Inhibition Of Akt Activation Research Articles

Perifosine enhances the potential antitumor effect of 5-fluorourasil and oxaliplatin in colon cancer cells harboring the PIK3CA mutation

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation in colon cancer contributes to the poor prognosis of the disease and chemoresistance of tumors. New therapies are needed; however, the lack of knowledge of the mechanism of chemoresistance has hindered progress. In this study, we investigated the mechanism of the reduced sensitivity of colon cancer cells to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP), and the effects of perifosine, an Akt inhibitor that enhances the cytotoxicity of 5-FU and L-OHP in colon cancer cells harboring the PIK3CA mutation. The use of 5-FU or L-OHP alone or in combination induced significant death of Caco-2 cells (PIK3CA wild type), but only weakly decreased the viability of DLD-1 and SW948 cells harboring the PIK3CA mutation. The use of 5-FU and L-OHP, either alone or in combination, strongly suppressed Akt activation, Survivin, Bcl-2, and Bcl-xL expression, and enhanced Puma, phospho-p53, and p53 expression in Caco-2 cells than in DLD-1 cells. In addition, perifosine enhanced the cytotoxicity of the 5-FU and L-OHP combination, inhibited Akt activation and the expression of Survivin, Bcl-2, and Bcl-xL, and increased the expression of Puma, phospho-p53, and p53 in DLD-1 cells. These results indicate that PIK3CA mutation contributes to reduced sensitivity to 5-FU and L-OHP via Akt activation in colon cancer cells. Perifosine increases the efficacy of 5-FU and L-OHP by suppressing Akt activation. Thus, the use of an Akt inhibitor in combination with 5-FU and L-OHP may be beneficial in colon cancer with cells harboring the PIK3CA mutation.

Retraction Note to: The Akt inhibitor KP372-1 suppresses Akt activity and cell proliferation and induces apoptosis in thyroid cancer cells.

The phosphatidylinositol 3′ kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten/Akt pathway, which is a critical regulator of cell proliferation and survival, is mutated or activated in a wide variety of cancers. Akt appears to be a key central node in this pathway and thus is an attractive target for targeted molecular therapy. We demonstrated that Akt is highly phosphorylated in thyroid cancer cell lines and human thyroid cancer specimens, and hypothesised that KP372-1, an Akt inhibitor, would block signalling through the PI3K pathway and inhibit cell proliferation while inducing apoptosis of thyroid cancer cells. KP372-1 blocked signalling downstream of Akt in thyroid tumour cells, leading to inhibition of cell proliferation and increased apoptosis. As thyroid cancer consistently expresses phosphorylated Akt and KP372-1 effectively blocks Akt signalling, further preclinical evaluation of this compound for treatment of thyroid cancer is warranted.

The expression patterns and prognostic significance of pleckstrin homology-like domain family A (PHLDA) in lung cancer and malignant mesothelioma.

BackgroundPleckstrin homology domain family A (PHLDA) genes play important roles in cancer cellular processes, including inhibiting Akt activation, repressing growth factor signaling, inhibiting the negative feedback of EGFR/ErbB2 signaling cells, and inducing apoptosis. However, the prognostic significance of PHLDA in non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MM) remains unclear. The present study investigates the associations between PHLDA expression patterns and their prognostic value in lung adenocarcinoma (LUAD) and MM.MethodsWe analyzed PHLDA family members at the genomic level in silico to explore their mRNA expression pattern and predictive significance in LUAD and MM. We then created a PHLDA–drug interaction network and a protein-protein interaction (PPI) network using different databases. Finally, we immunohistochemically assessed the protein expression of each PHLDA family member on tissue microarrays (TMAs) in both LUAD and MM cohorts with long-term follow-up.ResultsWhile PHLDA1 mRNA expression in both LUAD and MM was lower than that of normal tissue, PHLDA2 mRNA was significantly overexpressed in LUAD, and PHLDA3 mRNA was overexpressed in MM. In NSCLC, both low PHLDA1 mRNA expression and high PHLDA3 mRNA expression correlated with worse overall survival (OS) (P<0.01), whereas high PHLDA2 mRNA expression was associated with better OS (P<0.01). In MM, patients presenting high PHLDA1 and PHLDA2 mRNA expression had poor OS (P=0.01 and P<0.01, respectively). In addition, the PHLDA-drug interaction network indicated that several common drugs could potentially modulate PHLDA expression, and the PPI network suggested that PHLDA1 interacts with Notch family members, whereas PHLDA3 interacts with TP53. Our results also showed that the expression of PHLDA2 and PHLDA3 was significantly higher in LUAD and MM than that of PHLDA1 (P<0.05) and was associated with the risk of death. While patients with PHLDA2 >85.09 cells/mm2 had a low risk of death (P=0.01) and a median survival time of 48 months, those with PHLDA3 <70.38 cells/mm2 had a high risk of death (P=0.03) and a median survival time of 34 months.ConclusionsWe shed light on the role of the PHLDA family as promising predictive biomarkers and potential therapeutic targets in LUAD and MM.

