PDK1 Knockdown Research Articles

Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes.

Pyruvate dehydrogenase kinase (PDK) 1 is one of four isozymes that inhibit the oxidative decarboxylation of pyruvate to acetyl-CoA via pyruvate dehydrogenase. PDK activity is elevated in fasting or starvation conditions to conserve carbohydrate reserves. PDK has also been shown to increase mitochondrial fatty acid utilization. In cardiomyocytes, metabolic flexibility is crucial for the fulfillment of high energy requirements. The PDK1 isoform is abundant in cardiomyocytes, but its specific contribution to cardiomyocyte metabolism is unclear. Here we show that PDK1 regulates cardiomyocyte fuel preference by mediating triacylglycerol turnover in differentiated H9c2 myoblasts using lentiviral shRNA to knockdown Pdk1. Somewhat surprisingly, PDK1 loss did not affect overall PDH activity, basal glycolysis, or glucose oxidation revealed by oxygen consumption rate experiments and 13C6 glucose labelling. On the other hand, we observed decreased triacylglycerol turnover in H9c2 cells with PDK1 knockdown, which was accompanied by decreased mitochondrial fatty acid utilization following nutrient deprivation. 13C16 palmitate tracing of uniformly labelled acyl chains revealed minimal acyl chain shuffling within triacylglycerol, indicating that the triacylglycerol hydrolysis, and not re-esterification, was dysfunctional in PDK1 suppressed cells. Importantly, PDK1 loss did not significantly impact the cellular lipidome or triacylglycerol accumulation following palmitic acid treatment, suggesting that effects of PDK1 on lipid metabolism were specific to the nutrient-deprived state. We validated that PDK1 loss decreased triacylglycerol turnover in Pdk1 knockout mice. Together, these findings implicate a novel role for PDK1 in lipid metabolism in cardiomyocytes, independent of its canonical roles in glucose metabolism.

PDK1 upregulates PINK1-mediated pulmonary endothelial cell mitophagy during hypoxia-induced pulmonary vascular remodeling.

Hypoxic pulmonary hypertension (HPH) is a complication of lung diseases with pulmonary vascular remodeling, although the underlying molecular mechanisms have not been fully elucidated. This study investigated the underlying molecular events by using a rat HPH model and primary pulmonary microvascular endothelial cells (PMVECs). This study first established a rat HPH model and cultured PMVECs for transmission electron microscopic analysis and manipulation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) or phosphatase and tensin homolog-induced kinase 1 (PINK1) expression in vitro. After that, the cell viability was assessed and the expression of different proteins was assayed using cell viability and western blot assays, respectively. Reactive oxygen species production, apoptosis, NLR family pyrin domain containing 3 (NLRP3) expression, and the levels of interleukin (IL)-1β, IL-6, and IL-8 were also assessed, while the interaction of PDK1 and PINK1 was determined using co-immunoprecipitation/western blot assays. Hypoxia induced mitophagy in the PMVECs and upregulated PINK1/Parkin expression, whereas knockdown of PINK1 expression under hypoxic conditions inhibited cell proliferation but induced endothelial cell apoptosis in vitro, decreased reactive oxygen species production and NLRP3 expression, and reduced the levels of inflammatory factors in PMVECs. However, hypoxia induced PDK1 expression, whereas knockdown of PDK1 downregulated PINK1 expression. Furthermore, treatment of the model rats with the PDK1 inhibitor dichloroacetate (DCA) was able to decrease PINK1 expression. In addition, the PDK1 and PINK1 proteins could interact with each other in the mitochondria of PMVECs to regulate the cell viability. This study revealed that PDK1 induced PMVEC proliferation but inhibited their apoptosis to participate in pulmonary vascular remodeling, ultimately leading to HPH through regulation of PINK1-mediated mitophagy signaling. Therefore, PINK1 is a novel therapeutic target for the control of HPH.

Histone lactylation driven by mROS-mediated glycolytic shift promotes hypoxic pulmonary hypertension.

