Downstream AKT Signaling Research Articles

Activation of TrkB-Akt signaling rescues deficits in a mouse model of SCA6.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease resulting in motor coordination deficits and cerebellar pathology. Expression of brain-derived neurotrophic factor (BDNF) is reduced in postmortem tissue from SCA6 patients. Here, we show that levels of cerebellar BDNF and its receptor, tropomyosin receptor kinase B (TrkB), are reduced at an early disease stage in a mouse model of SCA6 (SCA684Q/84Q). One month of exercise elevated cerebellar BDNF expression and improved ataxia and cerebellar Purkinje cell firing rate deficits. A TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF), likewise improved motor coordination and Purkinje cell firing rate and elevated downstream Akt signaling. Prolonged 7,8-DHF administration persistently improved ataxia when treatment commenced near disease onset but was ineffective when treatment was started late. These data suggest that 7,8-DHF, which is orally bioavailable and crosses the blood-brain barrier, is a promising therapeutic for SCA6 and argue for the importance of early intervention for SCA6.

ADSCs stimulated by VEGF-C alleviate intestinal inflammation via dual mechanisms of enhancing lymphatic drainage by a VEGF-C/VEGFR-3-dependent mechanism and inhibiting the NF-κB pathway by the secretome

BackgroundAdipose-derived stem cells (ADSCs) have provided promising applications for Crohn’s disease (CD). However, the practical efficacy of ADSCs remains controversial, and their mechanism is still unclear. Based on the pathogenesis of dysregulated immune responses and abnormal lymphatic alterations in CD, vascular endothelial growth factor-C (VEGF-C) is thought to be a favourable growth factor to optimize ADSCs. We aimed to investigate the efficacy of VEGF-C-stimulated ADSCs and their dual mechanisms in both inhibiting inflammation “IN” and promoting inflammation “OUT” in the intestine.MethodsHuman stem cells isolated from adipose tissues were identified, pretreated with or without 100 ng/ml VEGF-C and analysed for the secretion of cell culture supernatants in vitro. Lymphatic endothelial cells (LECs) were treated with ADSCs-conditioned medium or co-cultured with ADSCs and VEGF-C stimulated ADSCs. Changes in LECs transmigration, and VEGF-C/VEGFR-3 mRNA levels were assessed by transwell chamber assay and qRT–PCR. ADSCs and VEGF-C-stimulated ADSCs were intraperitoneally injected into mice with TNBS-induced chronic colitis. ADSCs homing and lymphatic vessel density (LVD) were evaluated by immunofluorescence staining. Lymphatic drainage was assessed using Evans blue. Cytokines and growth factors expression was detected respectively by ELISA and qRT–PCR. The protein levels of VEGF-C/VEGFR-3-mediated downstream signals and the NF-κB pathway were assayed by western blot. Faecal microbiota was measured by 16S rRNA sequencing.ResultsADSCs stimulated with VEGF-C released higher levels of growth factors (VEGF-C, TGF-β1, and FGF-2) and lower expression of cytokines (IFN-γ and IL-6) in cell supernatants than ADSCs in vitro (all P < 0.05). Secretome released by VEGF-C stimulated ADSCs exhibited a stronger LEC migratory capability and led to elevated VEGF-C/VEGFR-3 expression, but these effects were markedly attenuated by VEGFR-3 inhibitor. VEGF-C-stimulated ADSCs homing to the inflamed colon and mesenteric lymph nodes (MLNs) can exert stronger efficacy in improving colitis symptoms, reducing inflammatory cell infiltration, and significantly enhancing lymphatic drainage. The mRNA levels and protein concentrations of anti-inflammatory cytokines and growth factors were markedly increased with decreased proinflammatory cytokines in the mice treated with VEGF-C-stimulated ADSCs. Systemic administration of VEGF-C-stimulated ADSCs upregulated the colonic VEGF-C/VEGFR-3 pathway and activated downstream AKT and ERK phosphorylation signalling, accompanied by decreased NF-κB p65 expression. A higher abundance of faecal p-Bacteroidetes and lower p-Firmicutes were detected in mice treated with VEGF-C-stimulated ADSCs (all P < 0.05).ConclusionVEGF-C-stimulated ADSCs improve chronic intestinal inflammation by promoting lymphatic drainage and enhancing paracrine signalling via activation of VEGF-C/VEGFR-3-mediated signalling and inhibition of the NF-κB pathway. Our study may provide a new insight into optimizing ADSCs treatment and investigating potential mechanisms in CD.