Slit/Robo signaling regulates Leydig cell steroidogenesis

BackgroundFirst identified as a regulator of neuronal axon guidance, Slit/Robo signaling has since been implicated in additional physiologic and pathologic processes, such as angiogenesis, organogenesis and cancer progression. However, its roles in the regulation of testis function have been little explored.MethodsImmunohistochemistry and RT-qPCR analyses were performed to detect the expression of Slit/Robo signaling effectors in the adult mouse testis. To identify the roles and mechanisms of Slit/Robo signaling in the regulation of steroidogenesis, RT-qPCR, immunoblotting and hormone measurements were carried out using Leydig cells (primary cultures and the MA10 cell line) treated with exogenous SLIT ligands, and testes from Robo1-null mice.ResultsSlit1, -2 and -3 and Robo1 and -2 expression was detected in the adult mouse testis, particularly in Leydig cells. In vitro treatment of Leydig cells with exogenous SLIT ligands led to a decrease in the expression of the steroidogenic genes Star, Cyp11a1, and Cyp17a1. SLIT2 treatment decreased the phosphorylation of the key steroidogenic gene regulator CREB, possibly in part by suppressing AKT activity. Furthermore, SLIT2 treatment reduced the responsiveness of MA10 cells to luteinizing hormone by decreasing the expression of Lhcgr. Consistent with these in vitro results, an increase in testicular Star mRNA levels and intra-testicular testosterone concentrations were found in Robo1-null mice. Finally, we showed that the expression of the Slit and Robo genes in Leydig cells is enhanced by testosterone treatment in vitro, by an AR-independent mechanism.ConclusionTaken together, these results suggest that Slit/Robo signaling represents a novel mechanism that regulates Leydig cell steroidogenesis. It may act in an autocrine/paracrine manner to mediate negative feedback by testosterone on its own synthesis.4VdBjKyUK_zz54UEEeomAMVideo

Virtual Screening of Natural Compounds as Potential PI3K-AKT1 Signaling Pathway Inhibitors and Experimental Validation.

A computational screening for natural compounds suitable to bind the AKT protein has been performed after the generation of a pharmacophore model based on the experimental structure of AKT1 complexed with IQO, a well-known inhibitor. The compounds resulted as being most suitable from the screening have been further investigated by molecular docking, ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analysis and toxicity profiles. Two compounds selected at the end of the computational analysis, i.e., ZINC2429155 (also named STL1) and ZINC1447881 (also named AC1), have been tested in an experimental assay, together with IQO as a positive control and quercetin as a negative control. Only STL1 clearly inhibited AKT activation negatively modulating the PI3K/AKT pathway.

Lipid-soluble polyphenols from sweet potato exert antitumor activity and enhance chemosensitivity in breast cancer

Polyphenols are abundant in vegetables and fruit. They have been shown to have various antitumor, antioxidant, and anti-inflammatory effects. Here, we extracted the lipid-soluble fraction of polyphenols from fermented sweet potato (Ipomoea batatas). These lipid-soluble polyphenols mainly contained caffeic acid derivatives with strong antioxidant ability, which we hypothesized to affect diseases for which oxidative stress is a factor, such as cancer. We therefore investigated the antitumor and chemo-sensitizing effects of lipid-soluble polyphenols on E0771 murine breast cancer cells. The lipid-soluble polyphenols accumulated in the cells’ cytoplasm due to its high lipophilicity, and reduced reactive oxygen species through its strong antioxidant activity. The lipid-soluble polyphenols also arrested the cell cycle at G0/G1 by suppressing Akt activity, and enhanced the cytotoxicity of anticancer agents. In this model, lipid-soluble polyphenols inhibited tumor growth and enhanced the efficacy of chemotherapy drugs. These results suggest the potential of lipid-soluble polyphenols as a functional food to support cancer therapy.