Increased mitochondrial reactive oxygen species (mROS) and glycolysis have been established in pulmonary hypertension (PH). However, the effect of elevated mROS on glycolytic shift and how increased glycolysis promotes hypoxic pulmonary artery smooth muscle cell (PASMC) proliferation and vascular remodeling remain elusive. Here, we reported that hypoxia-induced mROS inhibit HIF-1α hydroxylation and further trigger PASMC glycolytic switch through the upregulated HIF-1α/PDK1&PDK2/p-PDH-E1α axis, which facilitates lactate accumulation and histone lactylation. Through H3K18la and HIF-1α ChIP-seq analysis, we found that the enhanced histone lactylation of HIF-1α targets, such as Bmp5, Trpc5, and Kit, promotes PASMC proliferation. Knockdown of Pdk1&2 blunts lactate production, histone lactylation marks, and PASMC proliferation. Moreover, pharmacological intervention with lactate dehydrogenase inhibitor diminishes histone lactylation and ameliorates PASMC proliferation and vascular remodeling in hypoxic PH rats. Taken together, this study provides proof of concept for anti-remodeling therapy through lactate manipulation.

PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFβ1-promoted stem-like properties in head and neck cancer

BackgroundResistance to chemotherapeutic drugs is a key factor for cancer recurrence and metastases in head and neck cancer (HNC). Cancer stem cells (CSCs) in tumors have self-renewal, differentiation, and higher drug resistance capabilities, resulting in a poor prognosis for patients. In glucose metabolism, pyruvate dehydrogenase kinase (PDK) inhibits pyruvate dehydrogenase and impedes pyruvate from being metabolized into acetyl-CoA and entering the tricarboxylic acid cycle to generate energy. Studies have reported that PDK1 and PDK2 inhibition suppresses the growth, motility, and drug resistance of cancer cells. Furthermore, while TGFβ1 levels are persistently elevated in HNC patients with poor prognosis, the role of PDK isoforms in the TGFβ1-promoted progression and stem-like properties of HNC is unclear.MethodsLevels of PDK1 and PDK2 were evaluated in HNC tissue microarrays by immunohistochemistry to explore potential clinical relevance. PDK1 and PDK2 were knocked down by the lentivirus shRNA system to investigate their role in TGFβ1-promoted tumor progression in vitro.ResultsWe found that PDK2 levels were increased in the later stage of HNC tissues compared to constant PDK1 expression. After PDK1 and PDK2 knockdown, we discovered increased ATP production and decreased lactate production in TGFβ1-treated and untreated HNC cells. However, only PDK2 silencing significantly inhibited the clonogenic ability of HNC cells. We subsequently found that TGFβ1-promoted migration and invasion capabilities were decreased in PDK1 and PDK2 knockdown cells. The tumor spheroid-forming capability, motility, CSC genes, and multidrug-resistant genes were downregulated in PDK1 and PDK2 silencing CSCs. PDK1 and PDK2 inhibition reversed cisplatin and gemcitabine resistance of CSCs, but not paclitaxel resistance.ConclusionThe results demonstrated that the PDK1- and PDK2-mediated Warburg effect contributes to the TGFβ1-enhanced stemness properties of HNC. Therefore, PDK1 and PDK2 may serve as molecular targets for the combination therapy of HNC.

HIF-1α plays an essential role in BMP9-mediated osteoblast differentiation through the induction of a glycolytic enzyme, PDK1.

Bone homeostasis is regulated by bone morphogenic proteins (BMPs), among which BMP9 is one of the most osteogenic. Here, we have found that BMP9 rapidly increases the protein expression of hypoxia-inducible factor-1α (HIF-1α) in osteoblasts under normoxic conditions more efficiently than BMP2 or BMP4. A combination of BMP9 and hypoxia further increased HIF-1α protein expression. HIF-1α protein induction by BMP9 is not accompanied by messenger RNA (mRNA) increase and is inhibited by the activation of prolyl hydroxylase domain (PHD)-containing protein, indicating that BMP9 induces HIF-1α protein expression by inhibiting PHD-mediated protein degradation. BMP9-induced HIF-1α protein increase was abrogated by inhibitors of phosphoinositide 3-kinase (PI3K) and protein kinase B(AKT) kinase, indicating that it is mediated by PI3K-AKT signaling pathway. BMP9 increased mRNA expression of pyruvate dehydrogenase kinase 1(PDK1), a glycolytic enzyme, and vascular endothelial growth factor-A(VEGF-A), an angiogenic factor, in osteoblasts. Notably, BMP9-induced mRNA expression of PDK1, but not that of VEGF-A, was significantly inhibited by small interference RNA-mediated knockdown of Hif-1α. BMP9-induced matrix mineralization and osteogenic marker gene expressions were significantly inhibited by chemical inhibition and gene knockdown of either Hif-1α or Pdk-1, respectively. Since increased glycolysis is an essential feature of differentiated osteoblasts, our findings indicate that HIF-1α expression is important in BMP9-mediated osteoblast differentiation through the induction of PDK1.

