Akt In Response Research Articles

Cdon ablation in motor neurons causes age-related motor neuron degeneration and impaired sciatic nerve repair.

The functional deterioration and loss of motor neurons are tightly associated with degenerative motor neuron diseases and aging-related muscle wasting. Motor neuron diseases or aging-related muscle wasting in turn contribute to increased risk of adverse health outcomes in the elderly. Cdon (cell adhesion molecule-downregulated oncogene) belongs to the immunoglobulin superfamily of cell adhesion molecule and plays essential roles in multiple signalling pathways, including sonic hedgehog (Shh), netrin, and cadherin-mediated signalling. Cdon as a Shh coreceptor plays a critical role in motor neuron specification during embryonic development. However, its role in adult motor neuron function is unknown. Hb9-Cre recombinase-driven motor neuron-specific Cdon deficient mice (mnKO) and a compound mutant mice (mnKO::SOD1G93A ) were generated to investigate the role of Cdon in motor neuron degeneration. Motor neuron regeneration was examined by using a sciatic nerve crush injury model. To investigate the phenotype, physical activity, compound muscle action potential, immunostaining, and transmission electron microscopy were carried out. In the mechanism study, RNA sequencing and RNA/protein analyses were employed. Mice lacking Cdon in motor neurons exhibited middle age onset lethality and aging-related decline in motor function. In the sciatic nerve crush injury model, mnKO mice exhibited an impairment in motor function recovery evident by prolonged compound muscle action potential duration (4.63±0.35 vs. 3.93±0.22s for f/f, P<0.01) and physical activity. Consistently, neuromuscular junctions of mnKO muscles were incompletely occupied (49.79±5.74 vs. 79.39±3.77% fully occupied neuromuscular junctions for f/f, P<0.0001), suggesting an impaired reinnervation. The transmission electron microscopy analysis revealed that mnKO sciatic nerves had smaller axon diameter (0.88±0.13 vs. 1.43±0.48μm for f/f, P<0.0001) and myelination defects. RNA sequencing of mnKO lumbar spinal cords showed alteration in genes related to neurogenesis, inflammation and cell death. Among the altered genes, ErbB4 and FgfR expressions were significantly altered in mnKO as well as in Cdon-depleted NSC34 motor neuron cells. Consistently, Cdon-depleted NSC34 cells exhibited elevated levels of cleaved Caspase3 and γH2AX proteins, as well as Bax transcription. Cdon-depleted NSC34 cells also exhibited impaired activation of Akt in response to neuregulin-1 (NRG1) treatment. Our current data demonstrate the functional importance of Cdon in motor neuron function and nerve repair. Cdon ablation causes alterations in neurotrophin signalling that leads to motor neuron degeneration.

Kidins220 sets the threshold for survival of neural stem cells and progenitors to sustain adult neurogenesis

In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. Current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects accompanied by increased neuroblast death and high loss of newly formed neurons in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. We also show that NSCs with Kidins220 can survive with lower concentrations of EGF than the ones lacking this molecule. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs growth and expansion. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair.

MicroR-9c-5p and novel-mir50 co-target Akt to regulate Lasioderma serricorne reproduction.

High fecundity is a common characteristic of insect pests which increases the difficulty of population control. Serine/threonine kinase Akt is an indispensable component of the insulin signaling pathway. Silencing of LsAkt severely hinders reproduction in Lasioderma serricorne, a stored product insect pest. However, the post-transcriptional pathway of LsAkt in L. serricorne remains unknown. This study identified 2 binding sites of miR-9c-5p and novel-mir50 in the coding sequences of LsAkt. The expression profiles of 2 microRNAs (miRNAs) and LsAkt displayed an opposite pattern during the adult stages. Luciferase reporter assay showed that novel-mir50 and miR-9c-5p could downregulate the expression of LsAkt. Overexpression of miR-9c-5p and novel-mir50 by injection of mimics inhibited the expression of LsAkt and reduced oviposition, decreased egg hatchability, and blocked ovarian development. It also decreased the expression of genes involved in ovarian development (LsVg and LsVgR) and the nutritional signaling pathway (LsTOR, LsS6K, and Ls4EBP), and reduced the phosphorylation of Akt. Conversely, injection of miR-9c-5p and novel-mir50 inhibitors induced the expressions of LsAkt, LsVg, LsVgR, LsTOR, LsS6K, and Ls4EBP, enhanced Akt phosphorylation level, and accelerated ovarian development. Injection of bovine insulin downregulated the expression of miR-9c-5p and novel-mir50 and upregulated the LsAkt expression. It also rescued the reproductive development defects associated with miR-9c-5p/novel-mir50 overexpression, forming a positive regulatory loop of insulin signaling. These results indicate that miR-9c-5p/novel-mir50 regulates the female reproduction of L. serricorne by targeting Akt in response to insulin signaling. The data also demonstrate the effects of the insulin/miRNA/Akt regulatory axis in insect reproduction.