A phase Ib/II study of xentuzumab, an IGF-neutralising antibody, combined with exemestane and everolimus in hormone receptor-positive, HER2-negative locally advanced/metastatic breast cancer

BackgroundXentuzumab—a humanised IgG1 monoclonal antibody—binds IGF-1 and IGF-2, inhibiting their growth-promoting signalling and suppressing AKT activation by everolimus. This phase Ib/II exploratory trial evaluated xentuzumab plus everolimus and exemestane in hormone receptor-positive, locally advanced and/or metastatic breast cancer (LA/MBC).MethodsPatients with hormone receptor-positive/HER2-negative LA/MBC resistant to non-steroidal aromatase inhibitors were enrolled. Maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of xentuzumab/everolimus/exemestane were determined in phase I (single-arm, dose-escalation). In phase II (open-label), patients were randomised 1:1 to the RP2D of xentuzumab/everolimus/exemestane or everolimus/exemestane alone. Randomisation was stratified by the presence of visceral metastases. Primary endpoint was progression-free survival (PFS).ResultsMTD was determined as xentuzumab 1000 mg weekly plus everolimus 10 mg/day and exemestane 25 mg/day. A total of 140 patients were enrolled in phase II (70 to each arm). Further recruitment was stopped following an unfavourable benefit-risk assessment by the internal Data Monitoring Committee appointed by the sponsor. Xentuzumab was discontinued; patients could receive everolimus/exemestane if clinically indicated. Median PFS was 7.3 months (95% CI 3.3–not calculable) in the xentuzumab/everolimus/exemestane group and 5.6 months (3.7–9.1) in the everolimus/exemestane group (hazard ratio 0.97, 95% CI 0.57–1.65; P = 0.9057). In a pre-specified subgroup of patients without visceral metastases at screening, xentuzumab/everolimus/exemestane showed evidence of PFS benefit versus everolimus/exemestane (hazard ratio 0.21 [0.05–0.98]; P = 0.0293). Most common any-cause adverse events in phase II were diarrhoea (29 [41.4%] in the xentuzumab/everolimus/exemestane group versus 20 [29.0%] in the everolimus/exemestane group), mucosal inflammation (27 [38.6%] versus 21 [30.4%]), stomatitis (24 [34.3%] versus 24 [34.8%]), and asthenia (21 [30.0%] versus 24 [34.8%]).ConclusionsAddition of xentuzumab to everolimus/exemestane did not improve PFS in the overall population, leading to early discontinuation of the trial. Evidence of PFS benefit was observed in patients without visceral metastases when treated with xentuzumab/everolimus/exemestane, leading to initiation of the phase II XENERA™-1 trial (NCT03659136).Trial registrationClinicalTrials.gov, NCT02123823. Prospectively registered, 8 March 2013.

AGEs promote calcification of HASMCs by mediating Pi3k/AKT-GSK3β signaling.

This study aimed to investigate the effects of advanced glycation end products (AGEs) on the calcification of human arterial smooth muscle cells (HASMCs) and to explore whether AGEs can promote the calcification of HASMCs by activating the phosphoinositide 3-kinase (PI3K)/AKT-glycogen synthase kinase 3 beta (GSK3-β) axis. Cultured HASMCs were divided into five groups: blank control group, dimethyl sulfoxide (vehicle) group, AGEs group, LY294002 (AKT inhibitor) group, and TWS119 (GSK3-β inhibitor) group. Cells were pretreated with either vehicle, LY294002, or TWS119 for 2 hours followed by incubation with AGEs (25 μg/mL) for 5 days, and the expression levels of proteins in each group were analyzed by western blotting. AGE treatment promoted HASMC calcification, which coincided with increased expression of p-AKT and p-GSK3-β (serine 9). Also, AGEs upregulated the expression of osteoprotegerin and bone morphogenetic protein, and these effects were suppressed by LY294002 but enhanced by TWS119. In conclusion, AGEs promote calcification of HASMCs, and this effect is ameliorated by inhibition of AKT activity but potentiated by inhibition of GSK3-β activity. Hence, AGEs trigger HASMC calcification by regulating PI3K/AKT-GSK3-β signaling.

