Phosphorylation Of PDK1 Research Articles

Suppression of TBCK enhances TRAIL-mediated apoptosis by causing the inactivation of the akt signaling pathway in human renal carcinoma Caki-1 cells.

TBC1 domain-containing kinase (TBCK) protein functions as a growth suppressor in certain cell types and as a tumor promoter in others. Although TBCK knockdown increases the responsiveness of cancer cells to anticancer drugs, the detailed mechanisms by which TBCK knockdown increases susceptibility to anticancer drugs remain unknown. This study analyzed the role of TBCK in sensitivities to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and doxorubicin in human renal cancer cells. Flow cytometry was employed to evaluate the extent of apoptosis. Western blotting, transient transfection, and lentiviral infection techniques were conducted to investigate the impact of TBCK on apoptosis-related protein expression and mitogen-activated protein kinase (MAPK). TBCK knockdown in renal cancer cells inhibits ERK and Akt signaling pathways and increases TRAIL and doxorubicin sensitivity. In TBCK-knockdown Caki-1 cells, ERK and Akt phosphorylation was suppressed compared to control cell lines, and TRAIL and doxorubicin sensitivities were increased in these cells. In addition, the phosphorylation of PDK1 was suppressed in TBCK-suppressed cells, indicating that TBCK may be involved in the PDK1 and Akt signaling pathways. The introduction of dominantly active Akt into TBCK-suppressed cells restored their sensitivity to TRAIL. In addition, TBCK downregulation enhanced TRAIL sensitivity in different renal cancer cell lines. These data suggest that TBCK could potentially have a crucial function in influencing the effects of anti-cancer drugs including TRAIL by modulating the signaling pathway involving Akt and PDK1 in human renal cancer cells.

The Ypk1 protein kinase signaling pathway is rewired and not essential for viability in Candida albicans.

Protein kinases are central components of almost all signaling pathways that control cellular activities. In the model organism Saccharomyces cerevisiae, the paralogous protein kinases Ypk1 and Ypk2, which control membrane lipid homeostasis, are essential for viability, and previous studies strongly indicated that this is also the case for their single ortholog Ypk1 in the pathogenic yeast Candida albicans. Here, using FLP-mediated inducible gene deletion, we reveal that C. albicans ypk1Δ mutants are viable but slow-growing, explaining prior failures to obtain null mutants. Phenotypic analyses of the mutants showed that the functions of Ypk1 in regulating sphingolipid biosynthesis and cell membrane lipid asymmetry are conserved, but the consequences of YPK1 deletion are milder than in S. cerevisiae. Mutational studies demonstrated that the highly conserved PDK1 phosphorylation site T548 in its activation loop is essential for Ypk1 function, whereas the TORC2 phosphorylation sites S687 and T705 at the C-terminus are important for Ypk1-dependent resistance to membrane stress. Unexpectedly, Pkh1, the single C. albicans orthologue of Pkh1/Pkh2, which mediate Ypk1 phosphorylation at the PDK1 site in S. cerevisiae, was not required for normal growth of C. albicans under nonstressed conditions, and Ypk1 phosphorylation at T548 was only slightly reduced in pkh1Δ mutants. We found that another protein kinase, Pkh3, whose ortholog in S. cerevisiae cannot substitute Pkh1/2, acts redundantly with Pkh1 to activate Ypk1 in C. albicans. No phenotypic effects were observed in cells lacking Pkh3 alone, but pkh1Δ pkh3Δ double mutants had a severe growth defect and Ypk1 phosphorylation at T548 was completely abolished. These results establish that Ypk1 is not essential for viability in C. albicans and that, despite its generally conserved function, the Ypk1 signaling pathway is rewired in this pathogenic yeast and includes a novel upstream kinase to activate Ypk1 by phosphorylation at the PDK1 site.

Abstract 2553: Elucidation of the downstream signaling of PIK3CA genomic alterations in serous ovarian cancer