Chronic Intermittent Hypoxia during Sleep Causes Browning of Interscapular Adipose Tissue Accompanied by Local Insulin Resistance in Mice

Obstructive sleep apnea (OSA) is a highly prevalent condition, characterized by intermittent hypoxia (IH), sleep disruption, and altered autonomic nervous system function. OSA has been independently associated with dyslipidemia, insulin resistance, and metabolic syndrome. Brown adipose tissue (BAT) has been suggested as a modulator of systemic glucose tolerance through adaptive thermogenesis. Reductions in BAT mass have been associated with obesity and metabolic syndrome. No studies have systematically characterized the effects of chronic IH on BAT. Thus, we aimed to delineate IH effects on BAT and concomitant metabolic changes. C57BL/6J 8-week-old male mice were randomly assigned to IH during sleep (alternating 90 s cycles of 6.5% FIO2 followed by 21% FIO2) or normoxia (room air, RA) for 10 weeks. Mice were subjected to glucose tolerance testing and 18F-FDG PET-MRI towards the end of the exposures followed by BAT tissues analyses for morphological and global transcriptomic changes. Animals exposed to IH were glucose intolerant despite lower total body weight and adiposity. BAT tissues in IH-exposed mice demonstrated characteristic changes associated with "browning"-smaller lipids, increased vascularity, and a trend towards higher protein levels of UCP1. Conversely, mitochondrial DNA content and protein levels of respiratory chain complex III were reduced. Pro-inflammatory macrophages were more abundant in IH-exposed BAT. Transcriptomic analysis revealed increases in fatty acid oxidation and oxidative stress pathways in IH-exposed BAT, along with a reduction in pathways related to myogenesis, hypoxia, and IL-4 anti-inflammatory response. Functionally, IH-exposed BAT demonstrated reduced absorption of glucose on PET scans and reduced phosphorylation of AKT in response to insulin. Current studies provide initial evidence for the presence of a maladaptive response of interscapular BAT in response to chronic IH mimicking OSA, resulting in a paradoxical divergence, namely, BAT browning but tissue-specific and systemic insulin resistance. We postulate that oxidative stress, mitochondrial dysfunction, and inflammation may underlie these dichotomous outcomes in BAT.

Consequences of in vitro benzyl butyl phthalate exposure for blubber gene expression and insulin-induced Akt activation in juvenile grey seals

Plastic and plasticiser pollution of marine environments is a growing concern. Although phthalates, one group of plasticisers, are rapidly metabolised by mammals, they are found ubiquitously in humans and have been linked with metabolic disorders and altered adipose function. Phthalates may also present a threat to marine mammals, which need to rapidly accumulate and mobilise their large fat depots. High molecular weight (HMW) phthalates may be most problematic because they can accumulate in adipose. We used blubber explants from juvenile grey seals to examine the effects of overnight exposure to the HMW, adipogenic phthalate, benzyl butyl phthalate (BBzP) on expression of key adipose-specific genes and on phosphorylation of Akt in response to insulin. We found substantial differences in transcript abundance of Pparγ, Insig2, Fasn, Scd, Adipoq and Lep between moult stages, when animals were also experiencing differing mass changes, and between tissue depths, which likely reflect differences in blubber function. Akt abundance was higher in inner compared to outer blubber, consistent with greater metabolic activity in adipose closer to muscle than skin, and its phosphorylation was stimulated by insulin. Transcript abundance of Pparγ and Fasn (and Adipoq in some animals) were increased by short term (30 min) insulin exposure. In addition, overnight in vitro BBzP exposure altered insulin-induced changes in Pparγ (and Adipoq in some animals) transcript abundance, in a tissue depth and moult stage-specific manner. Basal or insulin-induced Akt phosphorylation was not changed. BBzP thus acted rapidly on the transcript abundance of key adipose genes in an Akt-independent manner. Our data suggest phthalate exposure could alter seal blubber development or function, although the whole animal consequences of these changes are not yet understood. Knowledge of typical phthalate exposures and toxicokinetics would help to contextualise these findings in terms of phthalate-induced metabolic disruption risk and consequences for marine mammal health.