MicroRNA-193b impairs muscle growth in mouse models of type 2 diabetes by targeting the PDK1/Akt signalling pathway

Aims/hypothesisType 2 diabetes is associated with a reduction in skeletal muscle mass; however, how the progression of sarcopenia is induced and regulated remains largely unknown. We aimed to find out whether a specific microRNA (miR) may contribute to skeletal muscle atrophy in type 2 diabetes.MethodsAdeno-associated virus (AAV)-mediated skeletal muscle miR-193b overexpression in C57BLKS/J mice, and skeletal muscle miR-193b deficiency in db/db mice were used to explore the function of miR-193b in muscle loss. In C57BL/6 J mice, tibialis anterior-specific deletion of 3-phosphoinositide-dependent protein kinase-1 (PDK1), mediated by in situ AAV injection, was used to confirm whether miR-193b regulates muscle growth through PDK1. Serum miR-193b levels were also analysed in healthy individuals (n = 20) and those with type 2 diabetes (n = 20), and correlations of miR-193b levels with HbA1c, fasting blood glucose (FBG), body composition, triacylglycerols and C-peptide were assessed.ResultsIn this study, we found that serum miR-193b levels increased in individuals with type 2 diabetes and negatively correlated with muscle mass in these participants. Functional studies further showed that AAV-mediated overexpression of miR-193b induced muscle loss and dysfunction in healthy mice. In contrast, suppression of miR-193b attenuated muscle loss and dysfunction in db/db mice. Mechanistic analysis revealed that miR-193b could target Pdk1 expression to inactivate the Akt/mammalian target of rapamycin (mTOR)/p70S6 kinase (S6K) pathway, thereby inhibiting protein synthesis. Therefore, knockdown of PDK1 in healthy mice blocked miR-193b-induced inactivation of the Akt/mTOR/S6K pathway and impairment of muscle growth.Conclusions/interpretationOur results identified a previously unrecognised role of miR-193b in muscle function and mass that could be a potential therapeutic target for treating sarcopenia.Graphical abstract

Abstract P236: Targeting AXL/PDK1 signaling axis activates immunogenic cell death in liver cancer

Abstract Hepatocellular carcinoma (HCC) is the most malignant form of primary liver cancers with poor prognosis. Majority of the cases are diagnosed in advanced stages where therapeutic options are limited and the response rates are low. There is an unmet clinical need for more effective therapeutic treatment. AXL is a receptor tyrosine kinase which belongs to the family of TAM receptors. Overexpression of AXL has been reported in multiple cancers, including HCC. We and others have also reported an up-regulation of AXL in sorafenib-resistant HCC. However, its role in regulating anti-tumor immunity in HCC is not fully understood. In this study, we observed an immunosuppressive gene signature in sorafenib-resistant HCC by RNA-Seq and pathway enrichment analysis. Genetic manipulation and pharmacological inhibition of AXL promoted immunogenic cell death in sorafenib-resistant HCC cells, as evident by increased surface calreticulin and HMGB1 secretion. PDK1, a key modulatory kinase downstream of AXL in the PI3K/Akt pathway, was also up-regulated in sorafenib-resistant HCC. Knockdown of PDK1 similarly led to an increased immunogenic cell death. In a sorafenib-resistant immunocompetent mouse HCC model, blockade of AXL with a selective AXL inhibitor increased tumor-infiltrating activated and proliferating CD8+ T cells, resulting in tumor suppression and prolonged survival of the mice. Taken together, our findings suggest that targeting the AXL/PDK1 signaling axis could be a viable therapeutic option which enhances anti-tumor immunity for the treatment of HCC. Citation Format: Yunong Xie, Lei Zhou, Cheuk Yin Lin, Terence Lee, Jin Ding, Stephanie Ma, Man Tong. Targeting AXL/PDK1 signaling axis activates immunogenic cell death in liver cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P236.

MiR-877-5p targets PDK-1 to promote aspirin-induced apoptosis in gastric mucosal cells.

