S6 Protein Research Articles

P.1.g.045 - Effect of individual Valeriana officinalis and Melissa officinalis extracts and a combination on learning, memory and locomotor activity of rats

Polysaccharides derived from Echinacea have historically been shown to be immunostimulatory. We describe in this work however the anti-inflammatory effect of a water extract of Echinacea purpurea roots (EPRW) that inhibited Pam3Csk4 stimulated production of TNFα by human monocytic THP-1 cells. The polyphenols and alkylamides typically found in Echinacea extracts were absent in EPRW suggesting that the anti-inflammatory component(s) was a polysaccharide. This anti-inflammatory activity was shown to be mediated by the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway as chemical inhibition of PI3K abolished the EPRW anti-inflammatory effect. Demonstration of phosphorylation of Akt and ribosomal S6 proteins, downstream targets of PI3K confirmed EPRW-mediated activation of this pathway. In conclusion, this observation suggests that non-alkylamide/non-polyphenolic phytochemicals from Echinacea may contribute in part to some of the anti-inflammatory therapeutic effects such as reduced severity of symptoms that have been observed in vivo in the treatment of upper respiratory tract infections with Echinacea.

Growth inhibitory and apoptosis-inducing effects of allergen-free Rhus verniciflua Stokes extract on A549 human lung cancer cells

Evidence suggests that Rhusverniciflua Stokes (RVS) or its extract has the potential to be used for the treatment of inflammatory and neoplastic diseases. However, direct use of RVS or its extract as a herbal medicine has been limited due to the presence of urushiol, an allergenic toxin. In the present study, we prepared an extract of the allergen‑removed RVS (aRVS) based on a traditional method and investigated its inhibitory effect on the growth of various types of human cancer cells, including lung (A549), breast (MCF-7) and prostate (DU-145) cancer cell lines. Notably, among the cell lines tested, treatment with the aRVS extract strongly inhibited proliferation of the A549 cells at a 0.5mg/ml concentration for 24h that was not cytotoxic to normal human dermal fibroblasts. Furthermore, aRVS extract treatment largely reduced the survival and induced apoptosis of the A549 cells. At the mechanistic levels, treatment with the aRVS extract led to the downregulation of Bcl-2 and Mcl-1 proteins, the activation of caspase-9/-3 proteins, an increase in cytosolic cytochromec levels, the upregulation of Bax protein, an increase in phosphorylated p53 protein but a decrease in phosphorylated S6 protein in the A549 cells. Importantly, treatment with z-VAD‑fmk, a pan-caspase inhibitor attenuated aRVS extract-induced apoptosis in the A549 cells. These results demonstrate firstly that aRVS extract has growth inhibitory and apoptosis-inducing effects on A549 human lung cancer cells through modulation of the expression levels and/or activities of caspases, Bcl-2, Mcl-1, Bax, p53 and S6.

Abstract 080: mTORC1 as a Novel Regulator of Vascular Endothelial Function in Obese Mice and Humans

Obesity-induced hypertension is associated with vascular endothelial dysfunction. Recently, our laboratory has demonstrated a critical role of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in cardiovascular regulation. Here, we tested the hypothesis that dysregulation of mTORC1 signaling is involved in the endothelial dysfunction associated with obesity in mice and humans. We found that diet-induced obese (DIO) mice that display vascular endothelial dysfunction as compared to lean controls have increased mTORC1 signaling in aortic lysates indicated by the elevated (p<0.05) phosphorylated levels of mTOR and its downstream signaling targets S6-kinase and the ribosomal S6 protein measured by Western blot. Increased vascular mTORC1 signaling in DIO mice was associated with increased aortic NOX2 mRNA expression (2.0±0.2 vs. 1.0±0.3AU in lean controls; p<0.05). Isolated abdominal subcutaneous adipose arterioles from non-diabetic obese (BMI ≥30 kg/m 2 ; n=4; age 51±6 yrs; BMI 54±3 kg/m 2 ) humans exhibited a strong trend towards increased phosphorylated S6 protein compared to normal-weight (BMI <30kg/m 2 ; n=3; age 44±15 yrs; BMI 26±1 kg/m 2 ) individuals (5.0±1.9 vs 0.8±0.4AU; p=0.12), suggesting increased vascular mTORC1 signaling in human obesity. Next, we used an adenoviral construct of a constitutively active (CA) S6-kinase (Ad-CAS6K) to enhance mTORC1 signaling. In mouse endothelial cells, Ad-CAS6K increased mRNA expression of oxidative stress (NOX1and NOX2) and inflammatory markers (IL-1β) and decreased endothelial NOS expression (p<0.05). Transfection of aortic rings with the Ad-CAS6K resulted in impairment in acetylcholine-induced relaxation (Max. relaxation: 67± 5 vs. 81 ±3%; p<0.05) without altering the relaxation evoked by sodium nitroprusside (Max. relaxation: 90±1% vs. 90±2%) recapitulating the vascular phenotype in obese mice. Taken together, our data demonstrate a novel role of the mTORC1 signaling pathway in the regulation of vascular endothelial function. Our data also implicate dysregulation of the endothelial mTORC1 signaling pathway in the endothelial dysfunction associated with obesity.

