Akt Signaling Cascade Research Articles

Glycoprotein hormone α-subunit promotes cell proliferation and tumorigenesis in breast cancer.

The glycoprotein hormone α-subunit (CGA) is implicated in the occurrence and progression of a number of solid tumors. However, its role in breast cancer remains unclear. The present study aimed to investigate the biological functions and mechanisms of action of CGA in breast cancer. CGA protein expression was evaluated in clinical breast cancer specimens using immunohistochemistry. The association between CGA expression and patient prognosis was determined using the Kaplan-Meier method and Mantel-Cox test. At the same time, CGA mRNA and protein expression was explored in a normal mammary epithelial cell line and breast cancer cell lines. Breast cancer cell lines overexpressing or deficient in CGA were established, and the effect of CGA on cell proliferation was evaluated in vitro, and in vivo using a mouse xenograft tumor model. Intracellular signaling pathway activities were evaluated using western blotting in CGA-overexpressing or -depleted cells. Increased CGA protein expression was significantly associated with a poor prognosis in patients with breast cancer. Furthermore, while CGA mRNA and protein expression level was negligible in normal mammary epithelial cells, it was elevated in breast cancer cell lines. In vitro and in vivo experiments showed that CGA overexpression enhanced breast cancer cell proliferation via activation of the epidermal growth factor receptor, extracellular signal-regulated kinase 1/2 and serine/threonine kinase Akt signaling cascades. The present results suggest that CGA is upregulated in breast cancer tissues and that it is associated with a poor prognosis. CGA may serve as a candidate for developing targeted therapies for breast cancer.

Transforming growth factor β1 signaling links extracellular matrix remodeling to intracellular lipogenesis upon physiological feeding events

Adipose tissue dynamically changes its mass in response to external nutritional status, which plays an important role in maintaining the lipid homeostasis. Physiologically, feeding events are associated with the expansion of adipose tissue, but little is known about the detailed molecular mechanisms of this expansion. Here, using comprehensive transcriptome analysis, we found that levels of transforming growth factor β1 (TGF-β1), a key regulator of extracellular matrix (ECM) remodeling, were increased in adipose tissue under feeding conditions and associated with the lipogenic pathway. In addition, TGF-β receptors are highly expressed in adipose tissue, and pharmacological inhibition of TGF-β1 reduced adipose tissue mass and caused ectopic lipid accumulation in the liver. This reduced fat mass was associated with decreased gene expression in ECM remodeling and lipogenesis. Furthermore, similar results were observed in the adipose tissue of SMAD family member 3 knockout mice or upon systemic TGF-β neutralization, with significant reductions in both ECM remodeling and lipogenesis-related genes. Mechanistically, we found that insulin-induced TGF-β1 and cell-autonomous action remodels the ECM of adipocytes, which controls the downstream focal adhesion kinase–AKT signaling cascades and enhances the lipogenic pathway. Of note, destruction of collagens or matrix metalloproteinase/a disintegrin and metalloprotease activities, critical components of ECM remodeling, blocked TGF-β1-mediated focal adhesion kinase–AKT signaling and the lipogenic pathway. Taken together, this study identifies a previously unknown lipogenic role of TGF-β1 by which adipocytes can expand to adapt to physiological feeding events.

MTOR Targeted Cancer Chemoprevention by Flavonoids.

Over the past several decades, plant-derived products (phytochemicals) have been suggested to possess immense therapeutic potential. Among these phytochemicals, different flavonoids have been reported for their potent anticancer activity. To exhibit their anticancer potential, these flavonoids modulate different signaling pathways. Among these pathways, the mammalian target of rapamycin (mTOR) and associated phosphatidyl-inositol 3-kinase (PI3K)/protein kinase B (Akt) signaling cascade have been reported as a pivotal modulator of cell survival, proliferation, and death/apoptosis. Hence, targeting this cascade could be an ideal strategy to alleviate apoptosis and inhibit proliferation in different forms of cancer. The targeting of PI3K/Akt/mTOR by flavonoids have been well documented in the scientific literature. In the current study, we have studied the anticancer potential of various flavonoids, especially flavones, flavonols, and isoflavones that include apigenin, luteolin, baicalein, tangeretin, epigallocatechin- 3-gallate, genistein, and daidzein especially dealing with mTOR targeting.