Design, Synthesis, and Evaluation of New Mesenchymal-Epithelial Transition Factor (c-Met) Kinase Inhibitors with Dual Chiral Centers.

A series of tepotinib derivatives with two chiral centers was designed, synthesized, and evaluated as anticancer agents. The optimal compound (R, S)-12a strongly exhibited antiproliferative activity against MHCC97H cell lines with an IC50 value of 0.002 μM, compared to tepotinib (IC50 = 0.013 μM). Mechanistic studies revealed that compound (R, S)-12a significantly inhibited c-Met activation, as well as the downstream AKT signaling pathway, and suppressed wound closure. Moreover, compound (R, S)-12a induced cellular apoptosis and cell cycle arrest at the G1 phase in a dose-dependent fashion.

Methyltransferase-like 3 promotes the progression of lung cancer via activating PI3K/AKT/mTOR pathway.

Methyltransferase-like 3 (METTL3) catalyses N6-methyladenosine (m6 A) modification on messenger RNA (mRNA) and participates in a wide range of biological functions via epigenetically regulating gene expression. Recent studies suggested that dysregulation of METTL3 is associated with multiple human cancers; however, the role of METTL3 in lung cancer remains unclear. In the present study, through transcriptome analysis of lung cancer patients, we found that METTL3 is overexpressed in lung cancer patients and is associated with poor patient survival. More importantly, combining both in vitro and in vivo models, we revealed that in lung cancer cells, METTL3 overexpression activates PI3K/AKT/mTOR pathway and mTOR-mediated protein synthesis. Mechanistically, METTL3 promotes PI3K expression by introducing m6 A modification in PI3K 3' untranslated region (3' UTR). Elevated PI3K level then activates downstream AKT and mTOR signalling pathway and results in rapid cancer cell proliferation and metastasis. Taken together, our study reveals that METTL3-mediated m6 A methylation promotes lung cancer progression via activating PI3K/AKT/mTOR pathway.

Structure-based rational design enables efficient discovery of a new selective and potent AKT PROTAC degrader

AKT and associated signaling pathways have been recognized as promising therapeutic targets for decades, and growing evidence indicates that inhibition or degradation of cellular AKT are viable strategies to treat cancer. Guided by an in silico modeling approach for rational linker design and based on our previous work in this field, we herein efficiently synthesized a small group of cereblon-recruiting AKT PROTAC molecules and identified a highly potent AKT degrader B4. Compared to the existing AKT degraders, B4 has a structurally unique AKT targeting warhead derived from the pyrazole-furan conjugated piperidine derivatives. It induces selective degradation of all three isoforms of AKT and exhibits efficacious anti-proliferation against several human hematological cancers. Notably, B4 demonstrates potent inhibition of AKT downstream signaling superior to its parental inhibitor. Together with its active analogs, B4 expands the arsenal of AKT chemical degraders as a valuable probe to uncover AKTs new functions and as a potential drug candidate to treat cancer.

Smtnl2 regulates apoptotic germ cell clearance and lactate metabolism in mouse Sertoli cells

Smtnl2 is an epithelial Smoothelin that binds to actin filaments and is crucial for epithelial morphogenesis. We examined the role of Smtnl2 in Sertoli cells, which undergo dynamic cytoskeleton reorganization to phagocytose apoptotic germ cells, a process known as efferocytosis. We observed Smtnl2 expression in primary mouse Sertoli cells and the 15P1 Sertoli cell line. Smtnl2 expression increased in 15P1 cells committing efferocytosis. Smtnl2-deficient Sertoli cells exhibited defective ability to engulf apoptotic germ cells and importantly, the phenomenon occurred in the setting of an unaffected maturation of phagosome. We demonstrated that Smtnl2 regulates the engulfment process through the function of branched actin nucleation protein ARP3, an actin assembly dictator. Intriguingly, a shift in glucose metabolism that restricts lactate production in Sertoli cells was induced upon Smtnl2 depletion, leading to the activation of downstream AMPK and AKT signaling. Using an in vivo RNAi approach, we found that silencing of Smtnl2 in testis triggers an obvious disruption in cytoskeleton architecture and blood-testis barrier integrity across seminiferous epithelium, causing the detachment of massive germ cells from their nest, as evidenced by their exfoliation into the lumen. Overall, our study identifies Smtnl2 as a determinant for Sertoli cells’ functioning in supporting spermatogenesis.