Abstract Genomic aberrations of PIK3CA, which encodes the catalytic subunit p110α of the class IA phosphatidylinositol 3-kinase (PI3K), is frequently observed in cancers. In ovarian cancer of the serous subtype (the most common histotype of the disease), PIK3CA amplification is dominant over single nucleotide mutations in multiple patient cohorts, suggesting the possibility that PIK3CA amplification is an alternative to PIK3CA “hot spot” mutation for serous ovarian tumorigenesis. However, whether PIK3CA amplification and mutation exert the same functional consequences on serous ovarian tumorigenesis remains uncertain. This study aims to explore and compare the functional roles between the two types of PIK3CA gene aberrations on tumorigenicity, cellular signaling, and therapeutic responses in serous ovarian cancer. We confirmed that both PIK3CA amplification and E545K mutation could induce multiple tumorigenic phenotypes in CRISPR/Cas9-mediated E545K knock-in or wild-type PIK3CA overexpressing serous ovarian cancer cell lines. Signaling pathway analyses showed that E545K knock-in cells, but not PIK3CA-overexpressing cells, had the MAPK signaling pathway activated and were sensitive to MAPK pathway inhibition. An induction of AKT phosphorylation and PDK1 phosphorylation was observed in both E545K knock-in cells and wild-type PIK3CA-overexpressing cells. Accordingly, these cells displayed higher sensitivity towards p110α or AKT inhibitors. Intriguingly, in line with previous report which demonstrated YAP as AKT substrate, the activation of AKT associated with an increase in YAP S127 phosphorylation in the knock-in or PIK3CA-overexpressing cells. AKT inhibition suppressed YAP phosphorylation. Corroborating with the role of S127 in nuclear exclusion of YAP, YAP accumulated in the cytoplasm of these cells, leading to reduced transcriptional activity of YAP. While the functional significance of YAP cytoplasmic accumulation is yet to be investigated, our results reveal the common and distinct downstream pathways between PIK3CA amplification and mutation. These findings may be applicable to p110α-targeting therapies in serous ovarian cancer. Citation Format: Shibo Zhang, Hei Ip Hong, Victor CY Mak, Yuan Zhou, Panpan Wang, Lydia Wai Ting Cheung. Elucidation of the downstream signaling of PIK3CA genomic alterations in serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2553.

Penazaphilones J-L, Three New Hydrophilic Azaphilone Pigments from Penicillium sclerotiorum cib-411 and Their Anti-Inflammatory Activity.

Penazaphilones J-L (1-3), three new hydrophilic azaphilone pigments, as well as six known compounds, were discovered from the filamentous fungus Penicillium sclerotiorum cib-411. Compounds 1-3 were structurally elucidated by the detailed interpretation of their 1D and 2D NMR spectroscopic data. Compound 1 is an unprecedented hybrid of an azaphilone and a glycerophosphate choline. Compounds 2 and 3 each contain an intact amino acid moiety. The bioassay showed that compound 3 exhibited significant anti-inflammatory activity. Concretely, compound 3 significantly suppressed the NO production, the expression levels of COX-2, IL-6, IL-1β, and iNOS mRNA in LPS-stimulated RAW264.7 cells. Moreover, treatment of compound 3 prevented the translocation of NF-κB through inhibiting the phosphorylation of PI3K, PDK1, Akt, and GSK-3β. Thus, the inhibition of compound 3 against LPS-induced inflammation should rely on its inactivation on NF-κB.

Cardiomyocyte hyperplasia and immaturity but not hypertrophy are characteristic features of patients with RASopathies

AimsRASopathies are caused by mutations in genes that alter the MAP kinase pathway and are marked by several malformations with cardiovascular disorders as the predominant cause of mortality. Mechanistic insights in the underlying pathogenesis in affected cardiac tissue are rare. The aim of the study was to assess the impact of RASopathy causing mutations on the human heart. Methods and resultsUsing single cell approaches and histopathology we analyzed cardiac tissue from children with different RASopathy-associated mutations compared to age-matched dilated cardiomyopathy (DCM) and control hearts. The volume of cardiomyocytes was reduced in RASopathy conditions compared to controls and DCM patients, and the estimated number of cardiomyocytes per heart was ∼4–10 times higher. Single nuclei RNA sequencing of a 13-year-old RASopathy patient (carrying a PTPN11 c.1528C > G mutation) revealed that myocardial cell composition and transcriptional patterns were similar to <1 year old DCM hearts. Additionally, immaturity of cardiomyocytes is shown by an increased MYH6/MYH7 expression ratio and reduced expression of genes associated with fatty acid metabolism. In the patient with the PTPN11 mutation activation of the MAP kinase pathway was not evident in cardiomyocytes, whereas increased phosphorylation of PDK1 and its downstream kinase Akt was detected. ConclusionIn conclusion, an immature cardiomyocyte differentiation status appears to be preserved in juvenile RASopathy patients. The increased mass of the heart in such patients is due to an increase in cardiomyocyte number (hyperplasia) but not an enlargement of individual cardiomyocytes (hypertrophy).

Incarvillea compacta Maxim ameliorates inflammatory response via inhibiting PI3K/AKT pathway and NLRP3 activation.