Antcin K Inhibits TNF-α, IL-1β and IL-8 Expression in Synovial Fibroblasts and Ameliorates Cartilage Degradation: Implications for the Treatment of Rheumatoid Arthritis

Extracts from Taiwan’s traditional medicinal mushroom, Antrodia cinnamomea, exhibit anti-inflammatory activities in cellular and preclinical studies. However, this paper is the first to report that Antcin K, a triterpenoid isolated from A. cinnamomea, inhibits proinflammatory cytokine production in human rheumatoid synovial fibroblasts (RASFs), which are major players in rheumatoid arthritis (RA) disease. In our analysis of the mechanism of action, Antcin K inhibited the expression of three cytokines (tumor necrosis factor alpha [TNF-α], interleukin 1 beta [IL-1β] and IL-8) in human RASFs; cytokines that are crucial to RA synovial inflammation. Notably, incubation of RASFs with Antcin K reduced the phosphorylation of the focal adhesion kinase (FAK), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT) and nuclear factor-κB (NF-κB) signaling cascades, all of which promote cytokine production in RA. Intraperitoneal injections of Antcin K (10 mg/kg or 30 mg/kg) attenuated paw swelling, cartilage degradation and bone erosion, and decreased serum levels of TNF-α, IL-1β, IL-8 in collagen-induced arthritis (CIA) mice; in further experiments, IL-6 levels were similarly reduced. The inhibitory effects of Antcin K upon TNF-α, IL-1β and IL-8 expression in human RASFs was achieved through the downregulation of the FAK, PI3K, AKT and NF-κB signaling cascades. Our data support clinical investigations using Antcin K in RA disease.

IL-1 receptor signaling in podocytes limits susceptibility to glomerular damage.

Interleukin (IL)-1 receptor type 1 (IL-1R1) activation triggers a proinflammatory signaling cascade that can exacerbate kidney injury. However, the functions of podocyte IL-1R1 in glomerular disease remain unclear. To study the role of IL-1R1 signaling in podocytes, we selectively ablated podocyte IL-1R1 in mice (PKO mice). We then subjected PKO mice and wild-type controls to two glomerular injury models: nephrotoxic serum (NTS)- and adriamycin-induced nephropathy. Surprisingly, we found that IL-1R1 activation in podocytes limited albuminuria and podocyte injury during NTS- and adriamycin-induced nephropathy. Moreover, deletion of IL-1R1 in podocytes drove podocyte apoptosis and glomerular injury through diminishing Akt activation. Activation of Akt signaling abrogated the differences in albuminuria and podocyte injury between wild-type and PKO mice during NTS. Thus, IL-1R1 signaling in podocytes limits susceptibility to glomerular injury via an Akt-dependent signaling pathway. These data identify an unexpected protective role for IL-1R1 signaling in podocytes in the pathogenesis of glomerular disease.NEW & NOTEWORTHY The present study establishes that activation of the receptor for interleukin-1 limits susceptibility to damage to the kidney glomerulus in preclinical mouse models by stimulating Akt signaling cascades inside the podocyte.

Activation of PI3K/Akt Signaling Pathway in Rat Hypothalamus Induced by an Acute Oral Administration of D-Pinitol.

D-Pinitol (DPIN) is a natural occurring inositol capable of activating the insulin pathway in peripheral tissues, whereas this has not been thoroughly studied in the central nervous system. The present study assessed the potential regulatory effects of DPIN on the hypothalamic insulin signaling pathway. To this end we investigated the Phosphatidylinositol-3-kinase (PI3K)/Protein Kinase B (Akt) signaling cascade in a rat model following oral administration of DPIN. The PI3K/Akt-associated proteins were quantified by Western blot in terms of phosphorylation and total expression. Results indicate that the acute administration of DPIN induced time-dependent phosphorylation of PI3K/Akt and its related substrates within the hypothalamus, indicating an activation of the insulin signaling pathway. This profile is consistent with DPIN as an insulin sensitizer since we also found a decrease in the circulating concentration of this hormone. Overall, the present study shows the pharmacological action of DPIN in the hypothalamus through the PI3K/Akt pathway when giving in fasted animals. These findings suggest that DPIN might be a candidate to treat brain insulin-resistance associated disorders by activating insulin response beyond the insulin receptor.

Cell surface integrin α5ß1 clustering negatively regulates receptor tyrosine kinase signaling in colorectal cancer cells via glycogen synthase kinase 3.