The phosphoinositide-3-kinase (PI3K)-delta inhibitor seletalisib impairs monocyte-derived dendritic cells maturation, APC function, and promotes their migration to CCR7 and CXCR4 ligands.

PI3K pathway plays a crucial role in dendritic cells (DCs) functions, as it regulates different cellular processes, such as maturation and cytokines production. However, the specific role of PI3K p110δ isoform in human DCs has not been thoroughly addressed. In this study, we analyze the effects of seletalisib, a potent and specific inhibitor of PI3K p110δ, on phenotype and antigen-presenting functions of monocyte-derived DCs undergone maturation via LPS. Seletalisib treatment reduced membrane HLA-DR as well as CD83 and CD40 costimulatory molecules, whereas CD80 and CD86 expression was only partially affected. Additionally, DCs cultures showed reduced TNF-α, IL-10, and IL-12 and increased IL-23 secretion levels. This resulted ina reduced capacity of DCs to prime allogeneic T cells, with a strong decrease of Th1 differentiation. On the other hand, PI3K p110δ inhibitor seletalisib increased CXCR4 and CCR7 expression and augmented the DCs migration toward CCL19 and CXCL12 ligands. At molecular level, inhibition of PI3K p110δ isoform by seletalisib significantly down-regulated the phosphorylation of AKT and other downstream signaling molecules, such as ribosomal protein S6, 4E-BP1, and NF-κB p65. In contrast, seletalisib did not affect p38 MAP kinase phosphorylation or TLR-associated adapter molecule TIRAP in DCs. Our results indicate that PI3K p110δ can serve as an important regulatory signalfor DCs, and selective inhibition of PI3K p110δisoform by seletalisib could be used for the prevention of exaggerated and harmful immune responsesoccurring in pathologic conditions, such as autoimmune disorders.

Antrodia camphorata-Derived Antrodin C Inhibits Liver Fibrosis by Blocking TGF-Beta and PDGF Signaling Pathways.

Hepatic stellate cells (HSCs) play an essential role in the development of liver fibrosis. Antrodia camphorata (A. camphorata) is a medicinal fungus with hepatoprotective effect. This study investigated whether Antrodin C, an A. camphorata-fermented metabolite, could exert a protective role on liver fibrosis both in vitro and in vivo. The anti-fibrotic effect of Antrodin C was investigated in CFSC-8B cell (hepatic stellate cell) stimulated by transforming growth factor-β1 (TGF-β1) or platelet-derived growth factor-BB (PDGF-BB) in vitro and in CCl4 induced liver fibrosis in mice. Antrodin C (50 μM) inhibited TGF-β1 or PDGF-BB stimulated CFSC-8B cell activation, migration and extracellular matrix (ECM) accumulation (all p < 0.05). Antrodin C (3, 6 mg/kg/d) oral administration reduced the degree of liver fibrosis induced by CCl4 in mice. Antrodin C down-regulated the expression of α-smooth muscle actin (α-SMA) and collagen I in fibrotic livers. Furthermore, Antrodin C ameliorated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation in serum (all p < 0.05). Mechanistically, Antrodin C executes its anti-fibrotic activity through negatively modulate TGF-β1 downstream SMAD Family Member 2 (Smad2), AKT Serine/Threonine Kinase 1 (AKT), extracellular signal-regulated kinase (ERK), and P38 MAP Kinase (P38), as well as PDGF-BB downstream AKT and ERK signaling pathways. Antrodin C ameliorates the activation, migration, ECM production in HSCs and CCl4-induced liver fibrosis in mice, suggesting that Antrodin C could serve as a protective molecule against liver fibrosis.

