Down-regulation Of Akt Phosphorylation Research Articles

RETRACTED: Gu et al. Long Coding RNA XIST Contributes to Neuronal Apoptosis through the Downregulation of AKT Phosphorylation and Is Negatively Regulated by miR-494 in Rat Spinal Cord Injury. Int. J. Mol. Sci. 2017, 18, 732.

The journal retracts the article titled "Long Coding RNA XIST Contributes to Neuronal Apoptosis through the Downregulation of AKT Phosphorylation and Is Negatively Regulated by miR-494 in Rat Spinal Cord Injury" [...].

MiR-19b-3p inhibits cell viability and proliferation and promotes apoptosis by targeting IGF1 in KGN cells

Background Endometriosis (EM) is a major cause of infertility, but the pathogenesis and mechanisms are not yet fully elucidated. MiR-19b-3p is involved in many diseases, but its functional role in EM-associated infertility remains unexplored. This study aimed to examine miR-19b-3p abundance and IGF1 concentration in cumulus cells (CCs) and follicular fluid of EM-associated infertility patients, and to investigate the potential role of miR-19b-3p in KGN cells by identifying its target and elucidating the underlying mechanisms. Results The results from the case-control study indicated that, compared to the control group consisting of patients with tubal infertility, patients with EM-associated infertility exhibited a lower percentage of mature oocytes. MiR-19b-3p level was elevated in CCs from EM-associated infertility patients. IGF1 was identified as a direct target of miR-19b-3p and was negatively regulated by miR-19b-3p in KGN cells. Overexpression of miR-19b-3p significantly inhibited cell viability and proliferation, promoted apoptosis, and arrested the cell cycle at G0/G1 phase in KGN cells. The effects of miR-19b-3p were reversed by co-transfection of IGF1, and the biological effects of miR-19b-3p in KGN cells were mediated by IGF1. Additionally, miR-19b-3p targeted IGF1 to down-regulate AKT phosphorylation and participate in the apoptotic pathway in KGN cells. Conclusions This study demonstrates that miR-19b-3p level is elevated in CCs and IGF1 concentration is decreased in follicular fluid in patients with EM-associated infertility. MiR-19b-3p regulates the biological effects of KGN cells by targeting IGF1.

LncRNA MAGI2-AS3 enhances cisplatin sensitivity of non-small cell lung cancer cells by regulating the miR-1269a/PTEN/AKT pathway

To investigate the mechanism mediating the regulatory effect of lncRNA MAGI2-AS3 on cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC). MAGI2-AS3 and miR-1269a expression levels were detected by qRT-PCR in DDP-sensitive lung cancer cell lines (A549 and H1299) and their resistant counterparts (A549/DDP and H1299/DDP). In A549 and H1299 cells with MAGI2-AS3 silencing and A549/DDP and H1299/DDP cells overexpressing MAGI2-AS3, the effects of 20 μmol/L DDP on cell viability and apoptosis were examined with CCK-8 assay, colony formation assay, flow cytometry and Western blotting, and the changes in epithelial-mesenchymal transition (EMT) were assessed with wound healing and Transwell assays. The interaction between MAGI2-AS3, miR-1269a and PTEN was predicted using GEPIA, StarBase and miRDB and verified with luciferase reporter gene assay and radioimmunoprecipitation (RIP) assay. A miR-1269a mimic and pcDNA3.1-PTEN plasmid were used to perform the rescue assay. MAGI2-AS3 expression was significantly downregulated in lung cancer tissues (P < 0.05) in association with a poor prognosis (P < 0.05). In the two DDP-resistant lung cancer cell lines, MAGI2-AS3 expression was significantly lowered as compared with the sensitive cells. Silencing MAGI2-AS3 significantly enhanced cell viability and promoted EMT of A549 and H1299 cells irrespective of DDP treatment, and also decreased DDP-induced apoptosis of the cells. In A549/DDP and H1299/DDP cells, MAGI2-AS3 overexpression strongly repressed cell viability and EMT irrespective of DDP treatment and promoted DDP-induced cell apoptosis. Luciferase reporter gene and RIP assays confirmed the binding of MAGI2-AS3 with miR-1269a and the binding of miR-1269a with 3 '-UTR domain of PTEN. The rescue assay demonstrated that MAGI2-AS3 acted as a sponge for miR-1269a to promote PTEN expression and downregulate AKT phosphorylation, thus inhibiting EMT and promoting DDP-induced apoptosis of A549/DDP cells. MAGI2-AS3 enhances DDP sensitivity of NSCLC by targeted regulation of the miR-1269a/PTEN/AKT signaling axis.

Hispidin Increases Cell Apoptosis and Ferroptosis in Prostate Cancer Cells Through Phosphatidylinositol-3-Kinase and Mitogen-activated Protein Kinase Signaling Pathway.