The hVps34-SGK3 pathway alleviates sustained PI3K/Akt inhibition by stimulating mTORC1 and tumourgrowth.

We explore mechanisms that enable cancer cells to tolerate PI3K or Akt inhibitors. Prolonged treatment of breast cancer cells with PI3K or Akt inhibitors leads to increased expression and activation of a kinase termed SGK3 that is related to Akt. Under these conditions, SGK3 is controlled by hVps34 that generates PtdIns(3)P, which binds to the PX domain of SGK3 promoting phosphorylation and activation by its upstream PDK1 activator. Furthermore, under conditions of prolonged PI3K/Akt pathway inhibition, SGK3 substitutes for Akt by phosphorylating TSC2 to activate mTORC1. We characterise 14h, a compound that inhibits both SGK3 activity and activation in vivo, and show that a combination of Akt and SGK inhibitors induced marked regression of BT‐474 breast cancer cell‐derived tumours in a xenograft model. Finally, we present the kinome‐wide analysis of mRNA expression dynamics induced by PI3K/Akt inhibition. Our findings highlight the importance of the hVps34‐SGK3 pathway and suggest it represents a mechanism to counteract inhibition of PI3K/Akt signalling. The data support the potential of targeting both Akt and SGK as a cancer therapeutic.

Anorexic response to rapamycin does not appear to involve a central mechanism.

The authors have previously demonstrated that a low and intermittent peripheral dose of rapamycin (1mg/kg three times/week) to rats inhibited mTORC1 signalling, but avoided the hyperlipidemia and diabetes-like syndrome associated with higher doses of rapamycin. The dosing regimen reduced food intake, body weight, adiposity, serum leptin and triglycerides. mTORC1 signalling was inhibited in both liver and hypothalamus, suggesting some of the actions, in particular the decrease in food intake, may be the results of a central mechanism. To test this hypothesis, rapamycin (30μg/day for 4weeks) was infused into 23-25-month-old F344xBN rats by intracerebroventricular (icv) mini pumps. Our results demonstrated that central infusion did not alter food intake or body weight, although there was a tendency for a decrease in body weight towards the end of the study. mTORC1 signalling, evidenced by decreased phosphorylation of S6 protein at end of 4weeks, was not activated in liver, hypothalamus or hindbrain. Fat and lean mass, sum of white adipose tissues, brown adipose tissue, serum glucose, insulin and leptin levels remained unchanged. Thus, these data suggest that the anorexic and body weight responses evident with peripheral rapamycin are not the result of direct central action. The tendency for decreased body weight towards the end of study, suggests that there is either a slow transport of centrally administered rapamycin into the periphery, or that there is delayed action of rapamycin at sites in the brain.

Effect of confounding factors on a phospho-flow assay of ribosomal S6 protein for therapeutic drug monitoring of the mTOR-inhibitor everolimus in heart transplanted patients

Context: Several assays of monitoring immune cell function have been developed to enhance therapeutic drug monitoring.Objective: An in vitro-validated whole-blood assay of phosphorylated ribosomal protein S6 (pS6RP) was evaluated for confounders to monitor the mTOR-inhibitor everolimus (ERL).Materials and methods: Whole blood samples from 87 heart transplant recipients were analyzed for pS6RP-expression in CD3-positive T-cells by phospho-flow analysis.Results: ERL blood concentration, laboratory parameters, co-medications, demographic and clinical data were reviewed.Conclusion: Evaluating the pS6RP-assay revealed that pS6RP is influenced by cyclosporine A (CsA) blood concentration, duration of ERL treatment, co-medication with thiazide diuretics and different metabolic parameters.