This study aimed to investigate the role of miR-877-5p in aspirin-induced gastric mucosal injury. MiRNA microarray analysis was performed using paired gastric mucosal samples to find differentially expressed miRNAs. miR-877-5p was selected for subsequent analyses. Used as a model system, gastric epithelial cells (GES-1) were transfected with miR-877-5p mimic/inhibitor, then treated with aspirin. The expression of miR-877-5p in GES-1 cells was examined using quantitative real-time PCR (qRT-PCR). Flow cytometry analysis was used to detect cell apoptosis. Western blot assay was used to measure the protein levels of PDK1. The interaction between miR-877-5p and PDK1 was determined by luciferase reporter assay. The expression of miR-877-5p in gastric mucosal injury samples was higher than that in normal samples. Also, depletion of miR-877-5p reduced the apoptosis of GES-1 cells. Luciferase reporting assay confirmed that PDK1 was a target gene of miR-877-5p. PDK1 inhibited the apoptosis of GES-1 cells treated by aspirin. Moreover, this inhibitory effect was abrogated after PDK1 knockdown. Downregulation of miR-877-5p reduced the apoptosis by targeting PDK1 in GES-1 cells treated by aspirin, indicating that miR-877-5p may be a potential therapeutic target for gastric mucosal injury caused by aspirin.

PDK1 is a negative regulator of axon regeneration

Axon regeneration in the central nervous system is inefficient. However, the neurons in the peripheral nervous system display robust regeneration after injury, indicating that axonal regeneration is differentially controlled under various conditions. To identify those molecules regulating axon regeneration, comparative analysis from dorsal root ganglion neurons at embryonic or adult stages is utilized, which reveals that PDK1 is functions as a negative regulator of axon regeneration. PDK1 is downregulated in embryonic neurons after axotomy. In contrast, sciatic nerve axotomy upregulated PDK1 at protein levels from adult mice. The knockdown of PDK1 or the chemical inhibition of PDK1 promotes axon regeneration in vitro and in vivo. Here we present PDK1 as a new player to negatively regulate axon regeneration and as a potential target in the development of therapeutic applications.

The effects of PDK1-Akt signaling pathway intervention on cardiomyocyte HCN4 ion channels

Objective: To explore the effects of 3-phosphate dependent protein kinase 1-protein kinase B (PDK1-Akt) signaling pathway on the transcription, expression and function of cardiac hyperpolarized activated cyclic nucleotide gated 4 (HCN4) ion channels. Methods: Atrial myocytes were obtained from healthy male wild-type C57 mice and heart-specific PDK1 knockout mice (PDK1-KO) by enzymolysis. Then the atrial myocytes were divided into blank control group and PDK1-KO group. In further studies, the isolated atrial myocytes were cultured and further divided into drug control group (treated with dimethyl sulfoxide (DMSO)) and PDK1 knockdown group (treated with 1 μg/ml PDK1 short hairpin RNA (shRNA) interference plasmid), SC79 group (treated with 8 μmol/ml SC79), GSK2334470 group (treated with 10 nmol/L GSK2334470) and PDK1 knockdown+SC79 group (8 μmol/ml SC79 and 1 μg/ml PDK1 shRNA interference plasmid). Real time quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of PDK1 and HCN4, Western blot was used to detect the protein expression levels of PDK1, Akt and HCN4, the whole cell patch clamp was used to detecte the current density of HCN, and immunofluorescence was used to detecte the expression of HCN4 protein on atrial cells. Results: (1) the expression levels of HCN4 mRNA (1.46±0.03 vs. 0.99±0.01, P<0.001) and protein (1.14±0.02 vs. 1.00±0.06, P=0.017) in PDK1-KO group were higher than those in blank control group. The HCN current density in PDK1-KO group was higher than that in blank control group((-17.47±2.00) pA/pF vs. (-12.15±2.25) pA/pF, P=0.038). (2) The functions of PDK1 shRNA and specific Akt agonist SC79 were verified by comparing the PDK1 knockdown group and SC79 group with the drug control group. The results showed that the expression levels of PDK1 mRNA and protein in PDK1 knockdown group were lower than those in drug control group, and the expression level of phosphorylated Akt (Thr 308) protein in SC79 group was higher than that in drug control group. (3) The expression levels of HCN4 mRNA (3.61±0.46 vs. 1.00±0.08, P<0.001) and protein (2.33±0.11 vs. 1.00±0.05, P<0.001) in GSK2334470 group were higher than those in drug control group. (4) To reduce the effect of drug-miss target, the cultured atrial myocytes were transfected with shRNA plasmid of PDK1 and intervened with SC79. The results showed that the expression of HCN4 mRNA in PDK1 knockdown group was higher than that in the drug control group (1.76±0.11 vs. 1.00±0.06, P<0.001), and PDK1 knockdown+SC79 group (1.76±0.11 vs. 1.33±0.07, P=0.003). In PDK1 knockdown+SC79 group, the mRNA expression level was also higher than that in the drug control group (1.33±0.07 vs. 1.00±0.06, P<0.001). The expression level of HCN4 protein in PDK1 knockdown group was higher than that in drug control group (1.15±0.04 vs. 1.00±0.05, P=0.003). As for the The expression level of HCN4 protein, there was no significantly statistical difference between the PDK1 knockdown+SC79 group and the drug control group (P>0.05), but PDK1 knockdown+SC79 group was lower than PDK1 knockdown group (0.95±0.01 vs. 1.15±0.04, P<0.001). In patch clamp experiments, the results showed that the HCN current density was (-13.27±1.28) pA/pF in the drug control group, (-18.76±2.03) pA/pF in the PDK1 knockdown group, (-13.50±2.58) pA/pF in the PDK1 knockdown+SC79 group; the HCN current density of PDK1 knockdown group was higher than that of drug control group (P<0.001), but there was no significant difference between PDK1 knockdown+SC79 group and drug control group (P>0.05). (5) The results of immunofluorescence showed that the brightness of green fluorescence of PDK1 knockdown group was higher than that of drug control group, indicating that the expression of HCN4 localized on cell membrane was increased. However, the green fluorescence of PDK1 knockdown+SC79 group was lighter than that of PDK1 knockdown group, suggesting that the expression of HCN4 in PDK1-knockdown cell membrane decreased after further activating Akt. Conclusion: PDK1-Akt signaling pathway is involved in the regulation of HCN4 ion channel transcription, expression and function.