Incarvillea compacta Maxim is a traditional Tibetan medicine used to treat inflammation-related diseases, such as pneumonia, fever, jaundice, and otitis media. However, no studies have examined its anti-inflammatory mechanism. To validate the anti-inflammatory activity of I. compacta extract (ICE) and its protective effect on acute alcoholic gastritis, Phytochemicals of I. compacta were identified using Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Lipopolysaccharide (LPS)-induced RAW 264.7 macrophages were used in vitro along with an in vivo a mouse acute gastritis model. Pro-inflammatory mediators and cytokines were measured using the Griess reagent and Cytometric bead array (CBA) assay. Furthermore, inflammation-related molecules were analysed by Western blotting, RNA-Seq, and real-time quantitative PCR (RT-qPCR). The experimental results revealed that ICE decreased the nitric oxide (NO), IL-6, MCP-1, and TNF-α levels in LPS-stimulated RAW 264.7 cells, and downregulated the expression and phosphorylation of PDK1, AKT, and GSK3β. Moreover, ICE also downregulated the activation of NLRP3. The RNA-Seq analysis revealed that 340 differentially expressed genes (DEGs) response to ICE treatment was enriched in several inflammation-related biological processes. The results of the in vivo mouse acute gastritis model showed that ICE significantly reduced inflammatory lesions in the gastric mucosa and remarkably downregulated the expression of iNOS, TNF-α, IL-1β, and IL-6 mRNA in gastric tissue. Therefore, the results of this study obtained scientific evidence supporting the use of I. compacta.

Abstract 2569: Development of potent dual PDK1/AurA kinase inhibitors for Ewing sarcoma therapy

Abstract Background. It has been estimated that more than 50% of cancers are related to the dysregulation of the PI3K-PDK1-Akt signaling axis. Within this pathway, phosphoinositide-dependent kinase-1 (PDK1) and Aurora kinase A (AurA) have key roles. PDK1 is a phosphorylation-mediated activator of Akt, the main apoptotic regulator, thereby acting at the crossroads of several oncogenic pathways. Overexpression and/or aberrant activation of AurA have been functionally linked to neoplastic transformation principally through the development of centrosome amplification and chromosomal instability. In this study, the molecular mechanisms involved in the dual inhibitory capacity of PDK1- and AurA-targeting OXID-pyridonyl compounds were explored in a panel of tumor cell lines carrying PI3KCA, K-RAS, PTEN, RB-1, EGFR and/or TP53 mutations Materials and Methods. To evaluate the enzymatic inhibitory activity, the oxindole-based derivatives were subjected to FRET-based Z′-Lyte assay (Invitrogen™) against PDK1 and AurA. All in vitro assays for ADME-Tox profiling were performed as published previously[1]. Anti-proliferative effects of OXID-pyridonyl compounds (SA16, IB35, VI8, VI18) were assessed by soft agar clonogenic formation and MTT assays after 72-h exposures with increasing concentrations (from 0.014 to 30 µM) in a panel of 15 cell lines. Protein levels were analyzed by Western Blot using commercial antibodies. OSU-03012 (PDK-1 inhibitor) and MK-5108 (AurA inhibitor) were used as reference compounds. Results. Among the four evaluated compounds, SA16 displayed nanomolar inhibitory potency against both PDK1 and AurA. In vitro, drugs SA16 and VI8 showed to exert anti-tumor activity in the 6647, TC71, SKNMC Ewing sarcoma (ES) cells lines and in the HCT116 colon carcinoma-derived cells (IC50s values between 2.6 and 25 µM after 72-h exposures), when tested in a panel of 15 cancer cell lines. These results were confirmed by the evaluation of 3D-clonogenic capacity in soft agar. The effect of 1 µM SA16 at the protein level was evaluated after 5 to 120 min exposure in the ES cell line SKNMC. No modulation of Aur-A protein or PDK-1 phosphorylation (Ser241) were observed in SKNMC cells, whereas a decrease of downstream PDK1 targets such as p-AKT, p-RSK2, and p-p44/42 MAPK was observed after 30 min of exposure. In addition, SA16 displayed acceptable in vitro ADME-Tox properties. Our results on structural insights into the mode of binding[2], in vitro drug-intake kinetics and in vivo PK appear to indicate that SA16 presents adequate features as a scaffold for the design of PDK1/AurA dual-target molecules. Conclusion. We report the synthesis of novel first-in-class 2-oxindole-based derivatives as dual PDK1-AurA kinase inhibitors as a novel scaffold for the design and synthesis of a new multitarget strategy for Ewing sarcoma.[1] Runfola M. et al. Data Brief 29 (2020) 105206.[2] Sistito S. et al. Eu J Med Chem 226 (2021) 11389. Citation Format: Maria Eugenia Riveiro, Samuel Huguet, Ramiro Vazquez, Roberta Frapolli, Guido Puricelli, Simona Sestito, Olivier Madar, Keyvan Rezai, Gianpiero Garau, Simona Rapposelli. Development of potent dual PDK1/AurA kinase inhibitors for Ewing sarcoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2569.