Abstract 242: Deciphering the role of PTEN as a predictive biomarker to next generation isoform-selective PI3K inhibitors in PIK3CA mutant breast cancer cells

Abstract The phosphoinositide-3 kinase (PI3K) pathway is one of the most frequently activated signaling pathways in human cancer and represents a promising therapeutic target. Taselisib (GDC-0032), a potent and selective inhibitor of the PI3K alpha, delta and gamma isoforms, but less potent in the beta isoform, displays increased sensitivity in models that are driven by mutant PIK3CA (Ndubaku CO et al, J Med Chem, 2013). Selective pressure caused by, small molecules such as PI3K inhibitors will ultimately lead to clinical resistance and disease progression. Recently, a published mechanism of acquired resistance to the PI3K inhibitor alpelisib (BYL-719, a PI3K alpha-selective inhibitor) reported that genomic loss of the tumor suppressor PTEN led to clinical progression in a metastatic breast cancer patient harboring an activating PIK3CA mutation (Juric D et al, Nature, 2014). As taselisib and alpelisib are next generation PI3K inhibitors, we sought to determine if there are any differences to drug sensitivity in PIK3CA mutant cells that have concomitant loss of PTEN. To investigate this hypothesis, we first measured viability in breast cancer cells, each with differing genetic backgrounds treated with two PI3K inhibitors, taselisib and alpelisib. We found that PIK3CA mutant and PIK3CA mutant/PTEN null cells were sensitive to treatment when compared to wild-type and PTEN null cells. However, PIK3CA mutant/PTEN null cells displayed higher IC50 values for both inhibitors when compared to PIK3CA mutant cells. To control for differences in genetic backgrounds, we next tested whether knockdown (KD) of PTEN expression in PIK3CA mutant breast cancer cells would promote resistance and reactivate the PI3K signaling cascade even in the presence of a PI3K inhibitor. Transient, siRNA-targeted KD of PTEN in the PIK3CA mutant breast cancer cell lines EFM19 and T47D conferred resistance to alpelisib after 6 days of drug treatment, an observation that was less pronounced in taselisib treated cells. Additionally, PTEN KD in DMSO-treated cells led to an increase in cell proliferation and enhanced downstream PI3K signaling as measured by phosphorylated AKT and S6 proteins when compared to non-targeting siRNA controls. The reactivated, pro-survival signaling with PTEN KD was maintained upon treatment with both inhibitors when compared to control cells, an observation that was more pronounced in alpelisib treated cells. These data suggest that PIK3CB can compensate for PIK3CA inhibition and that PTEN loss could lead to clinical resistance to alpelisib, but may not constitute a resistance mechanism to taselisib. As both molecules have entered phase 3 studies in estrogen receptor positive breast cancer, understanding how biomarker-defined genotypes respond to next generation PI3K inhibitors is critical for identifying patients most likely to gain therapeutic benefit. Citation Format: Heather M. Moore, Heidi M. Savage, Mark R. Lackner, Timothy R. Wilson. Deciphering the role of PTEN as a predictive biomarker to next generation isoform-selective PI3K inhibitors in PIK3CA mutant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 242.

Abstract LB-215: Combination studies using PI3K isoform-selective inhibitors in PTEN-deficient cancer cells