A novel function of artesunate on inhibiting migration and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients

IntroductionAnti-malarial drug artesunate can suppress inflammation and prevent cartilage and bone destruction in collagen-induced arthritis model in rats—suggesting it may be a potent drug for rheumatoid arthritis (RA) therapy. We aimed to investigate its effect on the invasive property of fibroblast-like synoviocytes (FLS) from patients with RA.MethodsSynovial tissues were obtained by closed needle biopsy from active RA patients, and FLS were isolated and cultured in vitro. RA-FLS were treated with artesunate at various concentrations, while methotrexate or hydroxychloroquine was employed as comparator drugs. Cell viability, proliferation, cell cycle, apoptosis, migration, invasion, and pseudopodium formation of RA-FLS were assessed by CCK-8 assays, EdU staining, Annexin V-FITC/PI staining, transwell assays, or F-actin staining, respectively. Further, relative changes of expressed proteases were analyzed by Proteome profiler human protease array and verified by quantitative real-time PCR (qPCR), Western blot, and ELISA. The expression of signaling molecules of MAPK, NF-κB, AP-1, and PI3K/Akt pathways were measured by qPCR and Western blot. PDK-1 knockdown by specific inhibitor AR-12 or siRNA transfection was used to verify the pharmacological mechanism of artesunate on RA-FLS.ResultsArtesunate significantly inhibited the migration and invasion of RA-FLS in a dose-dependent manner with or without TNF-α stimulation. The effect was mediated through artesunate inhibition of MMP-2 and MMP-9 production, and pre-treatment with exogenous MMP-9 reversed the inhibitory effect of artesunate on RA-FLS invasion. Artesunate had a stronger inhibitory effect on migration and invasion of RA-FLS as well as greater anti-inflammatory effect than those of hydroxychloroquine. Similar inhibitory effect was detected between artesunate and methotrexate, and synergy was observed when combined. Mechanistically, artesunate significantly inhibited PDK-1 expression as well as Akt and RSK2 phosphorylation—in a similar manner to PDK-1-specific inhibitor AR-12 or PDK-1 knockdown by siRNA transfection. This inhibition results in suppression of RA-FLS migration and invasion as well as decreased MMP-2 and MMP-9 expression.ConclusionsOur study demonstrates artesunate is capable of inhibiting migration and invasion of RA-FLS through suppression of PDK1-induced activation of Akt and RSK2 phosphorylation—suggesting that artesunate may be a potential disease-modifying anti-rheumatic drug for RA.

PDK-1 mediated Hippo-YAP-IRS2 signaling pathway and involved in the apoptosis of non-small cell lung cancer cells.