Abstract P2-13-23: Activity and tolerability of combination of trastuzumab deruxtecan with the pan-AKT inhibitor capivasertib in preclinical HER2+ and HER2-low breast cancer models

Abstract Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate composed of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor approved for HER2+ metastatic breast and gastric cancer. Clinically, T-DXd has demonstrated significant antitumor activity in both HER2+ and HER2-low (IHC 1+ &amp; 2+/ISH-) breast cancer patients. The PI3K/AKT pathway is frequently activated in breast cancer, and mediates signals downstream of HER2. T-DXd has been shown to inhibit downstream AKT signaling driven by HER2. Capivasertib, a potent, selective inhibitor of all three AKT isoforms (AKT1/2/3) has shown clinical activity in breast cancer trials. Given the clinical activity of both agents, the anti-tumor activity of combined treatment with T-DXd with capivasertib, was explored preclinically in HER2+ and HER2-low models. Methods: The antiproliferative activity of the combination of T-DXd with capivasertib was assessed in a panel of 27 breast cancer cell lines using a 7 day viability assay. To determine whether the combination also translates in vivo, T-DXd (3 and 10mg/kg Q3W) and capivasertib (130mg/kg BID 4 days on/3 days off) were tested in KPL4 (HER2+). To determine whether the combination of T-DXd and capivasertib has a negative impact on normal tissue integrity, we further evaluated the combination in a human 2D in vitro bone marrow progenitor assay. Results: The combination treatment was more effective than each single agent in 6/27 of the models tested in vitro, resulting in increased cell kill in the viability assay. Two of these positive cell lines, HCC1569 (HER2+) and AU565 (HER2+) were PTEN altered, KPL4 (HER2+) and EFM19 (ER+) were mutant in PIK3CA, and HCC1419 (HER2+) and ZR-75-30 (ER+/HER2+) were neither PTEN or PIK3CA altered. In KPL4 xenografts, the combination of T-DXd and capivasertib showed modest increased activity over monotherapy T-DXd or capivasertib at 28d (Tumor Growth Inhibition (TGI) of T-DXd = 76%, capivasertib = 25%, and T-DXd+Capivasertib = 83%; p=0.3 combo vs. mono T-DXd) and more markedly at 46d (TGI of the combination vs T-DXd monotherapy was 65%). Importantly, the combination showed enhanced durability of response (stasis) in 100% mice while 38% (3/8) mice in the T-DXd mono therapy treatment group were actively re-growing at 46d. In the in vitro bone marrow assay, the combination demonstrated no increased interaction over monotherapy activity (average Loewe Synergy Score of -0.2). Conclusions: These results suggest combining T-DXd with capivasertib has potential to be active in breast cancer patients, with activity likely to be enriched in tumours with mutations in PIK3CA and PTEN, but also in tumours with no PI3K pathway activating mutations. Citation Format: Azadeh Cheraghchi Bashi, Theresa Proia, Suzanne Randle, Mark Anderton, Zeshaan Rasheed, J. Elizabeth Pease, Simon Barry, Danielle Carroll, Jerome Mettetal. Activity and tolerability of combination of trastuzumab deruxtecan with the pan-AKT inhibitor capivasertib in preclinical HER2+ and HER2-low breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-23.

7,8-Dihydroxyflavone alleviates apoptosis and inflammation induced by retinal ischemia-reperfusion injury via activating TrkB/Akt/NF-kB signaling pathway.

Retinal ischemia-reperfusion injury (RIRI) is of common occurrence in retinal and optic nerve diseases. The BDNF/TrkB signaling pathway has been examined to be neuroprotective in RIRI. In this study, we investigated the role of a potent selective TrkB agonist 7,8-dihydroxyfavone (DHF) in rat retinas with RIRI. Our results showed that RIRI inhibited the conversion of BDNF precursor (proBDNF) to mature BDNF (mBDNF) and increased the level of neuronal cell apoptosis. Compared with RIRI, DHF+RIRI reduced proBDNF level and at the same time increased mBDNF level. Moreover, DHF administration effectively activated TrkB signaling and and downstream Akt and Erk signaling pathways which increased nerve cell survival. The combined effects of mBDNF/proBDNF increase and TrkB signaling activation lead to reduction of apoptosis level and protection of retinas with RIRI. Moreover, it was also found that astrocytes labeled by GFAP were activated in RIRI and NF-kB mediated the increased expressions of inflammatory factors and these effects were partially reversed by DHF administration. Besides, we also used RNA sequencing to analyze the differently expressed genes (DEGs) and their enriched (Kyoto Encyclopedia of Genes and Genomes) KEGG pathways between Sham, RIRI, and DHF+RIRI. It was found that 1543 DEGs were differently expressed in RIRI and 619 DEGs were reversed in DHF+RIRI. The reversed DEGs were typically enriched in PI3K-Akt signaling pathway, Jak-STAT signaling pathway, NF-kB signaling pathway, and Apoptosis. To sum up, the DHF administration alleviated apoptosis and inflammation induced by RIRI via activating TrkB signaling pathway and may serve as a promising drug candidate for RIRI related ophthalmopathy.

