate mTORC1 for mesangial cell hypertrophy and matrix protein expression

High glucose milieu inhibits PTEN expression to activate Akt kinase and induces glomerular mesangial cell hypertrophy and matrix protein expression in diabetic nephropathy. Specific mechanism by which high glucose inhibits PTEN expression is not clear. We found that high glucose increased the expression of the microRNA-26a (miR-26a) in mesangial cells. Using a sensor plasmid with 3′UTR-driven luciferase, we showed PTEN as a target of miR-26a in response to high glucose. Overexpression of miR-26a reduced the PTEN protein levels resulting in increased Akt kinase activity similar to high glucose treatment. In contrast, anti-miR-26a reversed high glucose-induced suppression of PTEN with concomitant inhibition of Akt kinase activity. Akt-mediated phosphorylation of tuberin and PRAS40 regulates mTORC1, which is necessary for mesangial cell hypertrophy and matrix protein expression. Inhibition of high glucose-induced miR-26a blocked phosphorylation of tuberin and PRAS40, which lead to suppression of phosphorylation of S6 kinase and 4EBP-1, two substrates of mTORC1. Furthermore, we show that expression of miR-26a induced mesangial cell hypertrophy and increased fibronectin and collagen I (α2) expression similar to that observed with the cells incubated with high glucose. Anti-miR-26a inhibited these phenomena in response to high glucose. Together our results provide the first evidence for the involvement of miR-26a in high glucose-induced mesangial cell hypertrophy and matrix protein expression. These data indicate the potential therapeutic utility of anti-miR-26a for the complications of diabetic kidney disease.

Extracellular lumican inhibits pancreatic cancer cell growth and is associated with prolonged survival after surgery.

To evaluate the relevance between lumican expression patterns and the clinical course of patients with pancreatic ductal adenocarcinoma (PDAC), and to investigate the role of lumican in PDAC progression. One hundred thirty-one patient tumors were chosen for tissue microarray staining, and Cox regression analysis was used to test the associations between lumican expression and clinical, pathologic, and oncologic outcomes in all patients. Primary PDAC cells and recombinant human lumican protein were used to establish a working model to mimic the in vivo interactions between stromal lumican and PDAC cells. Using this model, we tested the effects of lumican on EGFR signaling via Akt and hypoxia-inducible factor-1α (HIF1α) and its subsequent influence on glucose consumption, lactate production, intracellular ATP, and apoptotic cell death. Lumican was present in the stroma surrounding PDAC cells in roughly one-half of primary tumors and the direct xenografts. Patients with stromal lumican were associated with a profound reduction in metastatic recurrence after surgery and 3-fold longer survival than patients without stromal lumican. In PDAC cells, extracellular lumican reduced EGFR expression and phosphorylation through enhanced dimerization and internalization of EGFR and the resultant inhibition of Akt kinase activity. Lumican also reduced HIF1α expression and activity via Akt. PDAC cells with enhanced HIF1α activity were resistant to lumican-induced inhibition of glucose consumption, lactate production, intracellular ATP, and apoptosis. There is a positive association between stromal lumican in primary PDAC tumors and prolonged survival after tumor resection. Lumican plays a restrictive role in EGFR-expressing pancreatic cancer progression.

Inhibition of Akt kinase activity suppresses entry and replication of influenza virus

The possibility of the pandemic spread of influenza viruses highlights the need for an effective cure for this life-threatening disease. Influenza A virus, belonging to a family of orthomyxoviruses, is a negative-strand RNA virus which encodes 11 viral proteins. A numbers of intracellular signaling pathways in the host cells interact with influenza the viral proteins, which affect various stages of viral infection and replication.In this study, we investigated how inhibition of Akt kinase activity impacts on influenza virus infection by using “Akt-in”, a peptide Akt inhibitor. In PR8 influenza-infected A549 cells, Akt interacted with the NS1 (Non structural protein 1), and hence increased phosphorylation of Akt kinase activity and NS1. Treatment of cells with either “TCL1- or TCL1b-based Akt-in” efficiently suppressed Akt kinase activity while decreasing the levels of phosphorylated NS1; this, in turn, inhibited viral replication in a dose- and time-dependent manner. The inhibitory effect on viral replication appears to not be due to inhibition of the production of inflammatory cytokines, including IL-6 and IL-8, in the host cells. Inhibition of Akt kinase activity in the host cells inhibited the efficiency of viral entry, which is associated with decreased levels of phosphorylated glycogen synthase kinase 3, a substrate of Akt. Thus inhibition of Akt kinase activity in host cells may have therapeutic advantages for influenza virus infection by inhibiting viral entry and replication.