Pyruvate dehydrogenase kinase-1 (PDK-1), a gatekeeper enzyme, was involved in cancer progression, such as tumor angiogenesis, cell survival, and growth. Recent evidence indicated that PDK-1 may be involved in lung cancer, however, the function and underlying mechanism of PDK-1 is remaining unclear. In the present study, our aim was to investigate the role and mechanisms of PDK-1 in human non-small cell lung cancer (NSCLC) cells. We first observed that PDK-1 was highly expressed in NSCLC cell lines. PDK-1 silence resulted in the inhibition of NSCLC cell survival. Also, cell apoptosis and caspase-3 activity were increased by PDK-1 knockdown in H1299 and A549 cells. Attenuation of PDK-1 expression blocked YAP and insulin receptor substrate 2 (IRS2) expression, and PDK-1 silence suppressed IRS2 expression dependent on Hippo–YAP signaling pathway. Moreover, further studies confirmed that YAP or IRS2 overexpression reversed the action of PDK-1 in NSCLC cells. In conclusion, our findings indicate that PDK1/Hippo–YAP/IRS2 signaling pathway plays a critical role in NSCLC cell survival and apoptosis.

Activation of Pyruvate Dehydrogenase by Sodium Dichloroacetate Shifts Metabolic Consumption from Amino Acids to Glucose in IPEC-J2 Cells and Intestinal Bacteria in Pigs.

The extensive metabolism of amino acids (AA) as fuel is an important reason for the low use efficiency of protein in pigs. In this study, we investigated whether regulation of the pyruvate dehydrogenase kinase (PDK)/pyruvate dehydrogenase alpha 1 (PDHA1) pathway affected AA consumption by porcine intestinal epithelial (IPEC-J2) cells and intestinal bacteria in pigs. The effects of knockdown of PDHA1 and PDK1 with small interfering RNA (siRNA) on nutrient consumption by IPEC-J2 cells were evaluated. IPEC-J2 cells were then cultured with sodium dichloroacetate (DCA) to quantify AA and glucose consumption and nutrient oxidative metabolism. The results showed that knockdown of PDHA1 using siRNA decreased glucose consumption but increased total AA (TAA) and glutamate (Glu) consumption by IPEC-J2 cells ( P < 0.05). Opposite effects were observed using siRNA targeting PDK1 ( P < 0.05). Additionally, culturing IPEC-J2 cells in the presence of 5 mM DCA markedly increased the phosphorylation of PDHA1 and PDH phosphatase 1, but inhibited PDK1 phosphorylation ( P < 0.05). DCA treatment also reduced TAA and Glu consumption and increased glucose depletion ( P < 0.05). These results indicated that PDH was the regulatory target for shifting from AA metabolism to glucose metabolism and that culturing cells with DCA decreased the consumption of AAs by increasing the depletion of glucose through PDH activation.

MiR-1271 negatively regulates AKT/MTOR signaling and promotes apoptosis via targeting PDK1 in pancreatic cancer.

Pancreatic cancer (PC) possesses a very poor prognosis, and its pathogenesis is not fully understood. Evidence has suggested that microRNAs play important roles in cancer development and progression, the present study was designed to study the function of miR-1271 in PC. PC tissues and adjacent normal tissues were collected from 17 patients. MiR-1271 and PDK1 expression were quantified by quantitative reverse-transcriptional polymerase chain reaction (RT-PCR). AKT/MTOR signaling activity and PDK1 protein expression were determined by Western blot. Cell viability and apoptosis were assessed by MTT assay and enzyme-linked immunosorbent assay (ELISA). Luciferase assay was used to verify whether miR-1271 directly targets PDK1. MiR-1271 was significantly down-regulated in PC tissues compared with that in the paired normal adjacent tissue, and its expression was up-regulated dose-dependently upon cisplatin treatment in PC cells. Overexpression of miR-1271 in these cells produced a pro-apoptotic effect, similar to what caused by cisplatin treatment. Moreover, overexpression of miR-1271 inhibited AKT/MTOR signaling, which was due to the targeting relationship between miR-1271 and PDK1. Finally, knockdown of PDK1 exerted a similar effect on apoptosis to that of miR-1271 overexpression. MiR-1271 is a potent tumor suppressor in PC, its pro-apoptotic function was partially mediated by reduced AKT/MTOR signaling. Targeting miR-1271 may represent an effective strategy for PC treatment.