Semi-Synthesis, Cytotoxic Evaluation, and Structure-Activity Relationships of Brefeldin A Derivatives with Antileukemia Activity.

Brefeldin A (1), a potent cytotoxic natural macrolactone, was produced by the marine fungus Penicillium sp. (HS-N-29) from the medicinal mangrove Acanthus ilicifolius. Series of its ester derivatives 2–16 were designed and semi-synthesized, and their structures were characterized by spectroscopic methods. Their cytotoxic activities were evaluated against human chronic myelogenous leukemia K562 cell line in vitro, and the preliminary structure–activity relationships revealed that the hydroxy group played an important role. Moreover, the monoester derivatives exhibited stronger cytotoxic activity than the diester derivatives. Among them, brefeldin A 7-O-2-chloro-4,5-difluorobenzoate (7) exhibited the strongest inhibitory effect on the proliferation of K562 cells with an IC50 value of 0.84 µM. Further evaluations indicated that 7 induced cell cycle arrest, stimulated cell apoptosis, inhibited phosphorylation of BCR-ABL, and thereby inactivated its downstream AKT signaling pathway. The expression of downstream signaling molecules in the AKT pathway, including mTOR and p70S6K, was also attenuated after 7-treatment in a dose-dependent manner. Furthermore, molecular modeling of 7 docked into 1 binding site of an ARF1–GDP-GEF complex represented well-tolerance. Taken together, 7 had the potential to be served as an effective antileukemia agent or lead compound for further exploration.

Mechanism of sensitivity to cisplatin, docetaxel, and 5-fluorouracil chemoagents and potential erbB2 alternatives in oral cancer with growth differentiation factor 15 overexpression.

The aim of this study was to: (a) explore the potential mechanism of cancer cell sensitivity to cisplatin, docetaxel, and 5‐fluorouracil (TPF) in oral squamous cell carcinoma (OSCC) patients overexpressing growth differentiation factor 15 (GDF15); and (b) identify potential alternative agents for patients who might not benefit from inductive TPF chemotherapy. The results indicated that OSCC cells overexpressing GDF15 were sensitive to TPF through a caspase‐9‐dependent pathway both in vitro and in vivo. Immunoprecipitation combined with mass spectrometry revealed that the erbB2 protein was a potential GDF15‐binding protein, which was verified by coimmunoprecipitation. Growth differentiation factor 15 overexpression promoted OSCC cell proliferation through erbB2 phosphorylation, as well as downstream AKT and Erk signaling pathways. When GDF15 expression was blocked, the phosphorylation of both the erbB2 and AKT/Erk pathways was downregulated. When OSCC cells with GDF15 overexpression were treated with the erbB2 phosphorylation inhibitor, CI‐1033, cell proliferation and xenograft growth colony formation were significantly blocked (P < .05). Thus, GDF15‐overexpressing OSCC tumors are sensitive to TPF chemoagents through caspase‐9‐dependent pathways. Growth differentiation factor 15 overexpression promotes OSCC proliferation through erbB2 phosphorylation. Thus, ErbB2 inhibitors could represent potential targeted drugs or an alternative therapy for OSCC patients with GDF15 overexpression.