Reducing PDK1/Akt Activity: An Effective Therapeutic Target in the Treatment of Alzheimer's Disease.

Alzheimer’s disease (AD) is a common age-related neurodegenerative disease that leads to memory loss and cognitive function damage due to intracerebral neurofibrillary tangles (NFTs) and amyloid-β (Aβ) protein deposition. The phosphoinositide-dependent protein kinase (PDK1)/protein kinase B (Akt) signaling pathway plays a significant role in neuronal differentiation, synaptic plasticity, neuronal survival, and neurotransmission via the axon–dendrite axis. The phosphorylation of PDK1 and Akt rises in the brain, resulting in phosphorylation of the TNF-α-converting enzyme (TACE) at its cytoplasmic tail (the C-terminal end), changing its internalization as well as its trafficking. The current review aimed to explain the mechanisms of the PDK1/Akt/TACE signaling axis that exerts its modulatory effect on AD physiopathology. We provide an overview of the neuropathological features, genetics, Aβ aggregation, Tau protein hyperphosphorylation, neuroinflammation, and aging in the AD brain. Additionally, we summarized the phosphoinositide 3-kinase (PI3K)/PDK1/Akt pathway-related features and its molecular mechanism that is dependent on TACE in the pathogenesis of AD. This study reviewed the relationship between the PDK1/Akt signaling pathway and AD, and discussed the role of PDK1/Akt in resisting neuronal toxicity by suppressing TACE expression in the cell membrane. This work also provides a perspective for developing new therapeutics targeting PDK1/Akt and TACE for the treatment of AD.

S6K1-mediated phosphorylation of PDK1 impairs AKT kinase activity and oncogenic functions

Functioning as a master kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1) plays a fundamental role in phosphorylating and activating protein kinases A, B and C (AGC) family kinases, including AKT. However, upstream regulation of PDK1 remains largely elusive. Here we report that ribosomal protein S6 kinase beta 1 (S6K1), a member of AGC kinases and downstream target of mechanistic target of rapamycin complex 1 (mTORC1), directly phosphorylates PDK1 at its pleckstrin homology (PH) domain, and impairs PDK1 interaction with and activation of AKT. Mechanistically, S6K1-mediated phosphorylation of PDK1 augments its interaction with 14-3-3 adaptor protein and homo-dimerization, subsequently dissociating PDK1 from phosphatidylinositol 3,4,5 triphosphate (PIP3) and retarding its interaction with AKT. Pathologically, tumor patient-associated PDK1 mutations, either attenuating S6K1-mediated PDK1 phosphorylation or impairing PDK1 interaction with 14-3-3, result in elevated AKT kinase activity and oncogenic functions. Taken together, our findings not only unravel a delicate feedback regulation of AKT signaling via S6K1-mediated PDK1 phosphorylation, but also highlight the potential strategy to combat mutant PDK1-driven cancers.

TRPM7 silencing modulates glucose metabolic reprogramming to inhibit the growth of ovarian cancer by enhancing AMPK activation to promote HIF-1\u03b1 degradation

BackgroundTumor cell metabolic reprogramming is crucial for the malignant behavior of cancer cells by promoting their proliferation. However, little is known on how transient receptor potential 7 (TRPM7) modulates metabolic reprogramming in ovarian cancer.MethodsThe effects of TRPM7 silencing on transcriptome profile, glucose uptake, lactic acid production, extracellular acidification rate (ECAR), oxygen consumption rate (OCR), intracellular ROS and ATP levels, and NAD+/NADH ratios in ovarian cancer cells were examined. The impacts of TRPM7 silencing on the levels of glycolysis-related HK2, PDK1 and oxidative phosphorylation (OXPHOS)-related IDH3B and UQCRC1, HIF-1α expression and AMPK phosphorylation were determined in ovarian cancer. The effect of AMPK activity on HIF-1α ubiquitination degradation was investigated in ovarian cancer cells.ResultsCompared with the control, TRPM7 silencing suppressed the proliferation of ovarian cancer cells by shifting preferable glycolysis to OXPHOS. In parallel, TRPM7 silencing decreased the glucose uptake of tumor-bearing mice and TRPM7 levels were negatively correlated with IDH3B and UQCRC1, but positively with HK2 and PDK1 expression in ovarian cancer tissues. Mechanistically, TRPM7 silencing significantly increased AMPK phosphorylation and decreased HIF-1α protein levels in ovarian cancer, particularly in HIF-1α silencing cells. The shifting from glycolysis to OXPHOS by TRPM7 silencing was abrogated by HIF-1α over-expression and impaired by inhibiting AMPK activity in ovarian cancer cells. Moreover, enhanced AMPK activation inhibited glycolysis, which was abrogated by HIF-1α over-expression in ovarian cancer cells. Moreover, the enhanced AMPK activation promoted HIF-1α ubiquitination degradation.ConclusionsTRPM7 silencing enhanced AMPK activation to shift glycolysis to oxidative phosphorylation by promoting HIF-1α ubiquitination degradation in ovarian cancer. Hence, TRPM7 may be a therapeutic target for intervention of ovarian cancer.