Chemotherapy is mainly used in the clinical treatment of prostate cancer. Different anticancer mechanisms can induce cell death in various cancers. Reactive oxygen species (ROS) play crucial roles in cell proliferation, differentiation, apoptosis, and signal transduction. It is widely accepted that ROS accumulation is closely related to chemical drug-induced cancer cell death. We utilized the MTT assay to detect changes in cell proliferation. Additionally, colony formation and wound healing assay were conducted to investigate the effect of hispidin on cell colony formation and migration ability. Fluorescence microscopy was used to detect intracellular and mitochondrial ROS levels, while western blot was used for detection of cell apoptosis. Hispidin treatment significantly decreased viability of PC3 and DU145 cancer cells but exhibited no cytotoxicity in WPMY-1 cells. Furthermore, hispidin treatment inhibited cell migration and colony formation and triggered cellular and mitochondrial ROS accumulation, leading to mitochondrial dysfunction and mitochondrion-dependent apoptosis. Moreover, hispidin treatment induced ferroptosis in PC3 cells. Scavenging of ROS with N-acetyl cysteine significantly inhibited hispidin-induced apoptosis by altering the expression of apoptosis-related proteins, such as cleaved caspase-3, 9, Bax, and Bcl2. Furthermore, hispidin treatment dramatically up-regulated MAPK (involving p38, ERK, and JNK proteins) and NF-kB signaling pathways while down-regulating AKT phosphorylation. Hispidin treatment also inhibited ferroptosis signaling pathways (involving P53, Nrf-2, and HO-1 proteins) in PC3 cells. In addition, inhibiting these signaling pathways via treatment with specific inhibitors significantly reversed hispidin-induced apoptosis, cellular ROS levels, mitochondrial dysfunction, and ferroptosis. Hispidin may represent a potential candidate for treating prostate cancer.

Anaplastic Lymphoma Kinase Receptor: Possible Involvement in Anorexia Nervosa

The pathophysiology of Anorexia Nervosa (AN) has not been fully elucidated. Anaplastic lymphoma kinase (ALK) receptor is a protein-tyrosine kinase mainly known as a key oncogenic driver. Recently, a genetic deletion of ALK in mice has been found to increase energy expenditure and confers resistance to obesity in these animals, suggesting its role in the regulation of thinness. Here, we investigated the expression of ALK and the downstream intracellular pathways in female rats subjected to the activity-based anorexia (ABA) model, which reproduces important features of human AN. In the hypothalamic lysates of ABA rats, we found a reduction in ALK receptor expression, a downregulation of Akt phosphorylation, and no change in the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. After the recovery from body weight loss, ALK receptor expression returned to the control baseline values, while it was again suppressed during a second cycle of ABA induction. Overall, this evidence suggests a possible involvement of the ALK receptor in the pathophysiology of AN, that may be implicated in its stabilization, resistance, and/or its exacerbation.

Abstract P3-11-04: Endoxifen Downregulates AKT Phosphorylation Through Protein Kinase C Beta 1 in ER+/HER2- Breast Cancer