Advancing host-directed therapy for tuberculosis/MDR-TB

Aim and Objective: Previous studies reported the implications of host-immune cell signalling pathways contributing towards survival of mycobacteria. Among the few, abrogation of PI3K-AKT-mTOR pathway has been shown to impact bacterial viability in macrophages. However, the intricate molecular mechanisms associated with such outcome at the juncture of host-pathogen interaction remain elusive. Methods: We have used novel AKT inhibitor against MTB infected macrophage and tested its survivability and its molecular mechanism of killing inside macrophage. Result: In the present work mouse peritoneal macrophages infected with pathogenic strain of M.tb (H37Rv) exhibited increased phosphorylation of AKT. Interestingly, suppression of AKT activity by a novel small molecule inhibitor during mycobacterial infection promoted phagolysosome fusion, apoptosis and autophagy, altogether contributing towards reduction in bacterial count in vitro. At the molecular level, we noticed that, small molecule inhibitor to AKT treatment deciphered the requirement of phosphorylation of AS160, Bad and mTOR by phosphorylated AKT in infected macrophages. Additionally, inhibition of phosphorylated AKT hampered nuclear translocation of NF-kB resulting in decreased cytokine expression. Conclusion: Taken together, our inhibitor can be utilized as an adjunctive therapy, as it shows high efficacy in decreasing bacterial count in combination with standard drugs to TB and also prevent the development of antibiotic resistance as host pathways are being targeted.

Isochromanoindolenines suppress triple-negative breast cancer cell proliferation partially via inhibiting Akt activation.

As the most malignant subtype of breast cancers, triple-negative breast cancer (TNBC) lacks effective targeted therapeutics clinically to date. In this study, one lead compound FZU-0025-065 with isochromanoindolenine scaffold was identified by a cell-based screening. Among nine breast cancer cell lines tested, TNBC are the most sensitive cell lines to FZU-0025-065. FZU-0025-065 inhibits TNBC cell growth in a time- and dosage-dependent manner. FZU-0025-065 suppresses the expression of cell cycle dependent kinase 4 (CDK4), Cyclin D1 and Cyclin B1; meanwhile, elevates the expression of cell cycle dependent kinase inhibitor p21 and p27. Importantly, we found that FZU-0025-065 suppresses AKT activation in a time- and dosage-dependent manner. Over-expression of constitutive active AKT partially rescues FZU-0025-065 induced cell growth inhibition in MDA-MB-468 cells, indicating FZU-0025-065 suppresses TNBC cell growth partially via inhibiting AKT activation. Finally, FZU-0025-065 suppresses TNBC cell growth in a xenograft mouse model. Taken together, our findings suggested that isochromanoindolenine derivative FZU-0025-065 inhibits TNBC via suppressing the AKT signaling and that FZU-0025-065 may be useful for TNBC treatment.

MigrationStimulating Factor (MSF): Its Role in the Tumour Microenvironment.

Migration Stimulating Factor(MSF) is a 70kDa truncated isoform of fibronectin (FN); its mRNA is generated from the FN gene by an unusual two-stage processing. Unlike full-length FN, MSF is not a matrix molecule but a soluble protein which displays cytokine-like activities not displayed by any other FN isoform due to steric hindrance. There are two isoforms of MSF; these are referred to as MSF+aa and MSF-aa, while the term MSF is used to include both.MSF was first identified as a motogen secreted by foetal and cancer-associated fibroblasts in tissue culture. It is also produced by sprouting (angiogenic) endothelial cells, tumour cells and activated macrophages. Keratinocytes and resting endothelial cells secrete inhibitors of MSF that have been identified as NGAL and IGFBP-7, respectively. MSF+aa and MSF-aa show distinct functionality in that only MSF+aa is inhibited by NGAL.MSF is present in 70-80% of all tumours examined, expressed by the tumour cells as well as by fibroblasts, endothelial cells and macrophages in the tumour microenvironment (TME). High MSF expression is associated with tumour progression and poor prognosis in all tumours examined, including breast carcinomas, non-small cell lung cancer (NSCLC), salivary gland tumours (SGT) and oral squamous cell carcinomas (OSCC). Epithelial and stromal MSF carry independent prognostic value. MSF is also expressed systemically in cancer patients, being detected in serum and produced by fibroblast from distal uninvolved skin. MSF-aa is the main isoform associated with cancer, whereas MSF+aa may be expressed by both normal and malignant tissues.The expression of MSF is not invariant; it may be switched on and off in a reversible manner, which requires precise interactions between soluble factors present in the TME and the extracellular matrix in contact with the cells. MSF expression in fibroblasts may be switched on by a transient exposure to several molecules, including TGFβ1 and MSF itself, indicating an auto-inductive capacity.Acting by both paracrine and autocrine mechanisms, MSF stimulates cell migration/invasion, induces angiogenesis and cell differentiation and alters the matrix and cellular composition of the TME. MSF is also a survival factor for sprouting endothelial cells. IGD tri- and tetra-peptides mimic the motogenic and angiogenic activities of MSF, with both molecules inhibiting AKT activity and requiring αvβ3 functionality. MSF is active at unprecedently low concentrations in a manner which is target cell specific. Thus, different bioactive motifs and extracellular matrix requirements apply to fibroblasts, endothelial cells and tumour cells. Unlike other motogenic and angiogenic factors, MSF does not affect cell proliferation but it stimulates tumour growth through its angiogenic effect and downstream mechanisms.The epithelial-stromal pattern of expression and range of bioactivities displayed puts MSF in the unique position of potentially promoting tumour progression from both the "seed" and the "soil" perspectives.