Mechanistic Study of Macranthoside B Effects on Apoptotic Cell Death in Human Cervical Adenocarcinoma Cells

Macranthoside B (MB) is a triterpenoid saponin extracted from Lonicera macranthoides, a traditional Chinese medicine. In the current study, we investigated the anticancer potential of MB in various cancer cells and elucidated its underlying mechanisms. MB exposure inhibited cell proliferation, induced mitochondrial membrane potential (MMP) loss, increased sub-G1 accumulation, and resulted in cleavage of caspase-3 and PARP, which are reflective of apoptosis. In HeLa cells, MB induced down-regulation of SOD2 and GPx1, phosphorylation of Akt and PDK1, and thus promoted ROS-mediated apoptosis. This was further supported by the protection of sub-G1 accumulation, MMP loss, cleavage of caspase-3 and PARP in the presence of N-acetylcys­teine (NAC). Additionally, MB induced cell death via down-regulation of ubiquitin-like with PHD and ring-finger domains 1 (UHRF1) and Bcl-xL. Taken together, this study provides a new insight into the apoptosis-inducing potential of MB, and its molecular mechanisms are associated with an increase in oxidative stress and inhibition of the PDK1/Akt pathway.

PSVII-7 Prematurity alters the regulation of Akt signaling in skeletal muscle of piglets

Abstract Objectives: Postnatal growth faltering is common after preterm birth. Recently we showed that premature birth in piglets impairs normal postnatal weight gain and skeletal muscle protein synthesis compared to piglets born at term. This response is associated with a reduction in the feeding-induced activation of Akt and subsequent downstream signaling, despite no change in insulin receptor activation. The aim of this study was to identify key regulators of Akt responsible for the blunted anabolic response in preterm muscle. Methods: Piglets delivered by cesarean section 11 d (preterm/PT) or 2 d (term/T) before term birth were fed by total parenteral nutrition. On day 3, after 4 h fasting, piglets were fasted one additional h or fed orally a sow milk replacer (per kg body weight: 31.5 kcal, 1.3 g carbohydrate, 2.7 g amino acids BW, 1.6 g lipid). Positive and negative regulators of Akt activity in longissimus dorsi muscle were determined by Western blot. Results: Phosphorylation of Akt1 and Akt2, but not Akt3, was lower in PT than in T pigs (P &amp;lt; 0.05). Phosphorylation of Akt activators, PDK1 and mTORC2, and the abundance of Ubl4A, a positive regulator of Akt, were lower in PT than in T (P &amp;lt; 0.05). Abundance of Akt inhibitors, PHLPP and SHIP2, but not PTEN, was higher in PT than in T (P &amp;lt; 0.05). Activation of the Akt phosphatase, PP2A, was unaffected by feeding in PT but inhibited by feeding in T pigs (P &amp;lt; 0.05). Conclusions: These results show that the feeding-induced activation of positive regulators of Akt is reduced by preterm birth, whereas the activation of negative regulators is enhanced. Our findings suggest that premature birth impairs the activation of Akt that is essential for channeling dietary nutrients for anabolism and likely contributes to the postnatal growth faltering of prematurity. Research Support: NIH and USDA.

PI3Kδ Sustains Keratinocyte Hyperproliferation and Epithelial Inflammation: Implications for a Topically Druggable Target in Psoriasis.

The phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway is aberrantly activated in psoriatic lesions and contributes to disease pathogenesis. Among PI3Ks enzymes, PI3Kα, β, and δ isoforms are known to bind the p85 regulatory subunit and mediate activation of AKT and other downstream effectors. In this study, we deepened our understanding of the expression and function of PI3Kδ in skin lesions of patients affected by psoriasis. For the first time, we found that PI3Kδ is overexpressed in psoriatic plaques, and its expression is not only confined to infiltrating immune cells but also accumulates in proliferating keratinocytes of the epidermal basal layer. We investigated the function of PI3Kδ in psoriatic skin by evaluating the impact of seletalisib, a newly developed selective PI3Kδ inhibitor, in both in vitro and in vivo experimental models of psoriasis. Of note, we found that PI3Kδ sustains keratinocyte hyperproliferation and impaired terminal differentiation induced by IL-22, as well as induces epithelial inflammation and resistance to apoptosis mediated by TNF-α in human keratinocytes. Mechanistically, PI3Kδ promotes PDK1 phosphorylation and signals through AKT-dependent or -independent pathways. It is worth mentioning that PI3Kδ inhibition by seletalisib attenuates the severity of psoriasiform phenotype induced in the Imiquimod-induced mouse model of psoriasis by restoring the physiological proliferation and differentiation programs in epidermal keratinocytes and contrasting the cutaneous inflammatory responses. Therefore, we suggest PI3Kδ as a potential topically druggable target in psoriasis and skin diseases characterized by epidermal hyperproliferation and skin inflammation.

SPOP-mediated ubiquitination and degradation of PDK1 suppresses AKT kinase activity and oncogenic functions

Background3-phosphoinositide-dependent protein kinase-1 (PDK1) acts as a master kinase of protein kinase A, G, and C family (AGC) kinase to predominantly govern cell survival, proliferation, and metabolic homeostasis. Although the regulations to PDK1 downstream substrates such as protein kinase B (AKT) and ribosomal protein S6 kinase beta (S6K) have been well established, the upstream regulators of PDK1, especially its degrader, has not been defined yet.MethodA clustered regularly interspaced short palindromic repeats (CRISPR)-based E3 ligase screening approach was employed to identify the E3 ubiquitin ligase for degrading PDK1. Western blotting, immunoprecipitation assays and immunofluorescence (IF) staining were performed to detect the interaction or location of PDK1 with speckle-type POZ protein (SPOP). Immunohistochemistry (IHC) staining was used to study the expression of PDK1 and SPOP in prostate cancer tissues. In vivo and in vitro ubiquitination assays were performed to measure the ubiquitination conjugation of PDK1 by SPOP. In vitro kinase assays and mass spectrometry approach were carried out to identify casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)-mediated PDK1 phosphorylation. The biological effects of PDK1 mutations and correlation with SPOP mutations were performed with colony formation, soft agar assays and in vivo xenograft mouse models.ResultsWe identified that PDK1 underwent SPOP-mediated ubiquitination and subsequent proteasome-dependent degradation. Specifically, SPOP directly bound PDK1 by the consensus degron in a CK1/GSK3β-mediated phosphorylation dependent manner. Pathologically, prostate cancer patients associated mutations of SPOP impaired PDK1 degradation and thus activated the AKT kinase, resulting in tumor malignancies. Meanwhile, mutations that occurred around or within the PDK1 degron, by either blocking SPOP to bind the degron or inhibiting CK1 or GSK3β-mediated PDK1 phosphorylation, could markedly evade SPOP-mediated PDK1 degradation, and played potently oncogenic roles via activating the AKT kinase.ConclusionsOur results not only reveal a physiological regulation of PDK1 by E3 ligase SPOP, but also highlight the oncogenic roles of loss-of-function mutations of SPOP or gain-of-function mutations of PDK1 in tumorigenesis through activating the AKT kinase.

Regulation of Akt Signaling in Skeletal Muscle Is Altered by Prematurity in a Neonatal Piglet Model

Abstract Objectives Preterm birth is a risk factor for growth faltering. We recently showed in a neonatal piglet model of prematurity that premature birth decreases weight gain and skeletal muscle protein synthesis. This is associated with reduced insulin-induced activation of signaling components downstream of Akt but with no change in the activation of the IR/IRS-1/PI3K axis upstream of Akt. The aim of this study was to identify key regulatory molecules involved in Akt activation that are responsible for the differential response of skeletal muscle to insulin in preterm compared to term pigs. Methods Piglets were delivered by cesarean section 11 d (preterm/PT) or 2 d (term/T) before term birth and a jugular vein catheter was placed for delivery of total parenteral nutrition. On day 3, after 4 h fasting, piglets were fasted one additional h or fed an elemental diet (31.5 kcal/kg, 1.3 g/kg carbohydrate, 2.7 g/kg amino acids, and 1.6 g/kg lipid), similar in composition to sow milk, via oral gavage. Longissimus dorsi muscle was collected following euthanasia (fasted state or 60 min after feeding). Positive and negative regulators of Akt activity were determined by immunoprecipitation and immunoblotting assays. Results Akt1 and Akt2 phosphorylation were lower in PT than in T pigs (P &amp;lt; 0.05), whereas Akt3 phosphorylation was not affected by prematurity. The phosphorylation of Akt activators PDK1 and mTORC2, but not FAK, was lower in PT than in T pigs (P &amp;lt; 0.05). The abundance of Ubl4A, a positive regulator of Akt, was lower in PT than in T pigs (P &amp;lt; 0.05). The abundance Akt inhibitors PHLPP and SHIP2, but not PTEN and IP6K1, was significantly higher in PT than in T pigs (P &amp;lt; 0.05). Activation of the phosphatase PP2A was lower in PT than in T pigs (P &amp;lt; 0.05), but its activation was not affected by feeding. However, PP2A activation was inhibited by feeding in T pigs (P &amp;lt; 0.05). Conclusions These results showed that following preterm birth, the postprandial activation of positive regulators of Akt is reduced whereas the activation of negative regulators of Akt is enhanced. Our findings suggest that premature birth impairs the activation of Akt that is essential for channeling dietary nutrients for anabolism and likely contributes to the extrauterine growth faltering of prematurity. Funding Sources NIH and USDA.