Abstract Background: In phase I/II clinical trials, the tamoxifen metabolite Z-endoxifen (ENDX) demonstrated substantial oral bioavailability and promising antitumor activity in endocrine-refractory estrogen-receptor positive breast cancer (ER+ BC) and other solid tumors, with ENDX plasma concentrations reaching as high as 5 µM, a concentration that far exceeds the ERα-targeting nanomolar concentrations. We previously identified protein kinase C beta 1 (PKCβ1), an oncogenic signaling kinase which regulates cell proliferation and tumorigenic transformation, as a novel ENDX receptor. ENDX-bound PKCβ1 (KD: 100 nM) and inhibited PKCβ1 kinase activity (IC50: 357 nM) in vitro, at concentrations achieved in phase I/II ENDX studies. Given the observations that ENDX induces antitumor activity in breast cancers resistant to endocrine therapies, including AIs, tamoxifen and fulvestrant, we postulated that ENDX may impact additional signaling pathways. Therefore, in this study we performed unbiased mass spectrometry to analyze the effects of ENDX on the phosphoproteome and total proteome. Methods: In estrogen unstimulated aromatase-expressing ER+/human epidermal growth factor 2 receptor negative (HER2-) MCF7AC1 breast cancer cells, the effects of ENDX on the phosphoproteome and total proteome were analyzed using ENDX concentrations achieved in tamoxifen treated patients (0.01 and 0.1 µM) and concentrations observed in the ENDX phase I/II trials (5 µM). NetWorKIN and RoKAI prediction tools were utilized to identify the upstream kinases of ENDX downregulated phosphoproteins. In estrogen unstimulated MCF7AC1 as well as in the ER+/HER2- T47D breast cancer cells, the ability of ENDX, tamoxifen, fulvestrant and PKC kinase inhibitor enzastaurin to block insulin or PKC agonist phorbol 12-myristate 13-acetate (PMA)-stimulated AKTSer473 phosphorylation was analyzed by immunoblot (IB) assay. Additionally, ENDX effects on AKTSer473 phosphorylation in the MCF7AC1 xenograft model in vivo was analyzed by IB assay. Further, the effects of PKCβ1 silencing in MCF7AC1 cells and the effects of AKT inhibitor MK-2206 treatment on growth and AKTSer473 phosphorylation in the MCF7AC1 as well as long-term estrogen deprived T47D (T47D-LTED) cell lines were analyzed by cell proliferation and IB assays. Results: In MCF7AC1 cells unstimulated with estrogen, ENDX at 5 µM but not at 0.01 and 0.1 µM concentrations inhibited growth, and induced apoptosis, suggesting an ERα-independent effect. Further, ENDX at 5 µM displayed three-fold greater effects in downregulating the phosphoproteome compared to the other concentrations, with minimal impact on the total proteome. Protein kinase C beta (PKCβ) and AKT1 were identified as the prevalent upstream kinases for ENDX downregulated protein phosphorylations. Notably, in estrogen unstimulated MCF7AC1 and T47D cells, ENDX at 5 µM attenuated phosphorylation of AKTSer473 and AKT substrates in vitro in the presence of insulin and in vivo. In MCF7AC1 cells, PMA-induced phosphorylation of PKCβ1 as well as AKTSer473 and AKT substrate phosphorylations were blocked by ENDX at 5 µM, with ENDX inducing PKCβ1 protein degradation both in the presence of insulin and PMA. Further, ENDX effects on growth and AKTSer473 phosphorylation were phenocopied by siRNA-mediated PKCβ1 knockdown as well as treatment with the pan-AKT inhibitor, MK-2206. Notably, the potent PKCβ1 kinase inhibitor, enzastaurin, had no effects either on PKCβ1 protein degradation nor on downstream AKT signaling. Conclusion: Taken together, these findings suggest that ENDX may exert anticancer activity via dual effects on blocking ERα as well as by targeting PKCβ1 for degradation, thus suppressing AKT signaling and induction of apoptosis. These preclinical findings will be studied in a planned neoadjuvant trial comparing ENDX with exemestane and ovarian function suppression (EVANGELINE) that will activate in the Fall of 2022. Citation Format: Swaathi Jayaraman, Xinyan Wu, Krishna R. Kalari, Xiaojia Tang, Mary Kuffel, Elizabeth S. Bruinsma, Santosh Renuse, James N. Ingle, Joel Reid, Matthew Schellenberg, John Hawse, Akhilesh Pandey, Matthew P. Goetz. Endoxifen Downregulates AKT Phosphorylation Through Protein Kinase C Beta 1 in ER+/HER2- Breast Cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-04.

Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway.

Xanthine oxidase (XO) is an important source of reactive oxygen species. This study investigated whether XO inhibition exerts renoprotective effects by inhibiting vascular endothelial growth factor (VEGF) and NADPH oxidase (NOX) in diabetic kidney disease (DKD). Febuxostat (5 mg/kg) was administered to streptozotocin (STZ)-treated 8-week-old male C57BL/6 mice via intraperitoneal injection for 8 weeks. The cytoprotective effects, its mechanism of XO inhibition, and usage of high-glucose (HG)-treated cultured human glomerular endothelial cells (GECs) were also investigated. Serum cystatin C, urine albumin/creatinine ratio, and mesangial area expansion were significantly improved in febuxostat-treated DKD mice. Febuxostat reduced serum uric acid, kidney XO levels, and xanthine dehydrogenase levels. Febuxostat suppressed the expression of VEGF mRNA, VEGF receptor (VEGFR)1 and VEGFR3, NOX1, NOX2, and NOX4, and mRNA levels of their catalytic subunits. Febuxostat caused downregulation of Akt phosphorylation, followed by the enhancement of dephosphorylation of transcription factor forkhead box O3a (FoxO3a) and the activation of endothelial nitric oxide synthase (eNOS). In an in vitro study, the antioxidant effects of febuxostat were abolished by a blockade of VEGFR1 or VEGFR3 via NOX-FoxO3a-eNOS signaling in HG-treated cultured human GECs. XO inhibition attenuated DKD by ameliorating oxidative stress through the inhibition of the VEGF/VEGFR axis. This was associated with NOX-FoxO3a-eNOS signaling.

LQB-118 Suppresses Migration and Invasion of Prostate Cancer Cells by Modulating the Akt/GSK3β Pathway and MMP-9/Reck Gene Expression.