Abstract 5410: First, second, and third generation mTOR pathways inhibitors for treatment of glioblastoma

Abstract The Mechanistic Target of Rapamycin (mTOR), which functions in two distinct multiprotein complexes, mTORC1 and mTORC2, is a downstream signaling kinase in the PI3K/AKT pathway. mTOR is often deregulated in glioblastoma (GB), the most aggressive primary brain tumor in adults, due to the frequent loss of the tumor suppressor, PTEN. mTORC1 and mTORC2 control cell growth, proliferation, migration, survival, and stem cell regulation in response to nutrients and growth factors. Nutrient-sensitive mTORC1 regulates protein synthesis through the downstream substrate, 4EBP1 (also called EIF4EBP1), p70S6K1, and p70S6K2. On the other hand, growth factor-sensitive mTORC2 is associated with Rapamycin-insensitivity. The presence of stem cells within the tumor mass and the surrounding areas precludes the possibility of the elimination of GB. Furthermore, the substrate of activated AKT, the proline-rich AKT substrate of 40 kDa (PRAS40), is a surrogate marker of activated AKT in response to mTOR inhibition therapy in GB patients that regulates mTORC1. The aim of this study is to evaluate the efficacy of first (Rapamycin (RAPA)), second (Torin1, Torin2, and XL388), and third (Rapalink-1) mTOR inhibitors to suppress cell growth, dissemination, overcome drug resistance of GB cells, and alter self-renewal properties of GB stem cells. Results demonstrated: a significant number of tumors showed an increased expression of pAKTSer473 and pmTORSer2448, suggesting an overactivation of the mTOR pathway; co-expression of mTOR and stem cell marker, nestin, was evident in GB tumors, implying its role in stem cell regulation; Torin2, but not Torin1 or XL388, suppressed mTORC1 activity completely as shown by the reduced expression of its pS6KSer235/236 in a dose-dependent manner; Torin1, Torin2, and XL388 inhibited the phosphorylation of 4E-BP1 and PRAS40; Torin1, Torin2, and XL388 suppressed dephosphorylation of pAKTSer473; functional analysis revealed that RAPA, Torin1, Torin2, XL388, and Rapalink-1 effectively suppressed proliferation and S-phase entry; RAPA, Torin1, Torin2 and Rapalink-1 inhibited GB cell migration, while XL388 was ineffective; RAPA, Torin1, Torin2, and Rapalink-1 suppressed the self-renewal of GB stem cells and inhibited their proliferation, XL388 remained ineffective; drug resistance analysis revealed that Torin2 was most effective in eradicating GB tumor cells. These results suggested that RAPA, Torin1, Torin2, and Rapalink-1, but not XL388, are useful in suppressing the mTORC1 and mTORC2 activities, thereby inhibiting GB cell proliferation, dissemination, and inhibiting stem cell self-renewal. Therefore, the use of these mTOR inhibitors presents a promising treatment strategy for Glioblastoma. Citation Format: Jose F. Dominguez, Haylen Rosberger, Paul Garell, Chirag D. Gandhi, Meena Jhanwar-Uniyal. First, second, and third generation mTOR pathways inhibitors for treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5410.

DNA-PK promotes activation of the survival kinase AKT in response to DNA damage through an mTORC2-ECT2 pathway.

The kinase AKT (also known as protein kinase B) is a key regulator of cell proliferation, survival, and metabolism. In addition to being activated by growth factors, AKT is activated in response to DNA damage. Here, we found that the DNA damage response kinase DNA-PK sustains cell survival through a phosphorylation event that leads to increased AKT activity. In various cancer and noncancer cells in culture, DNA damage caused by ionizing radiation or topoisomerase inhibitors triggered DNA-PK–dependent phosphorylation of the mTOR complex 2 (mTORC2) subunit Sin1, which enabled its interaction with the guanine nucleotide exchange factor ECT2. Depleting Sin1 or ECT2 or disrupting the protein interaction or catalytic function of ECT2 attenuated DNA damage–induced AKT activation, thereby enhancing cellular sensitivity to DNA-damaging agents. Our findings elucidate a mechanism mediating DNA damage–induced AKT activation and cell survival.

The regulatory subunits of PI3K, p85α and p85β, differentially affect BRD7-mediated regulation of insulin signaling.