Juglone prevents human platelet aggregation through inhibiting Akt and protein disulfide isomerase

Background/PurposeJuglone, a natural compound widely found in Juglandaceae plants, has been suggested as a potential drug candidate for treating cancer, inflammation, and diabetic vascular complications. In the present study, the antiplatelet effect and underlying mechanisms of juglone were investigated for the first time. Study design/methodsHuman platelet aggregation and activation were measured by turbidimetric aggregometry, flow cytometry, and Western blotting. In vitro antithrombotic activity of juglone was assessed using collagen-coated flow chambers under whole-blood flow conditions. The effect of juglone on protein disulfide isomerase (PDI) activity was determined by the dieosin glutathione disulfide assay. ResultsJuglone (1 – 5 μM) inhibited platelet aggregation and glycoprotein (GP) IIb/IIIa activation caused by various agonists. In a whole blood flow chamber system, juglone reduced thrombus formation on collagen-coated surfaces under arterial shear rates. Juglone abolished intracellular Ca2+ elevation and protein kinase C activation caused by collagen, but had no significant effect on that induced by G protein-coupled receptor agonists. In contrast, Akt activation caused by various agonists were inhibited in juglone-treated platelets. Additionally, juglone showed inhibitory effects on both recombinant human PDI and platelet surface PDI at concentrations similar to those needed to prevent platelet aggregation. ConclusionJuglone exhibits potent in vitro antiplatelet and antithrombotic effects that are associated with inhibition of Akt activation and platelet surface PDI activity.

Perphenazine exerts antitumor effects on HUT78 cells through Akt dephosphorylation by protein phosphatase 2A.

Sezary syndrome is a rare type of non-Hodgkin lymphoma. Protein phosphatase 2A (PP2A) is an important tumor suppressor whose activity is widely inhibited in a variety of tumors. Recently, reactivation of PP2A has attracted increasing attention as a promising approach for cancer therapy. Phenothiazine anti-psychotic perphenazine (PPZ) exerts antitumor effects by reactivating PP2A. The present study investigated the molecular mechanism underling the antitumor effects of PPZ in the neuroblastoma rat sarcoma oncogene (NRAS)-mutated Sezary syndrome cell line, HUT78. The results of the present study demonstrated that PPZ induced the dephosphorylation of Akt and ERK1/2, and triggered apoptosis in HUT78 cells. In addition, a PP2A inhibitor blocked the PPZ-mediated dephosphorylation of Akt but did not affect that of ERK1/2. The pharmacological inhibition of Akt and ERK1/2 signaling revealed that Akt activity serves an important role in the survival of HUT78 cells. The present data suggested that suppressing Akt activity by PP2A activation may be an attractive antitumor strategy for NRAS-mutated Sezary syndrome.

Loss of hepatic aldolase B activates Akt and promotes hepatocellular carcinogenesis by destabilizing the Aldob/Akt/PP2A protein complex.