Prostate cancer (PCa) is one of the most common malignancies in adult men. LQB-118 is a pterocarpanquinone with antitumor activity toward prostate cancer cells. It inhibits cell proliferation by down-regulating cyclins D1 and B1 and up-regulating p21. However, the effects of LQB-118 on PCa cell migration are still unclear. Herein, the LQB-118 effects on PCa metastatic cell migration/invasion and its mechanism of action were evaluated. PC3 cells were treated with LQB-118 or Paclitaxel (PTX), and cell migration (wound healing and Boyden chamber assays) and invasion (matrigel assay) were determined. The LQB-118 mechanisms were evaluated by αVβIII protein expression (flow cytometry), protein phosphorylation (Western blot), and mRNA expression (qPCR). LQB-118 impaired PCa cell migration and invasion, down-regulated Akt phosphorylation, and also reduced GSK3β phosphorylation, through a FAK-independent pathway. Also, it was observed that LQB-118 controlled the invasiveness behavior by reducing matrix metalloproteinase-9 (MMP-9) and up-regulating reversion-inducing cysteine rich protein with Kazal motifs (Reck) mRNA levels. Interestingly, LQB-118 increased integrin αvβIII expression, but this effect was not related to its activation, since the cell adhesion ability was reduced after LQB-118 treatment. These data highlight novel LQB-118 mechanisms in prostate cancer cells. LQB-118 acts as a negative regulator of the Akt/GSK3 signaling pathway and can modulate PCa cell proliferation, death, and migration/invasion. The results also support the use of LQB-118 for the treatment of metastatic PCa, alone or combined with another chemotherapeutic agent, due to its demonstrated pleiotropic activities.

Analysis of Differential Expression Profiles of Liver Cancer Cell Proteins After Treatment with Bile Acid

The pathogenesis of liver cancer has not been fully elucidated yet. Bile acids are components of bile, which are inorganic substances and regulate tumor progression. However, the differential expression profile of liver cancer cell proteins after bile acid treatment remains unclear. Human hepatoma cell line SMMC7721 was cultured and randomly assigned into control group and bile acid group followed by measuring the protein expression profile by protein fingerprinting. SMMC7721 cells were transfected with UGT2B or UGT2B, followed by analysis of UGT2B expression, cell proliferation, apoptosis, migration and PI3K/AKT signaling protein expression. The most obvious proteins with an increased expression after bile acid treatment were UGT2B, AAP, APLP2, LAPTM4B, NCOA4 with UGT2B being the most significant one. Overexpression of UGT2B decreased cell proliferation, promoted cell apoptosis, downregulated migration ability and AKT phosphorylation (P &lt;0.05). UGT2B siRNA transfection significantly down-regulated UGT2B expression, promoted cell proliferation, decreased apoptosis rate, increased migration ability and AKT phosphorylation (P &lt;0.05). In conclusion, bile acid can alter the protein expressions of liver cancer cells, with UGT2B being changed most obviously. UGT2B can affect liver cancer cell behaviors via modulating PI3K/AKT signaling.

P10.11.B Re-sensitizing cancer cells to Tumor Treating Fields (TTFields) through PI3K/AKT/mTOR pathway inhibition

Abstract Background Tumor Treating Fields (TTFields) are alternating electric fields disrupting cellular processes critical for cancer cell replication and tumor progression. TTFields therapy is approved for treatment of newly diagnosed glioblastoma (GBM), recurrent GBM, and unresectable malignant pleural mesothelioma, and is currently under clinical investigation for treating other solid tumors, such as ovarian cancer, non-small cell lung carcinoma (NSCLC), and hepatocellular carcinoma (HCC). The research herein aimed to identify potential treatment escape mechanisms and explore the possibility of targeted inhibition of these pathways for re-sensitizing the cells to TTFields. Material and Methods GBM U-87 MG, ovarian A2780, and NSCLC H1299 cells were treated with TTFields (1.7 V/cm RMS, 200 or 150 kHz) for continuous long-term application (7 or 13 days, specific conditions depending on the cell line). Changes in signaling pathways in these cells relative to cells exposed to short term TTFields application (3 or 7 days, depending on cell line) were examined by Luminex multiplex assay. Specific pathway markers were examined by immunohistochemistry of tumor sections from sham or TTFields-treated rats bearing N1S1 HCC tumors. TTFields were then co-applied with relevant pathway inhibitors, followed by cell count measurements and western blot examinations for specific pathway markers. The concomitant application of TTFields with a selected inhibitor was tested in mice inoculated orthotopically with MOSE-L firefly luciferase (FFL) ovarian cancer cells. Tumor volume was measured at study end by luciferin signal detection using the In Vivo Imaging System (IVIS). Results Cancer cells exposed to long-term application of TTFields displayed decreased sensitivity to TTFields. The PI3K/AKT/mTOR pathway was activated in these cells, with significant increases in AKT and RPS6 phosphorylation levels also observed in HCC tumors from rats treated with TTFields. PI3K inhibitors re-sensitized the cells to TTFields cytotoxicity, with associated down regulation of AKT phosphorylation. Application of TTFields concomitant with the PI3K inhibitor alpelisib resulted in enhanced efficacy in the ovarian cancer mouse model. Conclusions The current research demonstrated that PI3K/AKT/mTOR signaling pathway activation was involved in the response to long-term application of TTFields, with increased phosphorylation of key proteins observed both in vitro and in vivo. Concomitant treatment with PI3K inhibitors and TTFields re-sensitized the cells to TTFields, as it alleviated TTFields-induced AKT activation.