Akt activation by Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in ovarian cancer cells

Hyperactivation of Akt is associated with oncogenic changes in the growth, survival, and chemoresistance of cancer cells. The PI3K/phosphoinositide-dependent kinase (PDK) 1 pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr-308. We observed that Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (β) (CaMKK2) is highly expressed in high-grade serous ovarian cancer, and we investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, and Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and cell viability and in the regulation of Akt downstream targets involved in G1/S transition and apoptosis. CaMKK2 knockdown or inhibition decreased Akt phosphorylation at Thr-308 and Ser-473 to extents similar to those of PDK1 knockdown or PI3K inhibition. Combined CaMKK2 and PDK1 knockdown or CaMKK and PI3K inhibition, respectively, produced additive effects on p-Akt and cell growth, consistent with direct Akt phosphorylation by CaMKK2. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via p-Akt Thr-450, p-PDK1 Ser-241, or p-IRS1 Ser-636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr-308 in a Ca2+/CaM-dependent manner. In OVCa cells, p-Akt Thr-308 was significantly inhibited by intracellular Ca2+i chelation or CaM inhibition. Ionomycin-induced Ca2+ influx promoted p-Akt, an effect blocked by PDK1, and/or CaMKK2, siRNAs, and by PI3K and/or CaMKK inhibitors. CaMKK2 knockdown potentiated the effects of the chemotherapeutic drugs carboplatin and PX-866 to reduce proliferation and survival of OVCa cells.

Cyclin-dependent kinase 2 protects podocytes from apoptosis.

Loss of podocytes is an early feature of diabetic nephropathy (DN) and predicts its progression. We found that treatment of podocytes with sera from normoalbuminuric type 1 diabetes patients with high lipopolysaccharide (LPS) activity, known to predict progression of DN, downregulated CDK2 (cyclin-dependent kinase 2). LPS-treatment of mice also reduced CDK2 expression. LPS-induced downregulation of CDK2 was prevented in vitro and in vivo by inhibiting the Toll-like receptor (TLR) pathway using immunomodulatory agent GIT27. We also observed that CDK2 is downregulated in the glomeruli of obese Zucker rats before the onset of proteinuria. Knockdown of CDK2, or inhibiting its activity with roscovitine in podocytes increased apoptosis. CDK2 knockdown also reduced expression of PDK1, an activator of the cell survival kinase Akt, and reduced Akt phosphorylation. This suggests that CDK2 regulates the activity of the cell survival pathway via PDK1. Furthermore, PDK1 knockdown reduced the expression of CDK2 suggesting a regulatory loop between CDK2 and PDK1. Collectively, our data show that CDK2 protects podocytes from apoptosis and that reduced expression of CDK2 associates with the development of DN. Preventing downregulation of CDK2 by blocking the TLR pathway with GIT27 may provide a means to prevent podocyte apoptosis and progression of DN.

Abstract B13: Regulation of Akt activity, cell proliferation, and viability in ovarian cancer cells by calcium/calmodulin-dependent protein kinase kinase 2

Abstract Calcium/Calmodulin-dependent protein kinase kinase 2 (CaMKK2) has been implicated in the regulation of G1/S phase cell cycle progression in prostate and breast cancer but has not been studied in ovarian cancer (OvCa). We report here the following. CaMKK2 is abundantly expressed in high grade serous papillary cystadenocarcinoma. Knockdown (KD) of CaMKK2 expression using RNAi in OVCAR-3 OvCa cells resulted in slowing of cell growth, decreased cyclin D1 protein and mRNA, decreased phosphorylation of the tumor suppressor protein Rb at Ser807/811 and decreased DNA synthesis, indicating an arrest at the G1/S interface. CaMKK2 KD decreased cell viability, increased PARP cleavage and increased Caspase-3/7 activity suggesting apoptosis induction by CaMKK2 loss. CaMKK2 KD decreased p-Akt at Thr308 to a similar extent (59.3%) as that of PDK1 KD (50.4%) in OVCAR-3 cells. Combined CaMKK2 and PDK1 KDs led to an additive (80.7%) decrease in p-Akt suggesting that CaMKK2 regulates Akt activating phosphorylation independently of the canonical-PDK1 pathway. Additive effects of combined PDK1/CaMKK2 KD on p-Akt Thr308 in SKOV-3 OvCa cells were also obtained. The ability of baculovirus expressed and purified Akt to phosphorylate a specific peptide substrate was enhanced at least 9-fold by purified CaMKK2, whereas kinase activity of Akt T308A/S473A was not increased by CaMKK2. Direct phosphorylation of baculovirus expressed Akt at Thr308 by CaMKK2 was confirmed by Western blotting using Akt phospho-specific antibodies. Altogether these data show that CaMKK2 regulates cell growth and viability in OvCa cells and directly phosphorylates Akt to promote its activity via a non-PDK1-dependent pathway. These data indicate that CaMKK2 may be a novel therapeutic target for OvCa in combination with a PI3K inhibitor. Citation Format: Angela M. Gocher, Thomas F. Franke, Gissou Azabdaftari, Loukia G. Karacosta, Arthur M. Edelman. Regulation of Akt activity, cell proliferation, and viability in ovarian cancer cells by calcium/calmodulin-dependent protein kinase kinase 2. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B13.