Bromodomain-containing protein 7 (BRD7) has been shown to interact with the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85, in the insulin signaling pathway. Here, we show that upregulation of hepatic BRD7 improves glucose homeostasis even in the absence of either p85 isoform, p85α or p85β. However, BRD7 leads to differential activation of downstream effector proteins in the insulin signaling pathway depending on which isoform of p85 is present. In the presence of only p85α, BRD7 overexpression increases phosphorylation of insulin receptor (IR) upon insulin stimulation, without increasing the recruitment of p85 to IR substrate. Overexpression of BRD7 also increases activation of Akt in response to insulin, but does not affect basal phosphorylation levels of Akt. Meanwhile, the phosphorylation of glycogen synthase kinase 3β (GSK3β) is increased by overexpression of BRD7. On the other hand, in the presence of only p85β, BRD7 overexpression does not affect phosphorylation levels of IR, and Akt phosphorylation is not affected by insulin stimulation following BRD7 upregulation. However, BRD7 overexpression leads to increased basal phosphorylation levels of Akt and GSK3β. These data demonstrate that BRD7’s action on glucose homeostasis does not require the presence of both p85 isoforms, and p85α and p85β have unique roles in insulin signaling in the liver.

Therapeutic Suppression of FAK-AKT Signaling Overcomes Resistance to SHP2 Inhibition in Colorectal Carcinoma.

SHP2 mediates signaling from multiple receptor tyrosine kinases (RTKs) to extracellular signal-regulated kinase (ERK) and Ser and Thr kinase AKT, and its inhibitors offer an unprecedented opportunity for cancer treatment. Although the ERK signaling variation after SHP2 inhibition has been well investigated, the AKT signaling variation in colorectal carcinoma (CRC) is still unknown. Therefore, we performed immunohistochemistry and bioinformatics analyses to explore the significance of p-SHP2 in CRC. A panel of CRC cell lines with the SHP2 inhibitor, SHP099, was used to assess the effects on viability and signaling. The inhibitors of AKT and focal adhesion kinase (FAK) signaling were examined in combination with SHP099 as potential strategies to enhance the efficacy and overcome resistance. Frequent resistance to the SHP2 inhibitor was observed in CRC cells, even in those without RAS mutations. We observed rapid adaptive reactivation of the AKT pathway in response to SHP2 inhibition, possibly driven by the reactivation of RTKs or released p-FAK. High baseline p-FAK may also be associated with CRC cell resistance to SHP2 inhibition. Co-inhibition of FAK abrogated the feedback reactivation of AKT in response to SHP2 inhibition. Moreover, the combined inhibition of SHP2 with AKT or FAK resulted in sustained AKT pathway suppression and improved antitumor efficacy in vitro and in vivo. Our study found that reactivation of the AKT pathway is a key mechanism of adaptive resistance to SHP2 inhibition, highlighting the potential significance of AKT and FAK inhibition strategies to enhance the efficacy of SHP2 inhibitors in CRC treatment.

ELMO1 Regulates RANKL-Stimulated Differentiation and Bone Resorption of Osteoclasts.

Bone homeostasis is a metabolic balance between the new bone formation by osteoblasts and old bone resorption by osteoclasts. Excessive osteoclastic bone resorption results in low bone mass, which is the major cause of bone diseases such as rheumatoid arthritis. Small GTPases Rac1 is a key regulator of osteoclast differentiation, but its exact mechanism is not fully understood. ELMO and DOCK proteins form complexes that function as guanine nucleotide exchange factors for Rac activation. Here, we report that ELMO1 plays an important role in differentiation and bone resorption of osteoclasts. Osteoclast precursors derived from bone marrow monocytes (BMMs) of Elmo1–/– mice display defective adhesion and migration during differentiation. The cells also have a reduced activation of Rac1, p38, JNK, and AKT in response to RANKL stimulation. Importantly, we show that bone erosion is alleviated in Elmo1–/– mice in a rheumatoid arthritis mouse model. Taken together, our results suggest that ELMO1, as a regulator of Rac1, regulates osteoclast differentiation and bone resorption both in vitro and in vivo.