6]-Shogaol inhibits growth and induces apoptosis of non-small cell lung cancer cells by directly regulating Akt1/2

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. Despite progress in developing chemotherapeutics for the treatment of NSCLC, primary and secondary resistance limits therapeutic success. NSCLC cells exhibit multiple mutations in the epidermal growth factor receptor (EGFR), which cause aberrant activation of diverse cell signaling pathways. Therefore, suppression of the inappropriate amplification of EGFR downstream signaling cascades is considered to be a rational therapeutic and preventive strategy for the management of NSCLC. Our initial molecular target-oriented virtual screening revealed that the ginger components, including [6]-shogaol, [6]-paradol and [6]-gingerol, seem to be potential candidates for the prevention and treatment of NSCLC. Among the compounds, [6]-shogaol showed the greatest inhibitory effects on the NSCLC cell proliferation and anchorage-independent growth. [6]-Shogaol induced cell cycle arrest (G1 or G2/M) and apoptosis. Furthermore, [6]-shogaol inhibited Akt kinase activity, a downstream mediator of EGFR signaling, by binding with an allosteric site of Akt. In NCI-H1650 lung cancer cells, [6]-shogaol reduced the constitutive phosphorylation of signal transducer and activator of transcription-3 (STAT3) and decreased the expression of cyclin D1/3, which are target proteins in the Akt signaling pathway. The induction of apoptosis in NCI-H1650 cells by [6]-shogaol corresponded with the cleavage of caspase-3 and caspase-7. Moreover, intraperitoneal administration of [6]-shogaol inhibited the growth of NCI-H1650 cells as tumor xenografts in nude mice. [6]-Shogaol suppressed the expression of Ki-67, cyclin D1 and phosphorylated Akt and STAT3 and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positivity in xenograft tumors. The current study clearly indicates that [6]-shogaol can be exploited for the prevention and/or treatment of NSCLC.

The B Cell Receptor Antagonist R406 Alters The Balance Between Anti-Apoptotic and Pro-Apoptotic Signals To Strongly Synergize With ABT-199 In Diffuse Large B Cell Lymphoma Cells

SYK plays an essential role in B cell receptor (BCR) mediated survival in Diffuse Large B Cell Lymphoma (DLBCL). In a previous clinical investigation, fostamatinib, a potent Syk inhibitor, showed evidence of anti-tumour activity with an overall response rate of ∼20% in a cohort of DLBCL patients (JW Friedburg et al Blood. 2010, 115:2578-85). This finding prompted us to investigate the mechanism of action of fostamatinib in DLBCL. Our previous work demonstrated R406, the metabolic active form of fostamatinib, functions as a BCR antagonist in DLBCL cells. Treatment with R406 reduced MCL1 and down-regulated Bfl-1 expression, leading to apoptosis induction in sensitive cells. Due to R406's impact on apoptosis machinery, here we explored the ability of R406 to combine with a selective Bcl2 inhibitor, ABT-199 in DLBCL cells.Drug combinations were tested in DLBCL cells and synergy scores were determined using a 6x6 dose matrix followed by analysis via Chalice software (Zalicus). ABT-199 strongly synergized with R788, the prodrug of R406, in multiple R406 sensitive, but not resistant cell lines. In contrast, little effect was observed in combinations of R788 with other reagents, including ibrutinib (PCI-32765), bortezomib, CAL-101 and dasatinib. Flow-cytometry apoptosis analysis confirmed that R406 enhanced ABT-199-induced apoptosis in both ABC subtype and GCB subtype DLBCL cells. Further studies demonstrated R406 not only decreased anti-apoptotic proteins, but also activated pro-apoptotic proteins. Specifically, R406 suppressed ERK-dependent phosphorylation of BIM on S69, resulted in stabilization of BIM. In addition, R406 induced dephosphorylation of BAD at S136 in sensitive but not resistant cells, likely through inhibition of AKT kinase activity. Furthermore, treatment of R406 also upregulated other pro-apoptotic BH3-only proteins such as HRK1 and BMF1, which may partially contribute to the observed synergy.In conclusion, we have demonstrated R406 strongly synergizes with ABT-199 in vitro, likely by altering the balance between anti-apoptotic and pro-apoptotic signals in DLBCL cells. This may provide a potential combination therapy to be tested in DLBCL. Disclosures:Zhang:AstraZeneca: Employment, Possible shareholder Other. Francoise:AstraZeneca: Employment, Possible shareholder Other. Devereaux:AstraZeneca: Employment, Possible shareholder Other. Passino:AstraZeneca: Possible shareholder Other. Parmentier:AstraZeneca: Possible shareholder Other. Byth:AstraZeneca: Employment, Possible shareholder Other.