Loss of hepatic fructose-1, 6-bisphosphate aldolase B (Aldob) leads to a paradoxical up-regulation of glucose metabolism to favor hepatocellular carcinogenesis (HCC), but the upstream signaling events remain poorly defined. Akt is highly activated in HCC, and targeting Akt is being explored as a potential therapy for HCC. Herein, we demonstrate that Aldob suppresses Akt activity and tumor growth through a protein complex containing Aldob, Akt, and protein phosphatase 2A (PP2A), leading to inhibition of cell viability, cell cycle progression, glucose uptake, and metabolism. Interestingly, Aldob directly interacts with phosphorylated Akt (p-Akt) and promotes the recruitment of PP2A to dephosphorylate p-Akt, and this scaffolding effect of Aldob is independent of its enzymatic activity. Loss of Aldob or disruption of Aldob/Akt interaction in Aldob R304A mutant restores Akt activity and tumor-promoting effects. Consistently, Aldob and p-Akt expression are inversely correlated in human HCC tissues, and Aldob down-regulation coupled with p-Akt up-regulation predicts a poor prognosis for HCC. We have further discovered that Akt inhibition or a specific small-molecule activator of PP2A (SMAP) efficiently attenuates HCC tumorigenesis in xenograft mouse models. Our work reveals a novel nonenzymatic role of Aldob in negative regulation of Akt activation, suggesting that directly inhibiting Akt activity or through reactivating PP2A may be a potential therapeutic approach for HCC treatment.

Flunarizine, a drug approved for treating migraine and vertigo, exhibits cytotoxicity in GBM cells

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with a poor prognosis. The current treatment regimen, including surgical resection, radiation, and temozolomide (TMZ) chemotherapy, is still not curative. Therefore, there is an emerging need to develop a drug to treat GBM or synergistic enhance TMZ effect on GBM cells. Flunarizine (FLN), a drug approved for treating migraine and vertigo, was analyzed for its cytotoxicity and synergistic effect with TMZ on GBM cells in this study. Cell proliferation, clonogenic assay, flow cytometry, and Western blotting were used to determine the effects of FLN on three GBM cells, U-87 MG, LN-229, and U-118 MG cells. We found that FLN induced GBM cell death. FLN also interfered with U-87 MG cell cycle progression. Flow cytometric analysis showed an increase of apoptotic cells after FLN treatment. Caspase 9, caspase 3, and Poly (ADP-ribose) polymerase (PARP) activation were involved in apoptosis induction in U-87 MG and LN-229, suggesting the possible involvement of an intrinsic apoptotic pathway. We found that FLN treatment inhibited Akt pathway activation in U-87 MG cells, and synergistically increased the cytotoxicity of three GBM cells when combined with TMZ treatment. In conclusion, our current data suggested that FLN inhibited cell viability by inducing apoptosis. FLN inhibited Akt activation and enhanced the sensitivity of GBM cells to TMZ. These findings may provide important information regarding the application of FLN in GBM treatment in the future.

Silencing KIF14 reverses acquired resistance to sorafenib in hepatocellular carcinoma.

For nearly a decade, sorafenib has served as a first-line chemotherapeutic drug for the treatment of hepatocellular carcinoma (HCC), but it displays only limited efficacy against advanced drug-resistant HCC. Regorafenib, the first second-line drug approved for treatment after sorafenib failure, can reverse resistance to sorafenib. We used bioinformatics methods to identify genes whose expression was differentially induced by sorafenib and regorafenib in HCC. We identified KIF14 as an oncogene involved in the acquired resistance to sorafenib in HCC and investigated its potential as a target for reversing this resistance. Sustained exposure of resistant HCC cells to sorafenib activated the AKT pathway, which in turn upregulated KIF14 expression by increasing expression of the transcription factor ETS1. Silencing KIF14 reversed the acquired resistance to sorafenib by inhibiting AKT activation and downregulating ETS1 expression by blocking the AKT–ETS1–KIF14 positive feedback loop. Moreover, injection of siKIF14 with sorafenib suppressed growth of sorafenib-resistant HCC tumors in mice. These results demonstrate that targeting KIF14 could be an effective means of reversing sorafenib failure or strengthening sorafenib’s antitumor effects.