Expression of 3-phosphoinositide-dependent protein kinase 1 in colorectal cancer as a potential therapeutic target.

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is centrally involved in cancer progression, including proliferation, apoptosis and invasion. However, its expression pattern and possible cellular functions in human colorectal cancer remain unclear. In the present study, we show that PDK1 expression is up-regulated at both mRNA and protein levels in colorectal cancer clinical specimens and cell lines. Transient knockdown of PDK1 suppresses cellular growth, induces cellular apoptosis and causes abnormal cell cycle distribution. Meanwhile, decreased PDK1 level is closely associated with reduced Akt/cyclin D1 activity. Activating AKT activity and reintroducing cyclin D1 expression significantly compromised the oncogenic activity induced by PDK1. Together, our findings elucidate a key role for PDK1 in colorectal cellular functions trigged by the Akt/cyclin D1 pathway, thus providing a novel insight of PDK1 in colorectal carcinogenesis.

Podocyte apoptosis is prevented by blocking the Toll-like receptor pathway.

High serum lipopolysaccharide (LPS) activity in normoalbuminuric patients with type 1 diabetes (T1D) predicts the progression of diabetic nephropathy (DN), but the mechanisms behind this remain unclear. We observed that treatment of cultured human podocytes with sera from normoalbuminuric T1D patients with high LPS activity downregulated 3-phosphoinositide-dependent kinase-1 (PDK1), an activator of the Akt cell survival pathway, and induced apoptosis. Knockdown of PDK1 in cultured human podocytes inhibited antiapoptotic Akt pathway, stimulated proapoptotic p38 MAPK pathway, and increased apoptosis demonstrating an antiapoptotic role for PDK1 in podocytes. Interestingly, PDK1 was downregulated in the glomeruli of diabetic rats and patients with type 2 diabetes before the onset of proteinuria, further suggesting that reduced expression of PDK1 associates with podocyte injury and development of DN. Treatment of podocytes in vitro and mice in vivo with LPS reduced PDK1 expression and induced apoptosis, which were prevented by inhibiting the Toll-like receptor (TLR) signaling pathway with the immunomodulatory agent GIT27. Our data show that LPS downregulates the cell survival factor PDK1 and induces podocyte apoptosis, and that blocking the TLR pathway with GIT27 may provide a non-nephrotoxic means to prevent the progression of DN.

Upregulation of PDK1 associates with poor prognosis in esophageal squamous cell carcinoma with facilitating tumorigenicity in vitro.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) has been reported to be closely correlated with proliferation, apoptosis and invasion of esophageal cancer cells. However, its expression pattern and clinical significance in human esophageal squamous cell carcinoma (ESCC) remain unclear. To address this problem, immunohistochemistry was performed to detect the expression and the subcellular localization of PDK1 protein in 120 paired of formalin-fixed and paraffin-embedded cancerous and adjacent non-cancerous tissues. Then, its associations with tumor progression and patients' prognosis were statistically evaluated. After that, we performed migration and invasion assays of siRNA-targeted PDK1-transfected cells in vitro. As a result, immunohistochemistry analysis showed that the PDK1 protein was expressed in cytoplasm of the carcinoma cells in ESCC tissues, but not expressed in adjacent non-cancerous tissues. Statistical analysis indicated that the expression level of PDK1 protein in ESCC tissues was significantly higher than those in adjacent non-cancerous tissues (P < 0.001). In addition, high PDK1 expression was found to be closely correlated with advanced tumor stage (P = 0.006), positive lymph node metastasis (P = 0.01) and high histological grade (P = 0.02). Moreover, survival analysis revealed that PDK1 overexpression was significantly associated with shorter overall survival (P = 0.001). The multivariate analysis further identified PDK1 expression as an independent prognostic factor for ESCC patients. In ESCC cell lines, the knockdown of PDK1 using siRNA inhibited the cell migration and invasion in vitro. These findings support that PDK1 may contribute to the aggressive progression of ESCC. Of note, PDK1 may serve as an effective prognostic marker and provide a novel therapeutic strategy against human ESCC.