Genome-wide CRISPR-cas9 knockout screening identifies GRB7 as a driver for MEK inhibitor resistance in KRAS mutant colon cancer

Targeting the KRAS pathway is a promising but challenging approach for colorectal cancer therapy. Despite showing potent efficacy in BRAF-mutated melanoma, MEK inhibitors appeared to be tolerated by colorectal cancer cells due to their intrinsic compensatory signaling. Here, we performed genome-wide CRISPR/Cas9 screening in the presence of MEK inhibitor to identify genes that are synthetically lethal with MEK inhibition in CRC models harboring KRAS mutations. Several genes were identified as potential functional drivers, which were significantly enriched in the GRB7-mediated RTK pathway. Loss-of-function and gain-of-function assays validated that GRB7 potently rendered CRC cells primary resistance to MEK inhibitors through the RTK pathway. Mass spectrum analysis of GRB7 immunoprecipitates revealed that PLK1 was the predominant interacting kinase of GRB7. Inhibition of PLK1 suppressed downstream signaling of RTK, including FAK, STAT3, AKT, and 4EBP1. The combination of PLK1 and MEK inhibitors synergistically inhibited CRC cell proliferation and induced apoptosis in vitro and in vivo. In conclusion, we identified GRB7-PLK1 as a pivotal axis mediating RTKs, resulting in MEK inhibitor tolerance. PLK1 is therefore a promising target for synergizing MEK inhibitors in the clinical treatment of CRC patients harboring KRAS mutations.

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.

Increased abundance of Cbl E3 ligases alters PDGFR signaling in recessive dystrophic epidermolysis bullosa

In recessive dystrophic epidermolysis bullosa (RDEB), loss of collagen VII, the main component of anchoring fibrils critical for epidermal-dermal cohesion, affects several intracellular signaling pathways and leads to impaired wound healing and fibrosis. In skin fibroblasts, wound healing is also affected by platelet-derived growth factor receptor (PDGFR) signaling. To study a potential effect of loss of collagen VII on PDGFR signaling we performed unbiased disease phosphoproteomics. Whereas RDEB fibroblasts exhibited an overall weaker response to PDGF, Cbl E3 ubiquitin ligases, negative regulators of growth factor signaling, were stronger phosphorylated. This increase in phosphorylation was linked to higher Cbl mRNA and protein levels due to increased TGFβ signaling in RDEB. In turn, increased Cbl levels led to increased PDGFR ubiquitination, internalization, and degradation negatively affecting MAPK and AKT downstream signaling pathways. Thus, our results indicate that elevated TGFβ signaling leads to an attenuated response to growth factors, which contributes to impaired dermal wound healing in RDEB.

Linsitinib (OSI-906) modulates brain energy metabolism and seizure activity in the lithium-pilocarpine rat model

BackgroundEpileptic seizure is a process of energy accumulation, bursting, and depletion accompanied by the production, spread, and termination of epileptic discharges. The energy required for a seizure is mainly provided through mitochondrial production of ATP. Mitochondrial diseases often lead to epileptic seizures, and energy depletion caused by seizures can lead to mitochondrial dysfunction. The energy metabolism has become a key target for treatment of epileptic diseases.MethodThe effect of OSI-906, an insulin receptor (IR)/ insulin-like growth factor 1 receptor (IGF-1R) inhibitor, on behaviors and electroencephalographic activity in the lithium-pilocarpine rats were tested. 18F-FDG positron emission tomography (PET)/ computed tomography (CT) was performed to detect the relative whole-brain glucose uptake values. Electron microscopy was performed to observe the ultrastructure of neuronal and mitochondrial damage. The changes in blood glucose at different time points before and after the intervention were tested and the effects of OSI-906 on IR/IGF-1R and downstream Akt signaling in the context of seizures were evaluated.ResultsThe OSI-906 treatment applied 3 days before the pilocarpine-induced seizures significantly reduced the seizure severity, prolonged the seizure latency and decreased the EEG energy density. MicroPET/CT revealed that 50 mg/kg of OSI-906 inhibited the 18F-FDG glucose uptake after epileptic seizures, suggesting that OSI-906, through inhibiting IR/IGF-1R and the downstream AKT signaling, may regulate the excessive energy consumption of the epileptic brain. The OSI-906 treatment also reduced the mitochondrial damage caused by epileptic seizures.ConclusionThe IR/IGF-1R inhibitor OSI-906 can significantly reduce the sensitivity and severity of pilocarpine-induced seizures by inhibiting the IR/IGF-1R and the downstream Akt signaling pathway.

HBxAg promotes HBV replication and EGFR activation in human placental trophoblasts.