Marine-Derived Stichloroside C2 Inhibits Epithelial-Mesenchymal Transition and Induces Apoptosis through the Mitogen-Activated Protein Kinase Signalling Pathway in Triple-Negative Breast Cancer Cells.

Background Triterpenoid saponins from sea cucumbers exhibit significant antitumour, antifungal, and antibacterial activities. However, the associated molecular mechanisms have yet to be elucidated. In this study, we screened and explored the antitumour activity and underlying mechanisms of triterpenoid saponins isolated from Thelenota ananas. Methods We isolated and purified sea cucumber saponins, determined their chemical structures, and confirmed their function in vitro. We also screened and explored the antitumour activity and underlying mechanisms of triterpenoid saponins isolated from Thelenota ananas. Results Four saponins were discovered from sea cucumber Thelenota ananas collected from the South China Sea. We found that stichloroside C2 (STC2) inhibited the proliferation and clonogenesis of the human triple-negative breast cancer (TNBC) cell line MDA-MB-231 and mouse TNBC cell line 4 T1 in a dose-dependent manner and induced apoptosis and cycle arrest in these two TNBC cell lines. STC2 induced DNA damage in two TNBC cell lines and significantly increased the protein expression level of the DNA double-strand break marker γ-H2AX. STC2 downregulated the protein expression levels of phosphorylated cyclin-dependent kinase 1 (CDK1), cyclin B1, CDK2, and cyclin A2 in MDA-MB-231 and 4 T1 cells. STC2 upregulated Bax and cleaved PARP protein expression in two types of breast cancer cells. In addition, STC2 promoted E-cadherin expression; inhibited vimentin expression; upregulated the phosphorylation levels of the mitogen-activated protein kinase (MAPK) signalling pathway-related proteins p38, JNK, and ERK1/2; and downregulated Akt phosphorylation. Conclusions STC2 exerts anti-TNBC activity, inhibits epithelial–mesenchymal transition (EMT), and induces apoptosis by regulating the cell cycle, EMT-related proteins, and MAPK signalling pathway.

Turnover of the mTOR inhibitor, DEPTOR, and downstream AKT phosphorylation in multiple myeloma cells, is dependent on ERK1-mediated phosphorylation

DEPTOR is a 48 kDa protein upregulated in multiple myeloma (MM) cells. DEPTOR inhibits mTOR and, by repressing a negative feedback loop, promotes AKT activation. We previously identified a compound that binds to DEPTOR in MM cells and induces its proteasomal degradation. To identify the mechanism of degradation, here, we screened for drug-induced posttranslational modifications and identified reduced phosphorylation of DEPTOR on serine 235 (S235). We show that an S235 phosphomimetic DEPTOR mutant was resistant to degradation, confirming the importance of this posttranslational modification. In addition, a DEPTOR mutant with a serine-to-alanine substitution at S235 could only be expressed upon concurrent proteasome inhibition. Thus, S235 phosphorylation regulates DEPTOR stability. Screening the DEPTOR interactome identified that the association of USP-7 deubiquitinase with DEPTOR was dependent upon S235 phosphorylation. Inhibition of USP-7 activity resulted in DEPTOR polyubiquitination and degradation. A scansite search suggested that ERK1 may be responsible for S235 phosphorylation, which was confirmed through the use of inhibitors, ERK1 knockdown, and an in vitro kinase assay. Inhibition of ERK1 also downregulated AKT phosphorylation. To test if DEPTOR phosphorylation mediated this crosstalk, MM cells were transfected with WT or phosphomimetic DEPTOR and exposed to ERK inhibitors. Although WT DEPTOR had no effect on the inhibition of AKT phosphorylation, the phosphomimetic DEPTOR prevented inhibition. These results indicate that ERK1 maintains AKT activity in MM cells via phosphorylation of DEPTOR. We propose that DEPTOR-dependent crosstalk provides MM cells with a viability-promoting signal (through AKT) when proliferation is stimulated (through ERK).

Glucose transporter‑1 inhibition overcomes imatinib resistance in gastrointestinal stromal tumor cells.