Abstract A10: Withaferin-A, a natural compound inhibits AKT-induced metastatic colorectal cancer

Abstract The survival rate of metastatic colorectal carcinoma (CRC) is less than 5%. Use of 5-fluorouracil (5-FU), radiation, and surgery are the primary treatments; however, most metastatic cancers eventually cause death. Developing approaches to prevent progression to metastasis could therefore be part of an important therapeutic strategy, especially for high-risk patients, including those patients already diagnosed with high grade polyps or localized colon cancer. In our preclinical and clinical data, we hypothesized that accumulation of AKT (protein kinase-B) is a critical step in the aggressive phenotype of disease. The metastatic cascade of CRC involves the crucial process of the epithelial–mesenchymal transition (EMT), which endows cancer cells with migratory and invasive capabilities via acquisition of a mesenchymal phenotype. These characteristics also contribute to cellular resistance to chemotherapeutic drugs. Withaferin-A (WA) is a potent herbal molecule from withania sominefera herbal plant, inhibited CRC growth by down regulating AKT signaling. Furthermore, over expression of AKT significantly increased cell proliferation, aggressiveness and angiogenesis of colon tumors in xenograft models. However, WA overcomes AKT mediated growth in both cell culture and animal models by transcriptionally down regulating Epithelial Mesenchymal Transition (EMT) related transcription factors and genes such as Snail, Slug and β-catenin. We also found AKT expression is steadily increased in CRC patients in a stage specific manner, suggesting accumulation of AKT could be a critical step for CRC metastasis. We found that WA binds to AKT with a predicted affinity of -10.6 kcal/mol and inhibited its activation. We have identified several novel WA–derived analogs which may be more potent than the parental compound with high AKT binding affinity. We have identified a potent analog which inhibited AKT kinase activity and induced growth arrest (~30 times potent) more significantly than WA. Currently, we are testing the invivo efficacy of the potent analog of WA, which could be a used as a novel agent for chemoprevention and/or chemotherapy of colon cancer. Citation Format: Suman Suman, Trinath P. Das, Chendil Damodaran. Withaferin-A, a natural compound inhibits AKT-induced metastatic colorectal cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A10.

Protooncogene TCL1b functions as an Akt kinase co-activator that exhibits oncogenic potency in vivo

Protooncogene T-cell leukemia 1 (TCL1), which is implicated in human T-cell prolymphocytic leukemia (T-PLL), interacts with Akt and enhances its kinase activity, functioning as an Akt kinase co-activator. Two major isoforms of TCL1 Protooncogenes (TCL1 and TCL1b) are present adjacent to each other on human chromosome 14q.32. In human T-PLL, both TCL1 and TCL1b are activated by chromosomal translocation. Moreover, TCL1b-transgenic mice have never been created. Therefore, it remains unclear whether TCL1b itself, independent of TCL1, exhibits oncogenicity. In co-immunoprecipitation assays, both ectopic and endogenous TCL1b interacted with Akt. In in vitro Akt kinase assays, TCL1b enhanced Akt kinase activity in dose- and time-dependent manners. Bioinformatics approaches utilizing multiregression analysis, cluster analysis, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping, Venn diagrams and Gene Ontology (GO) demonstrated that TCL1b showed highly homologous gene-induction signatures similar to Myr-Akt or TCL1. TCL1b exhibited oncogenicity in in vitro colony-transformation assay. Further, two independent lines of β-actin promoter-driven TCL1b-transgenic mice developed angiosarcoma on the intestinal tract. Angiosarcoma is a rare form of cancer in humans with poor prognosis. Using immunohistochemistry, 11 out of 13 human angiosarcoma samples were positively stained with both anti-TCL1b and anti-phospho-Akt antibodies. Consistently, in various cancer tissues, 69 out of 146 samples were positively stained with anti-TCL1b, out of which 46 were positively stained with anti-phospho-Akt antibodies. Moreover, TCL1b structure-based inhibitor ‘TCL1b-Akt-in' inhibited Akt kinase activity in in vitro kinase assays and PDGF (platelet-derived growth factor)-induced Akt kinase activities—in turn, ‘TCL1b-Akt-in' inhibited cellular proliferation of sarcoma. The current study disclosed TCL1b bears oncogenicity and hence serves as a novel therapeutic target for human neoplastic diseases.

Candidate tumor suppressor and pVHL partner Jade-1 binds and inhibits AKT in renal cell carcinoma.