The innate immune kinase TBK1 directly increases mTORC2 activity and downstream signaling to Akt

TBK1 responds to microbes to initiate cellular responses critical for host innate immune defense. We found previously that TBK1 phosphorylates mTOR (mechanistic target of rapamycin) on S2159 to increase mTOR complex 1 (mTORC1) signaling in response to the growth factor EGF and the viral dsRNA mimetic poly(I:C). mTORC1 and the less well studied mTORC2 respond to diverse cues to control cellular metabolism, proliferation, and survival. Although TBK1 has been linked to Akt phosphorylation, a direct relationship between TBK1 and mTORC2, an Akt kinase, has not been described. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an Mtor S2159A knock-in allele (MtorA/A) using in vitro kinase assays and cell-based approaches, we demonstrate here that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation. We find that TBK1 and mTOR S2159 phosphorylation promotes mTOR-dependent phosphorylation of Akt in response to several growth factors and poly(I:C). Mechanistically, TBK1 coimmunoprecipitates with mTORC2 and phosphorylates mTOR S2159 within mTORC2 in cells. Kinase assays demonstrate that TBK1 and mTOR S2159 phosphorylation increase mTORC2 intrinsic catalytic activity. Growth factors failed to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal cross talk between TBK1 and mTOR, key regulatory nodes within two major signaling networks. As TBK1 and mTOR contribute to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings.

206-OR: RAGE-Dependent Macrophage Activation Aggravates Retrograde Axonal Transport via Attenuated Insulin Signaling of Peripheral Nerves in Experimental Diabetic Polyneuropathy

Advanced glycation end products (AGEs) activates its receptor, RAGE, resulting in a skewing of macrophage (Mϕ) polarity toward inflammatory phenotype (M1) and an impairment of the insulin signaling (IS) in adipose tissue. IS also regulates axonal transport (AT) in the peripheral nervous system. Although impaired IS and AT are assumed to be pathophysiology of diabetic polyneuropathy (DPN), the implication of Mϕ induced inflammation with RAGE activation in those factors is not fully understood. To this end, we generated bone marrow specific RAGE deficient mice to investigate the influence of RAGE signaling in Mϕ on the development of DPN. Bone marrow cells from C57BL/6 mice (W) or RAGE deficient mice (R) were transplanted into irradiated W (BMW and BMR). After 8 weeks of diabetes (D) induced by streptozotocin, nerve conduction velocities were delayed in BMWD compared to BMW, while those of BMRD were comparable to BMR. An increased infiltration of M1 was observed in the sciatic nerve (SN) in BMWD. Western blotting (WB) showed a lower phosphorylation of AKT in response to insulin in SN of BMWD, which suggested impaired IS, while it was maintained in BMRD. Retrograde AT (RAT) was evaluated with Fluoro-gold (FG) transportation from hindlimb-footpad to the dorsal root ganglion (DRG). Immunofluorescence revealed a lower positivity of FG in DRG of BMWD which suggested a deficit of RAT, while it was maintained in BMRD. Neuronal cells isolated from mice DRG were cocultured with a Mϕ cell line RAW264, which was polarized into M1 by AGEs. Intra-axonal vesicles were labeled fluorescently. Subsequently, AT was visualized by time-lapse imaging. The ratio and the velocity of RAT was decreased in the axon of the neurons cocultured with M1 compared with naïve Mϕ. In summary, our data suggested that the activation of RAGE signaling skewed Mϕ polarity toward M1, impairing IS and RAT through inflammation, which might trigger and develop DPN. Disclosure S. Osonoi: None. H. Mizukami: None. K. Kudo: None. Y. Yamamoto: None. Funding Japan Society for the Promotion of Science (18K16220)

TIR-Domain-Containing Adaptor-Inducing Interferon-β (TRIF) Is Involved in Glucose Metabolism in Adipose Tissue through the Insulin/AKT Signaling Pathway.

Obesity significantly increases the risk of developing type 2 diabetes mellitus and other metabolic diseases. Obesity is associated with chronic low-grade inflammation in white adipose tissues, which is thought to play an essential role in developing insulin resistance. Many lines of evidence indicate that toll-like receptors (TLRs) and their downstream signaling pathways are involved in development of chronic low-grade inflammation and insulin resistance, which are associated with obesity. Mice lacking molecules positively involved in the TLR signaling pathways are generally protected from high-fat diet-induced inflammation and insulin resistance. In this study, we have determined the effects of genetic deficiency of toll/interleukin-1 receptor-domain-containing adaptor-inducing interferon-β (TRIF) on food intake, bodyweight, glucose metabolism, adipose tissue macrophage polarization, and insulin signaling in normal chow diet-fed mice to investigate the role of the TRIF-dependent TLR signaling in adipose tissue metabolism and inflammation. TRIF deficiency (TRIF−/−) increased food intake and bodyweight. The significant increase in bodyweight in TRIF−/− mice was discernible as early as 24 weeks of age and sustained thereafter. TRIF−/− mice showed impaired glucose tolerance in glucose tolerance tests, but their insulin tolerance tests were similar to those in TRIF+/+ mice. Although no difference was found in the epididymal adipose mass between the two groups, the percentage of CD206+ M2 macrophages in epididymal adipose tissue decreased in TRIF−/− mice compared with those in TRIF+/+ mice. Furthermore, activation of epididymal adipose AKT in response to insulin stimulation was remarkably diminished in TRIF−/− mice compared with TRIF+/+ mice. Our results indicate that the TRIF-dependent TLR signaling contributes to maintaining insulin/AKT signaling and M2 macrophages in epididymal adipose tissue under a normal chow diet and provide new evidence that TLR4-targeted therapies for type 2 diabetes require caution.