As a key process within the tissue microenvironment, integrin signaling can influence cell functional responses to growth factor stimuli. We show here that clustering of integrin α5ß1 at the plasma membrane of colorectal cancer-derived epithelial cells modulates their ability to respond to stimulation by receptor tyrosine kinase (RTK)-activating growth factors EGF, NRG and HGF, through GSK3-mediated suppression of Akt pathway. We observed that integrin α5ß1 is lost from the membrane of poorly organized human colorectal tumors and that treatment with the integrin-clustering antibody P4G11 is sufficient to induce polarity in a mouse tumor xenograft model. While adding RTK growth factors (EGF, NRG and HGF) to polarized colorectal cancer cells induced invasion and loss of monolayer formation in 2D and 3D, this pathological behavior could be blocked by P4G11. Phosphorylation of ErbB family members as well as MET following EGF, NRG and HGF treatment was diminished in cells pretreated with P4G11. Focusing on EGFR, we found that blockade of integrin α5ß1 increased EGFR phosphorylation. Since activity of multiple downstream kinase pathways were altered by these various treatments, we employed computational machine learning techniques to ascertain the most important effects. Partial least-squares discriminant analysis identified GSK3 as a major regulator of EGFR pathway activities influenced by integrin α5ß1. Moreover, we used partial correlation analysis to examine signaling pathway crosstalk downstream of EGF stimulation and found that integrin α5ß1 acts as a negative regulator of the AKT signaling cascade downstream of EGFR, with GSK3 acting as a key mediator. We experimentally validated these computational inferences by confirming that blockade of GSK3 activity is sufficient to induce loss of polarity and increase of oncogenic signaling in the colonic epithelial cells.

Arginase II protein regulates Parkin-dependent p32 degradation that contributes to Ca2+-dependent eNOS activation in endothelial cells.

AimsArginase II (ArgII) plays a key role in the regulation of Ca2+ between the cytosol and mitochondria in a p32-dependent manner. p32 contributes to endothelial nitric oxide synthase (eNOS) activation through the Ca2+/CaMKII/AMPK/p38MAPK/Akt signalling cascade. Therefore, we investigated a novel function of ArgII in the regulation of p32 stability.Methods and resultsmRNA levels were measured by quantitative reverse transcription-PCR, and protein levels and activation were confirmed by western blot analysis. Ca2+ concentrations were measured by FACS analysis and a vascular tension assay was performed. ArgII bound to p32, and ArgII protein knockdown using siArgII facilitated the ubiquitin-dependent proteasomal degradation of p32. β-lactone, a proteasome inhibitor, inhibited the p32 degradation associated with endothelial dysfunction in a Ca2+-dependent manner. The amino acids Lys154, Lys 180, and Lys220 of the p32 protein were identified as putative ubiquitination sites. When these sites were mutated, p32 was resistant to degradation in the presence of siArgII, and endothelial function was impaired. Knockdown of Pink/Parkin as an E3-ubiquitin ligase with siRNAs resulted in increased p32, decreased [Ca2+]c, and attenuated CaMKII-dependent eNOS activation by siArgII. siArgII-dependent Parkin activation was attenuated by KN93, a CaMKII inhibitor. Knockdown of ArgII mRNA and its gene, but not inhibition of its activity, accelerated the interaction between p32 and Parkin and reduced p32 levels. In aortas of ArgII−/− mice, p32 levels were reduced by activated Parkin and inhibition of CaMKII attenuated Parkin-dependent p32 lysis. siParkin blunted the phosphorylation of the activated CaMKII/AMPK/p38MAPK/Akt/eNOS signalling cascade. However, ApoE−/− mice fed a high-cholesterol diet had greater ArgII activity, significantly attenuated phosphorylation of Parkin, and increased p32 levels. Incubation with siArgII augmented p32 ubiquitination through Parkin activation, and induced signalling cascade activation.ConclusionThe results suggest a novel function for ArgII protein in Parkin-dependent ubiquitination of p32 that is associated with Ca2+-mediated eNOS activation in endothelial cells.

IQGAP1 binds AMPK and is required for maximum AMPK activation

AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.