PRKAR1B-AS2 Long Noncoding RNA Promotes Tumorigenesis, Survival, and Chemoresistance via the PI3K/AKT/mTOR Pathway.

Many long noncoding RNAs have been implicated in tumorigenesis and chemoresistance; however, the underlying mechanisms are not well understood. We investigated the role of PRKAR1B-AS2 long noncoding RNA in ovarian cancer (OC) and chemoresistance and identified potential downstream molecular circuitry underlying its action. Analysis of The Cancer Genome Atlas OC dataset, in vitro experiments, proteomic analysis, and a xenograft OC mouse model were implemented. Our findings indicated that overexpression of PRKAR1B-AS2 is negatively correlated with overall survival in OC patients. Furthermore, PRKAR1B-AS2 knockdown-attenuated proliferation, migration, and invasion of OC cells and ameliorated cisplatin and alpelisib resistance in vitro. In proteomic analysis, silencing PRKAR1B-AS2 markedly inhibited protein expression of PI3K-110α and abrogated the phosphorylation of PDK1, AKT, and mTOR, with no significant effect on PTEN. The RNA immunoprecipitation detected a physical interaction between PRKAR1B-AS2 and PI3K-110α. Moreover, PRKAR1B-AS2 knockdown by systemic administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with PRKAR1B-AS2–specific small interfering RNA enhanced cisplatin sensitivity in a xenograft OC mouse model. In conclusion, PRKAR1B-AS2 promotes tumor growth and confers chemoresistance by modulating the PI3K/AKT/mTOR pathway. Thus, targeting PRKAR1B-AS2 may represent a novel therapeutic approach for the treatment of OC patients.

The Antileukemia Activity of ZSTK474 on U937 Cells

To investigate the antileukemia activity of phosphatidylinositol-3 kinase (PI3K) inhibitor ZSTK474 on human leukemia cell line U937. MTT, soft agar assay, flow cytometric analysis and western blot were used to detect the effect of ZSTK474 on U937 cell proliferation, tumorigenicity, cell cycle, cell apoptosis and phosphorylation levels of the key factor of PI3K/AKT pathway. Chou-Talalay method was used to evaluate the combination of ZSTK474 with Cytarabine or Homoharringtonine. PI3K inhibitor ZSTK474 could inhibit the proliferation and tumorigenicity of U937 cell, induce G1 cell cycle arrest and promote cell apoptosis, and enhance intracellular ROS production and decrease MMP, downregulate Cyclin D1, p-Rb, BCL-2 and upregulate p27, caspase-9, caspase-3, PARP and BAX. Furthermore, the phosphorylation of PDK1, GSK-3β, AKT and mTOR could be downregulated by ZSTK474. The combination of ZSTK474 with Homoharringtonine was synergistic. ZSTK474 can inhibit the pathway of PI3K/AKT, ZSTK474 alone or in combination with Homoharringtonine shows potential antileukemia activity on U937 cells.

Cancer-derived IgG involved in cisplatin resistance through PTP-BAS/Src/PDK1/AKT signaling pathway.