RETRACTED: G-5555 synergized miR-485-5p to alleviate cisplatin resistance in ovarian cancer cells via Pi3k/Akt signaling pathway

The present study was meant for the discovery of the underlying functions of miR-485-5p in ovarian cancer concerning cisplatin resistance in vitro. RT-qPCR assessed the miR-485-5p expression in ovarian cancer cell lines, normal cells and cisplatin-resistant Cell line OVCA433-CR. After OVCA433-CR treated with 0,3,5umol/L cisplatin, miR-485-5p expressions were determined. MTT observed the cell cytotoxicity in OVCA433-CR after regulation of miR-485-5p. Targets can predicted the putative binding between miR-485-5p and PAK1 and Luciferase Assay verified this. RT-qPCR decided the inhibitory effect in between. MTT tested the cytotoxicity in different combinations of miR-485-5p and PAK1. Western Blot tested the phosphorylation of Pi3k and Akt in response to miR-485-5p and PAK1 interplay. We evaluated the role of Pi3k/Akt signaling in regulation of miR-485-5p and cisplatin resistance with Wortmannin. miR-485-5p was lower expressed in ovarian cancer cells than normal ones and even lower in OVCA433-CR than OVCA433. As the cisplatin concerntration increased, miR-485-5p decreased. miR-485-5p mimics induced lower cisplatin resistance while miR-485-5p inhibitor caused higher resistance. PAK1 targeted miR-485-5p and inhibited miR-485-5p. PAK1 inhibitor helped to lower the resistance to cisplatin caused by miR-485-5p upregulation. miR-485-5p mimics silenced Pi3k/Akt signaling and PAK1 inhibitor aggravated the silencing. Inhibition of Pi3k/Akt signaling increased miR-485-5p, thereby decreasing the cisplatin-resistance in OVCA433-CR. miR-485-5p decreased cisplatin resistance in ovarian cancer cells via Pi3k/Akt signaling, suggesting that miR-485-5p upregulation might alleviate the cisplatin resistance in ovarian patients.

Effects of Lycopene on Growth Factor Response in Prostate Cancer Cells

Dietary and nutritional factors are considered critical modulating factors in prostate cancer development. Epidemiological data have suggested to the idea that dietary consumption of lycopene, found in tomatoes, may prevent prostate cancer. This study examined the mechanism by which lycopene interacts with prostate cancer cells, with the goal of discovering less toxic treatment and prevention options. The hypothesis that was addressed was that lycopene interferes with growth factor‐mediated signal transduction in prostate cancer cells. The effects of lycopene on PC‐3, a human prostate cancer cell line, were analyzed using proliferation assays, immunoblot analysis, and confocal microscopy. The growth factors used were lysophosphatidic acid (LPA) and epidermal growth factor (EGF). Lycopene (10 μM) inhibited LPA and EGF‐induced proliferation of PC‐3 cells, confirming previous unpublished results. Lycopene also inhibited activation of Akt in response to LPA and EGF as assessed by immunoblotting. Confocal immunofluorescence microscopy showed that the decrease in activated Akt was most prominent in the cell nucleus. These results confirm the ability of lycopene to inhibit growth factor response in human prostate cancer cells, and suggest new directions for future studies.Support or Funding InformationSupported by the ASPET Summer Undergraduate Research Program, College of Pharmacy and Pharmaceutical Sciences, Sue Harriet Monroe Mullen Innovative Cancer Prevention Research Fund, and the ASPET David Lehr Award.