Hepatitis B virus (HBV) infection is a global epidemic. The main transmission route of chronic HBV infection is from mother to child, yet the mechanisms underlying HBV intrauterine infection remain unclear. In the present study, the effect and the mechanism underlying hepatitis B virus X antigen (HBxAg) on HBV replication and EGFR activation in trophoblasts was investigated. Serum samples from pregnant women with HBV infection were used to infect trophoblasts and HBxAg expression was detected using ELISA. HBV plasmids carrying either full length hepatitis B virus X (HBx) or HBx with a deletion mutation (ΔHBx) were transfected into trophoblasts and expression levels of HBV DNA, hepatitis B e-antigen and pregenomic (pg)RNA, and structural maintenance of chromosomes (Smc) 5/6 were assessed. The association between HBx and EGFR promoters was characterized using a luciferase reporter assay and EGFR/PI3K/phosphorylated (p)-AKT expression and apoptosis rate were also monitored. The results of the present study indicated that HBxAg expression increased with the increasing titre of HBV DNA (P<0.05). Compared with the wild-type group, the amount of HBV DNA in the supernatant and cells was significantly reduced (P<0.05) in the ΔHBx group and the intracellular HBeAg and pgRNA levels were also significantly decreased (P<0.05). In addition, Smc5/6 expression was also significantly decreased (P<0.05) when the intracellular HBx protein was expressed compared with mock-transfected cells. Co-transfection of HBx and EGFR promoter plasmids in JEG-3 and HTR-8 cells significantly elevated EGFR promoter driven luciferase expression relative to the control group (P<0.01). In EGFR overexpressing cells, the expression of PI3K/p-AKT was significantly increased, whereas the apoptosis rate was significantly decreased (P<0.05). These results were reversed in the EGFR-knockdown group. In conclusion, the present study demonstrated that HBx promotes HBV replication in trophoblasts via downregulation of Smc5/6, activates the EGFR promoter and inhibits trophoblast apoptosis via the PI3K/p-AKT downstream signalling pathway, thereby increasing the risk of HBV intrauterine infection.

Cigarette Smoke Containing Acrolein Upregulates EGFR Signaling Contributing to Oral Tumorigenesis In Vitro and In Vivo.

Simple SummaryOral squamous cell carcinoma (OSCC) is one of the most common smoking-related cancer types in the world. Better understanding of the pathophysiology of OSCC would lead to the development of novel therapeutic options. The epidermal growth factor receptor (EGFR) pathway plays a crucial role in the development of OSCC, and aberrant EGFR expression levels have been associated with smoking. Cigarette smoke contains large amounts of aldehydes such as acrolein, which is a highly reactive environmental toxin. In this study, our results present that acrolein is important in oncogenic transformation through activating the EGFR signaling pathway, contributing to oral carcinogenesis. To the best of our knowledge, this is the first study to provide molecular evidence, showing that cigarette smoke containing acrolein contributes to EGFR amplification and activation of downstream signaling in OSCC. Thus, acrolein might be a novel target for early detection and prevention of oral cancer in the future.Oral squamous cell carcinoma (OSCC) accounts for 80–90% of all intraoral malignant neoplasms. The single greatest risk factor for oral cancer is tobacco use, including cigarettes, cigars, chewing tobacco, and snuff. Aberrations of the epidermal growth factor receptor (EGFR) pathway features prominently in oral tumorigenesis and progression. It was shown that cigarette smoking (CS) is associated with worse prognosis in OSCC patients and overexpression of EGFR in tumor tissue. However, the mechanism by which cigarette smoking induced EGFR pathway activation remains to be fully elucidated. Acrolein, an IARC group 2A carcinogen, is a highly reactive aldehyde found in CS. Here we report that acrolein is capable of inducing tumorigenic transformation in normal human oral keratinocytes (NOK). The acrolein-transformed NOK cells showed EGFR copy number amplification, increased EGFR expression, and activation of downstream ERK and AKT signaling pathway. No p53 mutations were observed in acrolein-transformed NOK cells. Inhibiting EGFR pathway using an anti-EGFR antibody, cetuximab, inhibits tumor growth. Furthermore, by examining tissue sample from patients, we found an increased EGFR copy number was positively associated with acrolein-induced DNA damages in OSCC patients. Taken together, our results indicate that acrolein is important in tumorigenic transformation through amplification of EGFR and activating the downstream signaling pathway, contributing to oral carcinogenesis. This is the first study to provide molecular evidence showing that CS containing acrolein contributes to EGFR amplification in OSCC.