CSIG-17. PRMT5 INHIBITION SENSITIZES GLIOBLASTOMA MODELS TO TRAMETINIB TREATMENT

Abstract INTRODUCTION Glioblastoma (GBM) is the most common malignant primary brain tumor. With limited effective therapeutic strategies, prognosis for GBM is very poor. Our previous study shows that the expression of Protein Arginine Methyltransferase 5 (PRMT5) is upregulated in GBM; its inhibition promotes anti-GBM effect through apoptosis and senescence of mature and immature tumor cells, respectively. In GBM, RAS-RAF- MEK-ERK signaling is aberrantly activated and promotes tumor growth. Therefore, MEK inhibitors, including trametinib are currently under investigation for GBM therapy. In this study, we tested whether inhibition of PRMT5 can enhance the anti-tumor efficacy of trametinib in GBM. METHODS Patient-derived primary GBM neurospheres (GBMNS) with transient PRMT5 knockdown were treated with trametinib and cell viability, proliferation, cell cycle progression, and western blot analysis were conducted. In vivo, PRMT5-intact and -depleted GBMNS were intracranially implanted in NSG mice and treated with trametinib by daily oral gavage, and tumor progression and mice survival rate were analyzed by MRI and Kaplan-Meier survival curve, respectively. RESULTS Trametinib treatment upregulated the expression of PRMT5 in GBMNS. Depletion of PRMT5 increased the cytotoxic effect of trametinib in GBMNS. In concurrence with the trametinib-induced PRMT5 upregulation, trametinib treatment increased the activity of AKT that was blocked with PRMT5 knockdown. In vivo, PRMT5-depletion extended the survival of the tumor bearing mice that further increased in combination with trametinib treatment. Interestingly, trametinib treatment alone had no survival benefit. CONCLUSION Trametinib treatment induces PRMT5 expression. Depletion of trametinib-induced PRMT5 expression sensitizes GBMNS for trametinib by inhibiting AKT activity.

Activating CD137 Signaling Promotes Sprouting Angiogenesis via Increased VEGFA Secretion and the VEGFR2/Akt/eNOS Pathway.

Combination of antiangiogenesis and immunotherapy may be an effective strategy for treatment of solid tumors. Our previous work reported that activation of CD137 signaling promotes intraplaque angiogenesis. A number of studies have demonstrated that vascular endothelial growth factor receptor 2 (VEGFR2) is a key target for angiogenesis. However, it is unknown whether CD137-mediated angiogenesis is related to VEGFR2. In this study, we investigated the effect of CD137 on the VEGFR2 expression and explored the underlying mechanisms of CD137-mediated angiogenesis. Knock-out of CD137 in ApoE−/− mice significantly decreased neovessel density in atherosclerotic plaques. CD137 silencing or inhibition attenuated endothelial cell (ECs) proliferation, migration, and tube formation. We found activation of CD137 signaling for increased VEGFR2 transcription and translation steadily. Moreover, CD137 signaling activated phosphorylated VEGFR2 (Tyr1175) and the downstream Akt/eNOS pathway, whereas neutralizing CD137 signaling weakened the activation of VEGFR2 and the downstream Akt/eNOS pathway. The aortic ring assay further demonstrated that CD137 signaling promoted ECc sprouting. Inhibition of VEGFR2 by siRNA or XL184 (cabozantinib) and inhibition of downstream signaling by LY294002 (inhibits AKT activation) and L-NAME (eNOS inhibitor) remarkably abolished proangiogenic effects of CD137 signaling both in vitro and ex vivo. In addition, the condition medium from CD137-activated ECs and vascular endothelial growth factor A (VEGFA) had similar effects on ECs that expressed high VEGFR2. Additionally, activating CD137 signaling promoted endothelial secretion of VEGFA, while blocking CD137 signaling attenuated VEGFA secretion. In conclusion, activation of CD137 signaling promoted sprouting angiogenesis by increased VEGFA secretion and the VEGFR2/Akt/eNOS pathway. These findings provide a basis for stabilizing intraplaque angiogenesis through VEGFR2 intervatioin, as well as cancer treatment via combination of CD137 agonists and specific VEGFR2 inhibitors.

Osteoprotegerin interacts with syndecan-1 to promote human endometrial stromal decidualization by decreasing Akt phosphorylation.