Cx43 phosphorylation–mediated effects on ERK and Akt protect against ischemia reperfusion injury and alter the stability of the stress-inducible protein NDRG1

Gap junctions contain intercellular channels that enable intercellular communication of small molecules while also serving as a signaling scaffold. Connexins, the proteins that form gap junctions in vertebrates, are highly regulated and typically have short (<2 h) half-lives. Connexin43 (Cx43), the predominate connexin in the myocardium and epithelial tissues, is phosphorylated on more than a dozen serine residues and interacts with a variety of protein kinases. These interactions regulate Cx43 and gap junction formation and stability. Casein kinase 1 (CK1)-mediated phosphorylation of Cx43 promotes gap junction assembly. Using murine knock-in technology and quantitative PCR, immunoblotting, and immunoprecipitation assays, we show here that mutation of the CK1 phosphorylation sites in Cx43 reduces the levels of total Cx43 in the myocardium and increases Cx43 phosphorylation on sites phosphorylated by extracellular signal-regulated kinase (ERK). In aged myocardium, we found that, compared with WT Cx43, mutant Cx43 expression increases ERK activation, phosphorylation of Akt substrates, and protection from ischemia-induced injury. Our findings also uncovered that Cx43 interacts with the hypoxia-inducible protein N-Myc downstream-regulated gene 1 protein (NDRG1) and that Cx43 phosphorylation status controls this interaction and dramatically affects NDRG1 stability. We propose that, in addition to altering gap junction stability, Cx43 phosphorylation directly and dynamically regulates cellular signaling through ERK and Akt in response to ischemic injury. We conclude that gap junction-dependent NDRG1 regulation might explain some cellular responses to hypoxia.

Cell surface heparan sulfate proteoglycans are involved in the extracellular Hsp90-stimulated migration and invasion of cancer cells.

The extracellular heat shock protein 90 (Hsp90) is known to participate in cell migration and invasion. Recently, we have shown that cell surface heparan sulfate proteoglycans (HSPGs) are involved in the binding and anchoring of extracellular Hsp90 to the plasma membrane, but the biological relevance of this finding was unclear. Here, we demonstrated that the digestion of heparan sulfate (HS) moieties of HSPGs with a heparinase I/III blend and the metabolic inhibition of the sulfation of HS chains by sodium chlorate considerably impair the migration and invasion of human glioblastoma A-172 and fibrosarcoma HT1080 cells stimulated by extracellular native Hsp90. Heparin, a polysaccharide closely related to HS, also reduced the Hsp90-stimulated migration and invasion of cells. Phorbol 12-myristate 13-acetate, an intracellular inducer of cell motility bypassing the ligand activation of receptors, restored the basal migration of heparinase- and chlorate-treated cells almost to the control level, suggesting that the cell motility machinery was insignificantly affected in cells with degraded and undersulfated HS chains. On the other hand, the downstream phosphorylation of AKT in response to extracellular Hsp90 was substantially impaired in heparinase- and chlorate-treated cells as compared to untreated cells. Taken together, our results demonstrated for the first time that cell surface HSPGs play an important role in the migration and invasion of cancer cells stimulated by extracellular Hsp90 and that plasma membrane-associated HSPGs are required for the efficient transmission of signal from extracellular Hsp90 into the cell.

Dorsal ruffles enhance activation of Akt by growth factors.

In fibroblasts, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) stimulate the formation of actin-rich, circular dorsal ruffles (CDRs) and phosphatidylinositol 3-kinase (PI3K)-dependent phosphorylation of Akt. To test the hypothesis that CDRs increase synthesis of phosphorylated Akt1 (pAkt), we analyzed the contributions of CDRs to Akt phosphorylation in response to PDGF and EGF. CDRs appeared within several minutes of growth factor addition, coincident with a peak of pAkt. Microtubule depolymerization with nocodazole blocked CDR formation and inhibited phosphorylation of Akt in response to EGF but not PDGF. Quantitative immunofluorescence showed increased concentrations of Akt, pAkt and phosphatidylinositol (3,4,5)-trisphosphate (PIP3), the phosphoinositide product of PI3K that activates Akt, concentrated in CDRs and ruffles. EGF stimulated lower maximal levels of pAkt than did PDGF, which suggests that Akt phosphorylation requires amplification in CDRs only when PI3K activities are low. Accordingly, stimulation with low concentrations of PDGF elicited lower levels of Akt phosphorylation, which, like responses to EGF, were inhibited by nocodazole. These results indicate that when receptor signaling generates low levels of PI3K activity, CDRs facilitate local amplification of PI3K and phosphorylation of Akt.This article has an associated First Person interview with the first author of the paper.