Contribution of glutaredoxin-1 to Fas s-glutathionylation and inflammation in ethanol-induced liver injury

AimsThe reversible protein S-glutathionylation (PSSG) modification of Fas augments apoptosis, which can be reversed by the cytosolic deglutathionylation enzyme glutaredoxin-1 (Grx1), but its roles in alcoholic liver injury remain unknown. Therefore, the objective of this study was to investigate the impact of genetic ablation of Grx1 on Fas S-glutathionylation (Fas-SSG) in regulating ethanol-induced injury. Materials and methodsWe evaluated the Grx1 activity and oxidative damage, hepatic injury related indicators, Fas-SSG, we also assess the nuclear factor-κB (NF-κB) signaling, its downstream signal, and Akt signaling cascades, Furthermore, the number of Kupffer cells and related proinflammatory cytokines between WT and Grx1- groups after alcohol exposure. Key findingsEthanol-fed mice had increased Grx1 activity and oxidative damage in the liver. Grx1-deficient mice had more serious liver damage when exposed to ethanol compared to that of wild-type mice, accompanied by increased alanine aminotransferase and aspartate aminotransferase levels, Fas-SSG, cleaved caspase-3 and hepatocyte apoptosis. Grx1 ablation resulted in the suppression of ethanol-induced NF-κB signaling, its downstream signal, and Akt signaling cascades, which are required for protection against Fas-mediated apoptosis. Accordingly, blocking NK-κB prevented Fas-induced apoptosis in WT mice but not Grx1−/− mice. Furthermore, the number of Kupffer cells and related proinflammatory cytokines, including Akt, were lower in Grx1−/− livers than those of the controls. SignificanceGrx1 is essential for adaptation to alcohol exposure-induced oxidative injury by modulating Fas-SSG and Fas-induced apoptosis.

High phosphate actively induces cytotoxicity by rewiring pro-survival and pro-apoptotic signaling networks in HEK293 and HeLa cells.

Inorganic phosphate (Pi) is an essential nutrient for human health. Due to the changes in our dietary pattern, dietary Pi overload engenders systemic phosphotoxicity, including excessive Pi-related vascular calcification and chronic tissue injury. The molecular mechanisms of the seemingly distinct phenotypes remain elusive. In this study, we investigated Pi-mediated cellular response in HEK293 and HeLa cells. We found that abnormally high Pi directly mediates diverse cellular toxicity in a dose-dependent manner. Up to 10mM extracellular Pi promotes cell proliferation by activating AKT signaling cascades and augmenting cell cycle progression. By introducing additional Pi, higher than the concentration of 40mM, we observed significant cell damage caused by the interwoven Pi-related biological processes. Elevated Pi activates mitogen-activated protein kinase (MAPK) signaling, encompassing extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and Jun amino-terminal kinase (JNK), which consequently potentiates Pi triggered lethal epithelial-mesenchymal transition (EMT). Synergistically, high Pi-caused endoplasmic reticulum (ER) stress also contributes to apparent apoptosis. To counteract, Pi-activated AKT signaling promotes cell survival by activating the mammalian target of rapamycin (mTOR) signaling and blocking ER stress. Pharmacologically or genetically abrogating Pi transport, the impact of high Pi-induced cytotoxicity could be reduced. Taken together, abnormally high extracellular Pi results in a broad spectrum of toxicity by rewiring complicated signaling networks that control cell growth, cell death, and homeostasis.

Galactose-NlGr11 inhibits AMPK phosphorylation by activating the PI3K-AKT-PKF-ATP signaling cascade via insulin receptor and Gβγ.

As ligands of the sugar gustatory receptors, sugars have been known to activate the insulin/insulin-like growth factor signaling pathway; however, the precise pathways that are activated by the sugar-bound gustatory receptors in insects remain unclear. In this study, we aimed to investigate the signaling cascades activated by NlGr11, a sugar gustatory receptor in the brown planthopper Nilaparvata lugens (Stål), and its ligand. Galactose-bound NlGr11 (galactose-NlGr11) activated the -phosphatidylinositol 3-kinase (PI3K)-AKT signaling cascade via insulin receptor (InR) and Gβγ in vitro. In addition, galactose-NlGr11 inhibited the adenosine monophosphate-activated protein kinase (AMPK) phosphorylation by activating the AKT-phosphofructokinase (PFK)-ATP signaling cascade in vitro. Importantly, the InR-PI3K-AKT-PFK-AKT signaling cascade was activated and the AMPK phosphorylation was inhibited after feeding the brown planthoppers with galactose solution. Collectively, these findings confirm that NlGr11 can inhibit AMPK phosphorylation by activating the PI3K-AKT-PFK-ATP signaling cascades via both InR and Gβγ when bound to galactose. Thus, our study provides novel insights into the signaling pathways regulated by the sugar gustatory receptors in insects.