This study aimed to explore whether knockdown of cancer-derived IgG (CIgG) could enhance cisplatin-induced anti-cancer effects. Cancer-derived IgG was knocked down by siRNA or Tet-on shRNA in the absence or presence of cisplatin in WSU-HN6 or CAL27 cells. Cell proliferation, apoptosis, and mobility were evaluated using CCK-8, flow cytometry, and transwell assays, respectively. Molecular events were investigated using real-time PCR and Western blot assays. Knockdown of CIgG significantly promoted cisplatin-induced apoptosis and inhibition of cell proliferation, migration, and invasion. Cisplatin upregulated CIgG expression and phosphorylation of AKT and PDK1, while knockdown of CIgG downregulated phosphorylation of AKT and PDK1, and blocked cisplatin-induced upregulation of AKT and PDK1 phosphorylation. Moreover, knockdown of CIgG blocked cisplatin-induced upregulation of Src phosphorylation, and knockdown of Src blocked cisplatin-induced upregulation of AKT and PDK1 phosphorylation. Overexpression of Src upregulated AKT and PDK1 phosphorylation. Furthermore, knockdown of CIgG upregulated PTP-BAS mRNA and protein expression, whereas cisplatin downregulated PTP-BAS protein, but not mRNA expression; knockdown of PTP-BAS upregulated phosphorylation of Src, PDK1, AKT, and blocked CIgG knockdown-mediated enhancement of cisplatin-induced inhibition of cell proliferation. Knockdown of CIgG enhanced the anti-cancer effects of cisplatin through PTP-BAS/Src/PDK1/AKT signaling pathway in oral squamous cell carcinoma.

Hypoxia induces tumor cell growth and angiogenesis in non-small cell lung carcinoma via the Akt-PDK1-HIF1α-YKL-40 pathway.

BackgroundAs one of the most common forms of cancer, non-small cell lung carcinoma (NSCLC), is characterized by oxygen deprivation (hypoxia). The transcription factor hypoxia-inducible factor (HIF)-1α is a major mediator which responds hypoxia and regulates many contributing factors. The various modes of hypoxia regulation are frequently the focus of research studies. With reference to previous published research, we hypothesized that hypoxia promotes the growth and angiogenesis of NSCLC via the Akt-PDK1-HIF1α-YKL-40 pathway, and verified it.MethodsWe mainly investigated changes in related factor expression between differently treated CL1-5 cells. We carried out overexpression and underexpression transfection, Western blot, rt-PCR and ELISA, and observed cellular biological behaviors by CCK-8 migration and invasion assay, and tube formation assay.ResultsA hypoxic environment significantly increased the phosphorylation of Akt and PDK1 in mitochondria. The hypoxia-induced accumulation of p-Akt in mitochondria activated PDK1 phosphorylation, promoted the expression of HIF1α, and the expression of YKL-40. The overexpression of YKL-40 promoted the proliferation, migration, invasion and tubule formation of CL1-5 cells.ConclusionsA hypoxic tumor microenvironment can promote the expansion and angiogenesis of NSCLC cells through the Akt-PDK1-HIF1α-YKL-40 pathway. This may provide a new mechanism and potential interventional target for anti-vascular lung cancer therapy.

TIPE regulates VEGFR2 expression and promotes angiogenesis in colorectal cancer.

Background: Metastasis is the leading cause of death in colorectal cancer (CRC) patients. It is regulated mainly by tumor cell angiogenesis, and angiogenesis is caused by the binding of vascular endothelial growth factor (VEGF) to vascular endothelial growth factor receptor 2 (VEGFR2). Tumor necrosis factor-α-induced protein 8 (TNFAIP8, hereto after TIPE) plays an important role in tumorigenesis, development, and prognosis. However, the relationship between TIPE and VEGFR2 in CRC angiogenesis and the mechanism of action remain unknown.Method: In this study, we used quantitative real-time PCR, Western blotting and immunohistochemistry to detect TIPE and VEGFR2 expression in 55 specimens from CRC patients. We also used HCT116 CRC cells and human umbilical vein endothelial cells (HUVECs) for in vitro experiments by stably transducing shTIPE and shRNA control lentivirus into HCT116 cells, detecting VEGFR2 expression after TIPE knockdown and repurposing the culture supernatant as conditioned medium to stimulate angiogenesis of HUVECs. In vivo experiments with chicken chorioallantoic membranes (CAMs) and a nude mouse matrix subcutaneous tumor model were performed to validate the effects of TIPE on angiogenesis. Additionally, we analyzed the expression and phosphorylation levels of PDK1 and blocked PDK1 expression using inhibitors to determine whether TIPE-induced changes in VEGFR2-mediated angiogenesis acted via the PI3K-Akt pathway.Results: We found that TIPE and VEGFR2 are highly expressed in CRC and act as oncogenes. TIPE knockdown also downregulated VEGFR2 expression, which resulted in simultaneous inhibition of cell proliferation, cell migration and angiogenesis. Then, in vivo experiments further demonstrated that TIPE promotes angiogenesis in CRC. Finally, we found that TIPE promotes VEGFR2-mediated angiogenesis by upregulating PDK1 expression and phosphorylation and that blocking PDK1 expression can inhibit this process.Conclusion: TIPE promotes angiogenesis in CRC by regulating the expression of VEGFR2, which may be a target for antiangiogenic cancer therapy.