Imatinib mesylate (imatinib) is the primary agent of choice used to treat gastrointestinal stromal tumors (GIST). However, drug resistance to imatinib poses a major obstacle to treatment efficacy. In addition, the relationship between imatinib resistance and glycolysis is poorly understood. Glucose transporter (GLUT)-1 is a key component of glycolysis. The present study aimed to assess the potential relationship between components in the glycolytic pathway and the acquisition of imatinib resistance by GIST cells, with particular focus on GLUT-1. An imatinib-resistant GIST cell line was established through the gradual and continuous imatinib treatment of the parental human GIST cell line GIST-T1. The expression of glycolysis-related molecules (GLUT-1, hexokinase 2, pyruvate kinase M2 and lactate dehydrogenase) was assessed in parental and imatinib-resistant cells by western blotting, reverse transcription-quantitative PCR and glucose and lactate measurement kits. In addition, clinical information and transcriptomic data obtained from the gene expression omnibus database (GSE15966) were used to confirm the in vitro results. The potential effects of GLUT-1 inhibition on the expression of proteins in the glycolysis (GLUT-1, hexokinase 2, pyruvate kinase M2 and lactate dehydrogenase) and apoptosis pathways (Bcl-2, cleaved PARP, caspase-3 and caspase-9) in imatinib-resistant cells were then investigated following gene silencing and treatment using the GLUT-1 inhibitor WZB117 by western blotting. For gene silencing, the mature siRNAs for SLC2A1 were used for cell transfection. Annexin V-FITC/PI double-staining followed by flow cytometry was used to measure apoptosis whereas three-dimensional culture experiments were used to create three-dimensional spheroid cells where cell viability and spheroid diameter were measured. Although imatinib treatment downregulated GLUT-1 expression and other glycolysis pathway components hexokinase 2, pyruvate kinase M2, and lactate dehydrogenase in parental GIST-T1 cells even at low concentrations. By contrast, expression of these glycolysis pathway components in imatinib-resistant cells were increased by imatinib treatment. WZB117 administration significantly downregulated AKT phosphorylation and Bcl-2 expression in imatinib-resistant cells, whereas the combined administration of imatinib and WZB117 conferred synergistic growth inhibition effects in apoptosis assay. WZB117 was found to exert additional inhibitory effects by inducing apoptosis in imatinib-resistant cells. Therefore, the present study suggests that GLUT-1 is involved in the acquisition of imatinib resistance by GIST cells, which can be overcome by combined treatment with WZB117 and imatinib.

Metformin accelerates wound healing by Akt phosphorylation of gingival fibroblasts in insulin-resistant prediabetes mice.

This study aimed to investigate the effects of metformin on gingival wound healing in insulin-resistant prediabetes. C57BL/6J mice were fed normal diet (ND) or high-fat diet (HFD) for 10weeks; half of the HFD mice were treated with metformin (HFD+ Met) for the last 2weeks. Insulin and glucose tolerance tests were performed. The palatal gingiva (2.0×0.5mm) was surgically removed adjacent to the maxillary molars. Post-surgical wound closure was histomorphometrically evaluated for 1week. The mRNA expression of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the tissue were quantified by real-time polymerase chain reaction. In vitro, the proliferation and migration of human gingival fibroblasts (HGFs) cultured under high-glucose or control conditions with/without metformin were analyzed. Akt phosphorylation and VEGF expression following the insulin stimulation were evaluated with/without metformin in high-glucose or control media. HFD mice showed significantly higher plasma glucose levels and insulin resistance than ND mice. Gingival wound healing was delayed in HFD group compared with ND group but significantly improved in HFD + MET group. The decreased expression of VEGF and eNOS in HFD group was significantly elevated in the HFD + MET group. The proliferation and migration of HGFs were significantly impaired in high-glucose conditions compared with control; metformin treatment partially attenuated these effects. Metformin treatment significantly recovered the downregulated Akt phosphorylation and VEGF expression in high-glucose conditions. Metformin improved delayed gingival wound healing in insulin-resistant prediabetes by accelerating HGFs proliferation and migration via Akt phosphorylation in insulin signaling pathway.

MO629XANTHINE OXIDASE INHIBITOR ATTENUATES RENAL OXIDATIVE STRESS AND ENDOTHELIAL DYSFUNCTION THROUGH THE INHIBITION OF VEGF-NADPH OXIDASES IN DIABETIC NEPHROPATHY

Abstract Background and Aims Xanthine oxidase (XO) is one of major source of reactive oxygen species, and a XO inhibitor, febuxostat has been reported to the protection of kidney diseases. We investigated whether febuxostat exerts renoprotective effects against diabetic nephropathy (DN). Method Eight-week Male C57BL/6 mice were divided into four groups: Control group (Cont), Febuxostat control group (FEB), streptozotocin treated group (STZ) and a febuxostat and STZ-treated diabetes group (STZ+FEB). STZ was used to induce diabetes (50 mg/kg/day, 5 days), and 5 mg/kg of febuxostat was treated to experimental mice for 8 weeks. Results STZ-treated diabetic mice were significantly decreased in serum and kidney XO levels, serum cystatin C and albuminuria by febuxostat treatment. Febuxostat treatment decreased renal hypertrophy and mesangial matrix expansion in STZ-treated diabetic mice. Febuxostat treatment suppressed the expression of vascular endothelial growth factor (VEGF)1 and 3, NADPH oxidase (NOX)1, 2, and 4, and the levels of their catalytic subunit mRNA in in STZ-treated diabetic mice. Febuxostat treatment was accompanied by the downregulation of Akt phosphorylation, followed by the suppression of transcription factor forkhead box O3a phosphorylation and the enhancement of endothelial nitric oxide synthase. Finally, febuxostat improved oxidative stress and resulted in decreased 8-hydroxy-2'-deoxyguanosine and kidney malondialdehyde levels, and increased superoxide dismutase activity in STZ-treated diabetic mice. Conclusions Febuxostat attenuated DN by modulating oxidative stress and endothelial function through VEGF–NADPH oxidase signaling pathway.