The von Hippel-Lindau (VHL) tumor suppressor pVHL is lost in the majority of clear-cell renal cell carcinomas (RCC). Activation of the PI3K/AKT/mTOR pathway is also common in RCC, with PTEN loss occurring in approximately 30% of the cases, but other mechanisms responsible for activating AKT at a wider level in this setting are undefined. Plant homeodomain protein Jade-1 (PHF17) is a candidate renal tumor suppressor stabilized by pVHL. Here, using kinase arrays, we identified phospho-AKT1 as an important target of Jade-1. Overexpressing or silencing Jade-1 in RCC cells increased or decreased levels of endogenous phospho-AKT/AKT1. Furthermore, reintroducing pVHL into RCC cells increased endogenous Jade-1 and suppressed endogenous levels of phospho-AKT, which colocalized with and bound to Jade-1. The N-terminus of Jade-1 bound both the catalytic domain and the C-terminal regulatory tail of AKT, suggesting a mechanism through which Jade-1 inhibited AKT kinase activity. Intriguingly, RCC precursor cells where Jade-1 was silenced exhibited an increased capacity for AKT-dependent anchorage-independent growth, in support of a tumor suppressor function for Jade-1 in RCC. In support of this concept, an in silico expression analysis suggested that reduced Jade-1 expression is a poor prognostic factor in clear-cell RCC that is associated with activation of an AKT1 target gene signature. Taken together, our results identify 2 mechanisms for Jade-1 fine control of AKT/AKT1 in RCC, through loss of pVHL, which decreases Jade-1 protein, or through attenuation in Jade-1 expression. These findings help explain the pathologic cooperativity in clear-cell RCC between PTEN inactivation and pVHL loss, which leads to decreased Jade-1 levels that superactivate AKT. In addition, they prompt further investigation of Jade-1 as a candidate biomarker and tumor suppressor in clear-cell RCC.

Abstract 154: [6]-Shogaol inhibits cell growth and induces apoptosis in non-small-cell lung cancer (NSCLC) cells by regulating multiple targets

Abstract Non-small-cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. Despite substantial progress in developing chemotherapeutics for the treatment of NSCLC, primary and secondary resistance limits the success of these therapies. NSCLC cells exhibit different EGFR mutations and EGFR has extensive cross-talk with other cell signaling pathways. Therefore, suppression of the inappropriate expression of multiple targets is considered to be a rational therapeutic and preventive strategy for the management of NSCLC. Our initial molecular-target oriented virtual screening revealed that the ingredients of ginger, including [6]-gingerol, [6]-shogaol and [6]-paradol appeared to be potential candidates for the prevention and therapy of NSCLC cells. Among the three compounds, [6]-shogaol showed the greatest inhibitory effect on growth and soft agar colony formation of NSCLC cells. [6]-Shogaol induced cell cycle arrest (G1 or G2/M) and apoptosis. Furthermore, [6]-shogaol interacted with PI3-K and AKT and inhibited AKT kinase activity. Finally, [6]-shogaol increased the expression of p27, which is one of the target proteins in the AKT signaling pathway. The present results clearly indicate that [6]-shogaol exerted a strong anti-cancer effect and can be regarded as useful tool for the treatment of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 154. doi:1538-7445.AM2012-154

Ursolic acid-induced apoptosis in K562 cells involving upregulation of PTEN gene expression and inactivation of the PI3K/Akt pathway

Ursolic acid (UA), a pentacyclic triterpenoid derived from a variety of medicinal plants, exhibits potent anticancer activity against many types of cancer cells. However, the anticancer mechanism of UA is not clearly understood. Suppression of phosphatase and a tensin homolog deleted on chromosome 10 (PTEN) gene expression leading to activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway has been observed in many cancers including leukemia, making the PTEN gene and PI3K/Akt pathway a central target for cancer therapy. Here, we demonstrated that UA was able to inhibit growth, induce apoptosis in a human chronic myelogenous leukemia cell line (K562 cells) via upregulation of PTEN gene expression, inhibit Akt kinase activity, change mitochondrial transmembrane potential and reduce the release of cytochrome c and the activity of caspases. These results suggest that UA may elicit its strong antitumor effects via upregulation of the PTEN gene and inhibition of the PI3K/Akt pathway.