Decyl caffeic acid inhibits the proliferation of colorectal cancer cells in an autophagy-dependent manner in vitro and in vivo.

The treatment of human colorectal cancer (CRC) cells through suppressing the abnormal survival signaling pathways has recently become a significant area of focus. In this study, our results demonstrated that decyl caffeic acid (DC), one of the novel caffeic acid derivatives, remarkedly suppressed the growth of CRC cells both in vitro and in vivo. The inhibitory effects of DC on CRC cells were investigated in an in vitro cell model and in vivo using a xenograft mouse model. CRC cells were treated with DC at various dosages (0, 10, 20 and 40 μM), and cell survival, the apoptotic index and the autophagy level were measured using an MTT assay and flow cytometry analysis, respectively. The signaling cascades in CRC were examined by Western blot assay. The anti-cancer effects of DC on tumor growth were examined by using CRC HCT-116 cells implanted in an animal model. Our results indicated that DC differentially suppressed the growth of CRC HT-29 and HCT-116 cells through an enhancement of cell-cycle arrest at the S phase. DC inhibited the expression of cell-cycle regulators, which include cyclin E and cyclin A proteins. The molecular mechanisms of action were correlated to the blockade of the STAT3 and Akt signaling cascades. Strikingly, a high dosage of DC prompted a self-protection action through inducing cell-dependent autophagy in HCT-116 cells. Suppression of autophagy induced cell death in the treatment of DC in HCT-116 cells. DC seemed to inhibit cell proliferation of CRC differentially, and the therapeutic advantage appeared to be autophagy dependent. Moreover, consumption of DC blocked the tumor growth of colorectal adenocarcinoma in an experimental animal model. In conclusion, our results suggested that DC could act as a therapeutic agent through the significant suppression of tumor growth of human CRC cells.

Etoxazole induces testicular malfunction in mice by dysregulating mitochondrial function and calcium homeostasis

Epidemiological relationships between pesticide use and male infertility have been suggested for a long time. Etoxazole (ETX), an oxazoline pesticide, has been extensively used for pest eradication. It is considered relatively safe and has low mammalian toxicity because it specifically inhibits chitin synthesis. However, ETX may have toxic effects on the reproductive system. In this study, we examined the effects of ETX on the reproductive system using mouse testis cell lines (TM3 for Leydig cells and TM4 for Sertoli cells) and C57BL/6 male mice. We confirmed that ETX has anti-proliferative effects on the TM3 and TM4 cell lines. Moreover, ETX induced mitochondrial dysfunction and hampers calcium homeostasis. Western blot analysis of MAPK and Akt signaling cascades was performed to demonstrate the mode of action of ETX at a molecular level. Moreover, ETX induced misregulation of genes related to testicular function. Upon oral administration of ETX in C57BL/6 male mice, testis weight was reduced and transcriptional expression related to testis function was altered. These results indicate that ETX induces testicular toxicity by inducing mitochondrial dysfunction and calcium imbalance and regulating gene expression.

LncRNA PVT1 Promotes Tumorigenesis of Colorectal Cancer by Stabilizing miR-16-5p and Interacting with the VEGFA/VEGFR1/AKT Axis

Recently, the long noncoding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) was reported to be involved in the pathogenesis of several cancers, including human colorectal cancer (CRC). However, the molecular basis for cancer initiation, development, and progression remains unclear. In this study, we observe that upregulated PVT1 is associated with poor prognosis and bad clinicopathological features of CRC patients. In vitro means of PVT1 loss in a CRC cell line inhibit cell proliferation, migration, and invasion. Furthermore, dual-luciferase reporter and RNA pull-down assays indicated that PVT1 binds to miR-16-5p, which has been shown to play strong tumor suppressive roles in CRC. Targeted loss of miR-16-5p partially rescues the suppressive effect induced by PVT1 knockdown. Vascular endothelial growth factor A (VEGFA), a direct downstream target of miR-16-5p, was suppressed by PVT1 knockdown in CRC cells. Overexpression of VEGFA is known to modulate the AKT signaling cascade by activating vascular endothelial growth factor receptor 1 (VEGFR1). We, therefore, show that PVT1 loss combined with miR-16-5p overexpression reduces tumor volume maximally when propagated within a mouse xenograft model. We conclude that the PVT1-miR-16-5p/VEGFA/VEGFR1/AKT axis directly coordinates the response in CRC pathogenesis and suggest PVT1 as a novel target for potential CRC therapy.