A TFEB nuclear export signal integrates amino acid supply and glucose availability

How cells coordinate the response to fluctuating carbon and nitrogen availability required to maintain effective homeostasis is a key issue. Amino acid limitation that inactivates mTORC1 promotes de-phosphorylation and nuclear translocation of Transcription Factor EB (TFEB), a key transcriptional regulator of lysosome biogenesis and autophagy that is deregulated in cancer and neurodegeneration. Beyond its cytoplasmic sequestration, how TFEB phosphorylation regulates its nuclear-cytoplasmic shuttling, and whether TFEB can coordinate amino acid supply with glucose availability is poorly understood. Here we show that TFEB phosphorylation on S142 primes for GSK3β phosphorylation on S138, and that phosphorylation of both sites but not either alone activates a previously unrecognized nuclear export signal (NES). Importantly, GSK3β is inactivated by AKT in response to mTORC2 signaling triggered by glucose limitation. Remarkably therefore, the TFEB NES integrates carbon (glucose) and nitrogen (amino acid) availability by controlling TFEB flux through a nuclear import-export cycle.

Abstract 4498: Lysyl oxidase like-2 mediates tumor to stromal cell communication in oral cancer

Abstract Introduction: The lysyl oxidase family consists of 5 members and oxidizes specific lysine residues in biosynthetic collagen and elastin maturation. Lysyl oxidase like-2 (LOXL2) is elevated in oral cancer and promotes metastasis and correlates with poor prognosis. The objective of this study is to determine the mechanism by which LOXL2 promotes the progression and invasiveness of oral squamous cell carcinoma. Methods: In vitro: To investigate functions of LOXL2 in oral cancer, the effects of LOXL2 inhibitor (PXS-S1C) on human gingival fibroblasts treated with tumor cell conditioned medium (CM) were investigated. Cell proliferation assays, signaling arrays and western blots were used to evaluate the effect of PXS-S1C on CM-treated fibroblasts. The effect of PXS-S1C on cancer cells expression of LOXL2 and proliferation was determined. To find potential LOXL2 substrates in gingival fibroblasts treated with CM +/- PXS-S1C, carbonyl-containing proteins were purified by affinity chromatography and identified by MS and western blot. In vivo: The effect of PXS-S1C on cancer growth and metastasis in vivo was investigated using an orthotopic oral tongue cancer mouse model in both immunodeficient and immunocompetent mice. PXS-S1C at 10 mg/kg and 30 mg/kg was injected immediately following tumor cell injections. Tumors were monitored by caliper measurements, and by in vivo imaging (IVIS). The mice were sacrificed and their organs were subjected to immunohistochemistry staining for proliferation markers. Results: PXS-S1C significantly inhibited gingival fibroblast proliferation triggered by tumor cell CM and attenuated phosphorylation of PDGFRβ Y771 and P-PDGFRβ Y857, but not PDGFRβ Y751 in response to CM. PXS-S1C inhibited ERK1/2-signaling in fibroblasts and not AKT in response to CM. PDGFR activation by oral tumor cells was mimicked by PDGF-AB, but not PDGF-BB. PXS-S1C decreased the expression of LOXL2 by 5-fold in HSC3 oral cancer cells in vitro, suggesting a positive autoregulatory loop. Assessing for direct LOXL2 substrates in fibroblasts with functional consequences identified both PDGFR and integrin αV. Caliper measurements, IVIS, and immunohistochemistry demonstrated that inhibition of LOXL2 significantly decreases oral cancer progression and metastasis in vivo. Mice without PXS-S1C treatment developed larger tongue volumes (p&amp;lt;0.05), and more mice developed larger lymph nodes (3 out 12) compared to the PXS-S1C-treated mice (1 out of 12). IVIS imaging revealed inhibition of metastasis by PXS-S1C treatment. The expression of proliferation marker (Ki-67) and LOXL2 was lower in tongue tumors treated with PXS-S1C (p&amp;lt;0.05). Conclusions: LOXL2 secreted by cancer cells stimulates fibroblasts by enhancing PDGFR signaling and proliferation, and by stimulating αV integrin function and cell adhesion. Inhibition of LOXL2 could provide therapeutic strategies to address oral cancer. Citation Format: Faranak Mahjour, Philip C. Trackman. Lysyl oxidase like-2 mediates tumor to stromal cell communication in oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4498.