Ring-opening of five-membered heterocycles conjugated 4-isopropylresorcinol scaffold-based benzamides as HSP90 inhibitors suppressing tumor growth invitro and invivo.

A series of ring-opened dihydroxybenzamides have been designed and synthesized as heat shock protein 90 inhibitors. One of derivatives, compound 6b ((N-ethyl-2,4-dihydroxy-5-isopropyl-N-(pyridin-3-yl)benzamide)) demonstrated remarkable antiproliferative activity against in human KRAS mutant A549 and EGFR T790M mutant H1975 lung cancer cell lines with GI50 values of 0.07 and 0.05μM, respectively. It is also active against in other cancer cell lines, such as colorectal HCT116 (GI50=0.09μM), liver Hep3B (GI50=0.20μM) and breast MDA-MB-231 (GI50=0.09μM), and shows no evidence of toxicity in normal cell line. Compound 6b has an IC50 of 110.18nM in HSP90α inhibitory activity, slightly better than reference compound 1 (17-AAG, IC50=141.62nM) and achieves the degradation of multiple HSP90 client proteins in a dose- and time-dependent manner and downstream signaling of Akt in a concentration- and time-dependent manner in the human A549 lung cancer cell line. In the Boyden chamber assay, compound 6b can efficiently inhibit the migration of A549cells when compared to the reference compound 1. It also induce significant activity through the apoptotic pathway. Treatment with 6b showed no vision toxicity (IC50>10μM) on 661w photoreceptor cells as compared to AUY922 (3a) with a 0.04μM values of IC50 and has no effect in hERG test. In a bidirectional Caco-2 permeability assay, compound 6b was classified as a highly permeable compound which is not a substrate of efflux transporters. In a pharmacokinetic study in rats, 6b showed an F=17.8% of oral bioavailability. The effect of metabolic stability of compound 6b in human hepatocytes showed a T1/2 of 67.59min. Compound 6b (50mg/kg, po, daily) exhibits antitumor activity with a 72% TGD (tumor growth delay) in human A549 lung xenograft. The combination of 6b and afatinib, orally administered, showed tumor growth suppression with 67.5% of TGI in lung H1975 xenograft model. Thus compound 6b is a lead compound for further development of potential agents to treat lung cancer.

Macrophage migration inhibitory factor facilitates the therapeutic efficacy of mesenchymal stem cells derived exosomes in acute myocardial infarction through upregulating miR-133a-3p

BackgroundExosome transplantation is a promising cell-free therapeutic approach for the treatment of ischemic heart disease. The purpose of this study was to explore whether exosomes derived from Macrophage migration inhibitory factor (MIF) engineered umbilical cord MSCs (ucMSCs) exhibit superior cardioprotective effects in a rat model of AMI and reveal the mechanisms underlying it.ResultsExosomes isolated from ucMSCs (MSC-Exo), MIF engineered ucMSCs (MIF-Exo) and MIF downregulated ucMSCs (siMIF-Exo) were used to investigate cellular protective function in human umbilical vein endothelial cells (HUVECs) and H9C2 cardiomyocytes under hypoxia and serum deprivation (H/SD) and infarcted hearts in rats. Compared with MSC-Exo and siMIF-Exo, MIF-Exo significantly enhanced proliferation, migration, and angiogenesis of HUVECs and inhibited H9C2 cardiomyocyte apoptosis under H/SD in vitro. MIF-Exo also significantly inhibited cardiomyocyte apoptosis, reduced fibrotic area, and improved cardiac function as measured by echocardiography in infarcted rats in vivo. Exosomal miRNAs sequencing and qRT-PCR confirmed miRNA-133a-3p significantly increased in MIF-Exo. The biological effects of HUVECs and H9C2 cardiomyocytes were attenuated with incubation of MIF-Exo and miR-133a-3p inhibitors. These effects were accentuated with incubation of siMIF-Exo and miR-133a-3p mimics that increased the phosphorylation of AKT protein in these cells.ConclusionMIF-Exo can provide cardioprotective effects by promoting angiogenesis, inhibiting apoptosis, reducing fibrosis, and preserving heart function in vitro and in vivo. The mechanism in the biological activities of MIF-Exo involves miR-133a-3p and the downstream AKT signaling pathway.