Environmental Pollutant Benzo[a]pyrene Upregulated Long Non-coding RNA HZ07 Inhibits Trophoblast Cell Migration by Inactivating PI3K/AKT/MMP2 Signaling Pathway in Recurrent Pregnancy Loss.

Benzo(a)pyrene (BaP) is a ubiquitous environmental endocrine-disrupting chemical that is known to have toxic effects on reproduction. However, the underlying mechanisms describing how BaP and its metabolite benzo[a]pyrene-7, 8-diol-9, 10-epoxide (BPDE) induce recurrent pregnancy loss (RPL) are still largely unclear. In this study, we identified a novel long non-coding RNA (lnc-HZ07, NCBI MT936329) that was upregulated in trophoblast cells after exposure to BPDE, and lnc-HZ07 expression was significantly higher in RPL villous tissues than that in control villous tissues. Knockdown of lnc-HZ07 promoted trophoblast cell migration, whereas overexpression of lnc-HZ07 inhibited trophoblast cell migration. Further study showed that lnc-HZ07 inhibited trophoblast migration by downregulating matrix metalloproteinase 2 (MMP2) expression via dephosphorylation of AKT. These results demonstrated a novel regulatory pathway in which BaP downregulated AKT phosphorylation and inhibited MMP2 expression by upregulating lnc-HZ07, suggesting that lnc-HZ07 could be considered as a potential pathological marker of BaP-induced RPL and therapeutic target for this disease.

Safrana l Prevents Prostate Cancer Recurrence by Blocking the Re-activation of Quiescent Cancer Cells via Downregulation of S-Phase Kinase-Associated Protein 2.

The re-proliferation of quiescent cancer cells is considered to be the primary contributor to prostate cancer (Pca) recurrence and progression. In this study, we investigated the inhibitory effect of safranal, a monoterpene aldehyde isolated from Crocus sativus (saffron), on the re-proliferation of quiescent Pca cells in vitro and in vivo. The results showed that safranal efficiently blocked the re-activation of quiescent Pca cells by downregulating the G0/G1 cell cycle regulatory proteins CDK2, CDK4, CDK6, and phospho-Rb at Ser807/811 and elevating the levels of cyclin-dependent kinase inhibitors, p21 and p27. Further investigation on the underlying mechanisms revealed that safranal suppressed the mRNA and protein expression levels of Skp2, possibly through the deregulation of the transcriptional activity of two major transcriptional factors, E2F1 and NF-κB subunits. Moreover, safranal inhibited AKT phosphorylation at Ser473 and deregulated both canonical and non-canonical NF-κB signaling pathways. Safranal suppressed the tumor growth of quiescent Pca cell xenografts in vivo. Furthermore, safranal-treated tumor tissues exhibited a reduction in Skp2, E2F1, NF-κB p65, p-IκBα (Ser32), c-MYC, p-Rb (Ser807), CDK4, CDK6, and CDK2 and an elevation of p27 and p21 protein levels. Therefore, our findings demonstrate that safranal suppresses cell cycle re-entry of quiescent Pca cells in vitro and in vivo plausibly by repressing the transcriptional activity of two major transcriptional activators of Skp2, namely, E2F1 and NF-κB, through the downregulation of AKT phosphorylation and NF-κB signaling pathways, respectively.

Curcumin represses mTORC1 signaling in Caco-2 cells by a two-sided mechanism involving the loss of IRS-1 and activation of AMPK

The mechanistic target of rapamycin complex 1 (mTORC1) is a central modulator of inflammation and tumorigenesis in the gastrointestinal tract. Growth factors upregulate mTORC1 via the PI3K/AKT and/or Ras/MAPK signal pathways. Curcumin (CUR), a polyphenol found in turmeric roots (Curcuma longa) can repress mTORC1 kinase activity in colon cancer cell lines; however, key aspects of CUR mechanism of action remain to be elucidated including its primary cellular target. We investigated the molecular effects of physiologically attainable concentration of CUR (20 μM) in the intestinal lumen on mTORC1 signaling in Caco-2 cells. CUR markedly inhibited mTORC1 kinase activity as determined by the decreased phosphorylation of p70S6K (Thr389, −99%, P < 0.0001) and S6 (Ser235/236, −92%, P < 0.0001). Mechanistically, CUR decreased IRS-1 protein abundance (−80%, P < 0.0001) thereby downregulating AKT phosphorylation (Ser473, −94%, P < 0.0001) and in turn PRAS40 phosphorylation (Thr246, −99%, P < 0.0001) while total PRAS40 abundance was unchanged. The use of proteasome inhibitor MG132 showed that CUR-mediated loss of IRS-1 involved proteasomal degradation. CUR lowered Raptor protein abundance, which combined with PRAS40 hypophosphorylation, suggests CUR repressed mTORC1 activity by inducing compositional changes that hinder the complex assembly. In addition, CUR activated AMPK (Thr172 phosphorylation, P < 0.0001), a recognized repressor of mTORC1, and AMPK upstream regulator LKB1. Although cargo adapter protein p62 was decreased by CUR (−49%, P < 0.004), CUR did not significantly induce autophagy. Inhibition of AKT/mTORC1 signaling by CUR may have lifted the cross-inhibition onto MAPK signaling, which became induced; p-ERK1/2 (+670%, P < 0.0001), p-p38 (+1433%, P < 0.0001). By concomitantly targeting IRS-1 and AMPK, CUR's mechanism of mTORC1 inhibition is distinct from that of rapamycin.