Does osteoprotegerin (OPG) promote human endometrial stromal decidualization? OPG is essential for human endometrial stromal decidualization through its interaction with syndecan-1 to decrease Akt phosphorylation. OPG (a cytokine receptor) levels are significantly increased in the circulation of pregnant women. However, the role and mechanism of OPG in human endometrial stromal cell (ESC) decidualization remain elusive. We analyzed the endometrial expression of OPG in endometrial tissue samples collected from women with regular menstrual cycles (ranging from 25 to 35 days), and decidual tissue samples collected from woman with normal early pregnancy or recurrent pregnancy loss (RPL) who visited the Department of Gynecology and Obstetrics at a tertiary care center from January to October 2018. None of the subjects had hormonal treatment for at least 3 months prior to the procedure. In total, 16 women with normal early pregnancy and 15 with RPL were selected as subjects for this study. The function of OPG in decidualization was explored in a human endometrial stromal cell (HESC) line and primary cultures of HESCs. We collected endometrial tissues (by biopsy) from the subjects during their menstrual cycle and decidual tissues from subjects with a normal early pregnancy and those with RPL at the time of dilation and curettage. The control group comprised randomly selected women who underwent termination of an apparently normal early pregnancy. The endometrial OPG expression was analyzed using immunohistochemical staining and quantitative RT-PCR (qRT-PCR). Immunofluorescence staining and western blot, and qRT-PCR were used to explore the mRNA and protein expression, respectively, of OPG in an immortalized HESC line and in primary cultures of HESC during proliferation and decidualization. siRNA-mediated knockdown experiments were performed to examine the function of OPG in HESC proliferation and decidualization. Flow cytometry and the cell proliferation MTS assay were performed to further examine the role of OPG in HESC proliferation. We also analyzed decidual marker gene expression by qRT-PCR to assess the consequences of OPG loss for HESC decidualization. A co-immunoprecipitation (IP) assay was used to determine the potential interaction between the OPG and Syndecan-1. Western blot analysis of the rescue experiments performed using the phosphatidylinositol 3-kinase (PI3K) signaling-specific inhibitor LY294002 was used to investigate the downstream signaling pathways through which OPG could mediate HESC decidualization. OPG was expressed in both the human endometrium and in vitro decidualized ESCs. Knockdown experiments revealed that OPG loss impaired the expression of IGF-binding protein-1 (IGFBP-1) (P < 0.05) and prolactin (PRL) (P < 0.05), two specific markers of decidualization, in HESC undergoing decidualization. We also uncovered that OPG knockdown induced the aberrant activation of Akt (protein kinase B) during HESC decidualization (P < 0.05). The inhibition of Akt activation could rescue the impaired expression of the decidual markers PRL (P < 0.05) and IGFBP-1 (P < 0.05) in response to OPG knockdown. Syndecan-1 was considered a potential receptor candidate, as it was expressed in both the endometrium and in vitro cultured stromal cells. Subsequent co-IP experiments demonstrated the interaction between OPG and Syndecan-1 during decidualization. In addition, Syndecan-1 knockdown not only clearly attenuated the decidualization markers PRL (P < 0.05) and IGFBP-1 (P < 0.05) but also induced the aberrant enhancement of Akt phosphorylation in decidualized cells, consistent with the phenotype of OPG knockdown cells. Finally, we revealed that the transcript and protein expression of both OPG and Syndecan-1 was significantly lower in the decidual samples of women with RPL than in those of women with normal pregnancy (P < 0.05). N/A. In this study, based on a number of approaches, it was demonstrated that OPG mediated the repression of Akt that occurs during human stromal cell decidualization, however, the molecular link between OPG and Akt signaling was not determined, and still requires further exploration. OPG is required for decidualization, and a decrease in OPG levels is associated with RPL. These findings provide a new candidate molecule for the diagnosis and potential treatment of RPL. This work was supported in part by the National Natural Science Foundation of China U1605223 (to G.S.), 81701457 (to Y.J.) and 81601349 (to Y.J.). The authors have no conflicts of interest to disclose.

Estrogen receptor β regulates AKT activity through up-regulation of INPP4B and inhibits migration of prostate cancer cell line PC-3

Loss of the tumor suppressor, PTEN, is one of the most common findings in prostate cancer (PCa). This loss leads to overactive Akt signaling, which is correlated with increased metastasis and androgen independence. However, another tumor suppressor, inositol-polyphosphate 4-phosphatase type II (INPP4B), can partially compensate for the loss of PTEN. INPP4B is up-regulated by androgens, and this suggests that androgen-deprivation therapy (ADT) would lead to hyperactivity of AKT. However, in the present study, we found that in PCa, samples from men treated with ADT, ERβ, and INPP4B expression were maintained in some samples. To investigate the role of ERβ1 in regulation of INPPB, we engineered the highly metastatic PCa cell line, PC3, to express ERβ1. In these cells, INPP4B was induced by ERβ ligands, and this induction was accompanied by inhibition of Akt activity and reduction in cell migration. These findings reveal that, in the absence of androgens, ERβ1 induces INPP4B to dampen AKT signaling. Since the endogenous ERβ ligand, 3β-Adiol, is lost upon long-term ADT, to obtain the beneficial effects of ERβ1 on AKT signaling, an ERβ agonist should be added along with ADT.