PDK1 is important lipid kinase for RANKL-induced osteoclast formation and function via the regulation of the Akt-GSK3β-NFATc1 signaling cascade.

Perturbations in the balanced process of osteoblast-mediated bone formation and osteoclast-mediated bone resorption leading to excessive osteoclast formation and/or activity is the cause of many pathological bone conditions such as osteoporosis. The osteoclast is the only cell in the body capable of resorbing and degrading the mineralized bone matrix. Osteoclast formation from monocytic precursors is governed by the actions of two key cytokines macrophage-colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). Binding of RANKL binding to receptor RANK initiates a series of downstream signaling responses leading to monocytic cell differentiation and fusion, and subsequent mature osteoclast bone resorption and survival. The phosphoinositide-3-kinase (PI3K)-protein kinase B (Akt) signaling cascade is one such pathway activated in response to RANKL. The 3-phosphoinositide-dependent protein kinase 1 (PDK1), is considered the master upstream lipid kinase of the PI3K-Akt cascade. PDK1 functions to phosphorylate and partially activate Akt, triggering the activation of downstream effectors. However, the role of PDK1 in osteoclasts has yet to be clearly defined. In this study, we specifically deleted the PDK1 gene in osteoclasts using the cathepsin-K promoter driven Cre-LoxP system. We found that the specific genetic ablation of PDK1 in osteoclasts leads to an osteoclast-poor osteopetrotic phenotype in mice. In vitro cellular assays further confirmed the impairment of osteoclast formation in response to RANKL by PDK1-deficient bone marrow macrophage (BMM) precursor cells. PDK1-deficient BMMs exhibited reduced ability to reorganize actin cytoskeleton to form a podosomal actin belt as a result of diminished capacity to fuse into giant multinucleated osteoclasts. Notably, biochemical analyses showed that PDK1 deficiency attenuated the phosphorylation of Akt and downstream effector GSK3β, and reduced induction of NFATc1. GSK3β is a reported negative regulator of NFATc1. GSK3β activity is inhibited by Akt-dependent phosphorylation. Thus, our data provide clear genetic and mechanistic insights into the important role for PDK1 in osteoclasts.

Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair traumatic spinal cord injury by shifting microglial M1/M2 polarization

BackgroundSpinal cord injury (SCI) can lead to severe motor and sensory dysfunction with high disability and mortality. In recent years, mesenchymal stem cell (MSC)-secreted nano-sized exosomes have shown great potential for promoting functional behavioral recovery following SCI. However, MSCs are usually exposed to normoxia in vitro, which differs greatly from the hypoxic micro-environment in vivo. Thus, the main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (HExos) exhibit greater effects on functional behavioral recovery than those under normoxia (Exos) following SCI in mice and to seek the underlying mechanism.MethodsElectron microscope, nanoparticle tracking analysis (NTA), and western blot were applied to characterize differences between Exos and HExos group. A SCI model in vivo and a series of in vitro experiments were performed to compare the therapeutic effects between the two groups. Next, a miRNA microarray analysis was performed and a series of rescue experiments were conducted to verify the role of hypoxic exosomal miRNA in SCI. Western blot, luciferase activity, and RNA-ChIP were used to investigate the underlying mechanisms.ResultsOur results indicate that HExos promote functional behavioral recovery by shifting microglial polarization from M1 to M2 phenotype in vivo and in vitro. A miRNA array showed miR-216a-5p to be the most enriched in HExos and potentially involved in HExos-mediated microglial polarization. TLR4 was identified as the target downstream gene of miR-216a-5p and the miR-216a-5p/TLR4 axis was confirmed by a series of gain- and loss-of-function experiments. Finally, we found that TLR4/NF-κB/PI3K/AKT signaling cascades may be involved in the modulation of microglial polarization by hypoxic exosomal miR-216a-5p.ConclusionHypoxia preconditioning represents a promising and effective approach to optimize the therapeutic actions of MSC-derived exosomes and a combination of MSC-derived exosomes and miRNAs may present a minimally invasive method for treating SCI.