Abstract 5262: Identifying the role of AXL in fusion kinase inhibitor resistant non-small cell lung cancer

Abstract Chromosomal rearrangements involving ALK, ROS1, RET and others generate novel fusion kinases which comprise approximately 10% of the oncogenic drivers in lung adenocarcinoma. Several tyrosine kinase inhibitors (TKIs) are approved or in development for each of these oncogenes and have shown marked clinical improvement for patients, but resistance inevitably occurs, a significant proportion of which is not currently druggable. AXL is a receptor tyrosine kinase that is often upregulated in resistant cancers in response to both chemotherapy and targeted therapies such as EGFR inhibitors. AXL can promote resistance to targeted therapy by signaling through the MAPK and Akt pathways or heterodimerizing with other receptors to maintain growth and survival signaling. However, the role of AXL in mediating resistance to ALK, ROS1, and RET TKIs is not well understood. In screening our unique panel of patient-derived lung adenocarcinoma cell lines harboring ALK, ROS1 or RET gene fusions, we found elevated AXL expression was correlated with resistance to cognate TKIs, as well as mesenchymal features including spindle morphology and vimentin expression. Combining the AXL inhibitor R428 with TKI treatment, such as crizotinib for ALK and ROS1 driven cell lines, was effective at inhibiting colony formation and wound healing in fusion kinase inhibitor resistant cell lines. Combination treatment downregulates Akt phosphorylation in resistant cells, and we found the resistant cell lines were also more sensitive to PI3K inhibition than cell lines that are still sensitive to their specific TKI. Interestingly, we also identified AXL cross talk with other receptor tyrosine kinases. In a RET inhibitor resistant model expression of both AXL and EGFR are elevated and the receptors interact, which can be disrupted upon EGF stimulation, to drive resistance. Collectively, we show that AXL upregulation is a common marker of TKI resistance among ALK, ROS1, and RET fusion kinase driven lung adenocarcinoma models, and provide rationale for AXL as a clinical target for combination therapy to overcome drug resistance. Citation Format: Tara Peters, Anastasios Dimou, Anh Le, Robert Doebele. Identifying the role of AXL in fusion kinase inhibitor resistant non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5262.

Ascofuranone suppresses invasion and F-actin cytoskeleton organization in cancer cells by inhibiting the mTOR complex 1 signaling pathway.

Ascofuranone is an antiviral antibiotic that is known to exert multiple anti-tumor effects, including cell cycle arrest, inhibition of mitochondrial respiration, and inhibition of angiogenesis. In this study, we investigated the molecular mechanisms underlying the anti-metastatic effects of ascofuranone in insulin-like growth factor-I (IGF-1)-responsive cancer cells. The inhibitory effect of ascofuranone on cancer cell migration and invasion was assessed using scratch wound healing and Matrigel invasion assays, respectively. F-actin cytoskeleton organization was assessed using FITC conjugated phalloidin staining. Target gene expression was evaluated using Western blotting and gene silencing was performed using siRNA transfections. Finally, the anti-metastatic effect of ascofuranone was investigated in vivo. We found that ascofuranone suppressed IGF-1-induced cell migration, invasion and motility in multiple cancer cell lines. The effects of ascofuranone on actin cytoskeleton organization were found to be mediated by suppression of the mTOR/p70S6K/4EBP1 pathway. Ascofuranone inhibited IGF-1-induced mTOR phosphorylation and actin cytoskeleton organization via upregulation of AMPK and downregulation of Akt phosphorylation. It also selectively suppressed the IGF-1-induced mTOR complex (mTORC)1 by phosphorylation of Raptor, but did not affect mTORC2. Furthermore, we found that focal adhesion kinase (FAK) activation decreased in response to ascofuranone, rapamycin, compound C and wortmannin treatment. Finally, we found that ascofuranone suppressed phosphorylation of FAK and mTOR and dephosphorylation of Raptor in cancerous metastatic lung tissues in vivo. Our data indicate that ascofuranone suppresses IGF-1-induced cancer cell migration and invasion by blocking actin cytoskeleton organization and FAK activation through inhibition of the mTORC1 pathway, and reveal a novel anti-metastatic function of this compound.