140 Inhibition of AKT Kinase Activity Decreases Replication of Human Respiratory Syncytial Virus

BackgroundHuman Respiratory Syncytial Virus (RSV) is a leading cause of pediatric pulmonary disease and severe RSV infection predisposes to wheezing later in life. RSV infection has also been shown to be an environmental trigger for asthma. We are investigating whether targeting host factors important for RSV infection is a viable antiviral strategy. Lowering viral burden through these therapies will result in decreased severity of infection and may also prevent the occurrence of pathologic sequelae.MethodsInhibition of AKT by chemical inhibitors, siRNA, or dominant-negative mutants, was tested for activity against RSV replication in cultured cells. We examined the effect of viral protein expression on Akt activation and downstream signal transduction by Western blot and promoter assay. In addition, we examined the effect of Akt on specific viral processes (entry, macromolecular synthesis, and assembly) and proteins both in vitro and in RSV-infected cells, using kinase assays, Western blotting, and qRT-PCR.ResultsWe found that AKT inhibition decreases RSV protein expression and viral titers. Expression of RSV NS2 protein activates AKT, leading to NFkB-dependent transcription, and inhibition of AKT blocks this effect. Activated AKT also phosphorylates RSV P protein at a specific site. Interestingly, AKT inhibitors that target the pleckstrin homology (PH) domain of AKT showed decreased efficacy against RSV compared to those that target AKT kinase activity.ConclusionsInhibition of AKT can effectively decrease RSV replication in culture, likely by decreasing P phosphorylation and thus viral protein transcription and expression. Activation of AKT during RSV infection likely involves the NS2 protein and does not depend on the PH domain of AKT. AKT inhibitors have been found to be safe and efficacious in clinical trials for a number of different cancers; thus, AKT inhibition may be a potential therapeutic treatment for severe RSV infection.

Inhibition of hemangioma development in a syngeneic mouse model correlates with bcl-2 suppression and the inhibition of Akt kinase activity

Hemangiomas are benign vascular tumors that are characterised by excessive angiogenesis. While there is no definitive treatment for these tumors, several angiogenesis inhibitors, including bleomycin, have been employed. To better understand the mechanism of bleomycin in accelerating haemangioma regression, we investigated the effects of the drug on hemangiomagenesis using a previously described mouse hemangioma model. The effects of bleomycin were tested in mice injected with endothelioma cells to induce hemangioma development. At termination, tissue samples from bleomycin-treated and control mice were stained with hematoxylin and eosin for histological examination. Bcl-2, flk-1 and vWF expression were studied by immunofluorescence microscopy. Hematological analysis was undertaken using a hemocounter. Akt activity was analyzed in tissue homogenates and endothelioma cells using ELISA. Also, caspase activity was analysed in endothelioma cells by ELISA. Bleomycin inhibited tumor growth in vivo in a dose-dependant manner. Our findings also revealed that bleomycin inhibited Akt activation and suppressed bcl-2. In vitro bleomycin increased caspase activation. Our observations reveal possible mechanisms for the inhibitory effects of bleomycin on hemangiomagenesis, and raise the possibility that bcl-2 might be an important therapeutic target in the treatment of hemangiomas.

Abstract C86: DC120, a novel and potent inhibitor of AKT kinase, induces tumor cells apoptosis and suppresses tumor growth.

Abstract PKB/AKT kinase is the core component of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway which is one of the most frequently hyperactivated signaling pathways in human cancers. We designed and synthesized a series of 2-pyrimidyl-5-amidothiazole coumpounds based on the ATP binding site of AKT kinase, and the most potent compound DC120 was identified to inhibit AKT kinase activity in vitro with EC50 of 153nM by a FRET-based Z'-Lyte assay. Then the growth inhibition of DC120 on human CNE2 and MDA-MB-453 cell lines was tested in vitro. The results showed DC120 could obviously inhibit the proliferation of these cancer cell lines via induction of apoptosis, with the evidence of increasing sub-G1 cells, characteristic morphologic changes of apoptosis in the nucleus, and cleaved caspase-3 and PARP. Further study showed that MDA-MB-453 cells transfected with constitutively activated AKT1 were more sensitive to DC120, while CNE2 cells with knock-down of AKT1 expression by shRNA were more resistant to DC120. More importantly, DC120 attenuated the phosphorylation levels of FKHR, FKHRL1, GSK-3 and mTOR in a dose-dependent and time-dependent fashion, and led to an increase in the nuclear accumulation of exogenous FKHR in cancer cells. Additionally, the in vivo experiments showed DC120 at 20 mg/kg/qd remarkably inhibited the CNE2 xenograft tumor growth with the T/C value of 36.1%, accompanied by increasing TUNEL-positve cells in tumor sample. These data provide validation for the development of DC120 to treat cancers displaying elevated levels of AKT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C86.