Self‐Assembled/Drug‐Composed Nanomedicine for Synergistic Photonic Hyperthermia and Targeted Therapy of Breast Cancer by Inhibiting ERK, AKT, and STAT3 Signaling Cascades

AbstractSuperior to chemotherapy, photonic hyperthermia and targeted therapy have made attractive impacts on cancer treatment by virtue of their profound advantages such as high specificity and minimal invasiveness, but the rational integration of corresponding therapeutic drugs for achieving concurrent photothermal ablation/targeted therapy is still challenging. Herein, a self‐assembled nanomedicine Anlotinib@IR820 is constructed with drug formulations for highly efficient and synergistic photonic hyperthermia and targeted therapy against breast cancer. Specifically, the constructed Anlotinib@IR820 nanomedicine presents high accumulation at the tumor site owing to the enhanced permeability and retention effect and simultaneously overcomes the obstacles of poor water solubility of Anlotinib (for targeted therapy) and the short lifetime of IR820 (for photonic ablation). The photothermal ablation as activated by near‐infrared laser can not only irradiate cancer cells but also promote the cellular uptake of Anlotinib, which presents a profound synergistic function both in vitro and in vivo. Mechanically, Anlotinib@IR820 nanomedicine can induce apoptosis and cause cell cycle arrest in breast cancer through inhibiting ERK, AKT, and STAT3 pathways. Therefore, the rationally designed drug‐composed Anlotinib@IR820 nanomedicine exhibits high clinical translation potential because of its therapeutic nanoformulation, which provides an alternative option for efficient combinational therapy of breast cancer.

Abstract 4312: Multiplex parallel perturbation of Akt and MAPk signaling cascades

Abstract PI3K/Akt and MAPk signal transduction pathways are frequently dysregulated in human cancers. Previous work described the molecular basis of pro-survival signals mediated by Akt and MAPk. Mutation in and alteration of these pathways lead to tumorigenesis. However, signal propagation varies across cell type and precise signaling kinetics is less well characterized. The goal of this study is to profile the functional state of the PI3k/Akt and MAPk signaling circuits by coupling cell viability to antibody microarray and immunofluorescence. Simultaneous detection of Akt and MAPk signaling nodes can characterize signaling parameters that correlate to localization patterns across cell type and drug treatment conditions. To examine how signal propagation varied between cell lines, staurosporine was utilized to broadly inhibit protein kinase activity in a cervical cancer cell line, HeLa, and an osteosarcoma cell line, U2-OS, as model systems. To investigate signaling pathways, each cell line was treated and analyzed in parallel by forward phase phospho-specific antibody microarray panels and high content analysis of immunofluorescent staining. Staurosporine inhibited cell proliferation in a time and concentration dependent manner in both cell lines. U2-OS cells had a higher IC50 compared to HeLa cells indicating markedly different signaling parameters between the two models. Our observations suggest detecting cell type specific signaling kinetics can be used as a framework to predict cell sensitivity to specific inhibitors and be used to monitor drug resistance. Note: This abstract was not presented at the meeting. Citation Format: Andrew R. Wagner, Megan Newby, Kary Oakleaf. Multiplex parallel perturbation of Akt and MAPk signaling cascades [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4312.

A Functional Landscape of Resistance to MEK1/2 and CDK4/6 Inhibition in NRAS-Mutant Melanoma.

Combinatorial inhibition of MEK1/2 and CDK4/6 is currently undergoing clinical investigation in NRAS-mutant melanoma. To prospectively map the landscape of resistance to this investigational regimen, we utilized a series of gain- and loss-of-function forward genetic screens to identify modulators of resistance to clinical inhibitors of MEK1/2 and CDK4/6 alone and in combination. First, we identified NRAS-mutant melanoma cell lines that were dependent on NRAS for proliferation and sensitive to MEK1/2 and CDK4/6 combination treatment. We then used a genome-scale ORF overexpression screen and a CRISPR knockout screen to identify modulators of resistance to each inhibitor alone or in combination. These orthogonal screening approaches revealed concordant means of achieving resistance to this therapeutic modality, including tyrosine kinases, RAF, RAS, AKT, and PI3K signaling. Activated KRAS was sufficient to cause resistance to combined MEK/CDK inhibition and to replace genetic depletion of oncogenic NRAS. In summary, our comprehensive functional genetic screening approach revealed modulation of resistance to the inhibition of MEK1/2, CDK4/6, or their combination in NRAS-mutant melanoma. SIGNIFICANCE: These findings reveal that NRAS-mutant melanomas can acquire resistance to genetic ablation of NRAS or combination MEK1/2 and CDK4/6 inhibition by upregulating activity of the RTK-RAS-RAF and RTK-PI3K-AKT signaling cascade.