Abstract A large and growing body of evidence indicates that dysregulation of phosphoinositide 3-kinase (PI3K) pathway signaling activity is necessary for cancer cell survival, most commonly as a result of genetic defects causing a deficiency of the tumor suppressor PTEN or activating mutations or amplification of PIK3CA (PI3Kα). In the context of PTEN-deficient cancer cells, it appears that in most cases PI3Kβ (PIK3CB) and not PI3Kα is required for PI3K survival signaling. GSK2636771 is a potent, orally bioavailable, selective ATP-competitive inhibitor of PI3Kβ with single digit nanomolar potency. It possesses >10-fold selectivity over PI3Kδ and >1000-fold selectivity over other lipid/atypical kinases including PI3Kα and mTOR. In this study, western blot analysis was performed to assess phosphorylation changes to AKT, ERK, and S6 proteins in 21 tumor cell lines of various histologies and genetic backgrounds treated with GSK2636771. PTEN deficiency (lack of PTEN protein expression) was the best marker to predict response to PI3Kβ inhibition based on reduction of phospho-AKT levels. Some of these “responsive” cells also harbored other genetic alterations such as amplified and/or mutated (V600E) BRAF, NF1 mutations, or EGFR amplification. For example, inhibition of PI3Kβ resulted in partial reduction of phospho-AKT and phospho-S6 levels in PTEN-deficient A2058, MDA-MB-468, and LNCAP cells, whereas PI3Kα inhibition by BYL719 treatment had no effect on AKT, ERK, and S6 phosphorylation. In PTEN-deficient, PIK3CA (E542K) mutant CAL-51 cells, the opposite was observed: GSK2636771 had no effect on phospho levels of AKT, ERK, and S6, but BYL719 treatment caused reductions of phospho-AKT and phospho-S6. Interestingly, when both isoforms were inhibited, the effects on phospho-AKT and phospho-S6 levels were more pronounced in all four cell lines, suggesting that these compounds may synergize to inhibit PI3K signaling. Combination treatment of both inhibitors had modest effects on cell growth in LNCAP cells, although it was observed that 6-day GSK2636771 treatment pulsed with BYL719 for 2 days in LNCAP provided similar antiproliferative effects as co-treatment of both molecules for 6 days. This may suggest that pulsing BYL719 with constant GSK2636771 treatment may be effective at inhibiting tumor growth in vivo while minimizing the on target toxicity that has been observed with sustained PI3Kα inhibition. Citation Format: Christina Blackwell, M. Phillip De Young. Combination studies using PI3K isoform-selective inhibitors in PTEN-deficient cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-215.

Abstract 2994: Orlistat decreases endometrial cancer cell proliferation: implications for a novel treatment strategy

Abstract Objectives: Fatty acid synthase (FAS) is a key enzyme involved in fatty acid synthesis. It also plays an important role in tumorigenesis and is highly expressed in many cancers, including endometrial cancer. Orlistat is a weight loss medication that has been shown to be a potent inhibitor of FAS. The goal of this study was to evaluate the anti-tumorigenic potential of orlistat in endometrial cancer cell lines. Methods: The endometrial cancer cell lines ECC-1 and KLE were treated with varying doses of orlistat (0.01-500 μM). Cell proliferation was assessed using the MTT assay. Cell cycle progression was evaluated by Cellometer and apoptosis was assessed using the Annexin V assay. Reactive oxygen species (ROS) was measured using the DCFH-DA assay. Western immunoblotting was performed to determine changes in FAS, cellular stress, cell cycle progression and the AMPK/mTOR pathways. Results: Orlistat inhibited cell proliferation in a dose-dependent manner in both cell lines (IC50 = 81.7 μM in ECC-1; IC50 = 145.5 μM in KLE). Treatment with orlistat resulted in G1 arrest but did not affect apoptosis. Orlistat increased ROS at high doses (500 uM, p = 0.008 for ECC-1 cells and p = 0.012 for KLE cells) and induced expression of BIP and PERK. Western immunoblot analysis demonstrated that orlistat decreased expression of FAS, acetyl-CoA carboxylase (ACC), and carnitine palmitoyltransferase 1A (CPT1A), important proteins in fatty acid metabolism. Treatment with orlistat also increased expression of phosphorylated AMPK and decreased expression of the downstream S6 protein. Conclusions: Orlistat suppressed cell growth in endometrial cancer cell lines through inhibition of fatty acid metabolism, induction of cell cycle G1 arrest, and modulation of the AMPK/mTOR pathway. Given that patients with endometrial cancer have high rates of obesity, orlistat should be further investigated as a novel strategy for endometrial cancer treatment and prevention. Citation Format: Weiya Z. Wysham, Dario R. Roque, Jianjun Han, Hui Guo, Lu Zhang, Paola A. Gehrig, Chunxiao Zhou, Victoria L. Bae-Jump. Orlistat decreases endometrial cancer cell proliferation: implications for a novel treatment strategy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2994.

S6 protein kinase activates Juvenile Hormone and vitellogenin production in the cockroach Blattella germanica

AbstractNutritional status regulates different processes, such as growth and development, through TOR (target of rapamycin) and insulin receptor signalling pathways. The ribosomal S6 protein kinase (S6K) is a downstream element of both pathways. Using cDNA from the German cockroach Blattella germanica (L.), two S6K isoform sequences (BgS6K) are cloned. The long isoform differs from the short one by the insertion of a 22‐amino acid duplication, involving the key phosphorylation position Thr390. As a result of this, the long isoform presents a new, potentially regulatory phosphorylation site. RNA interference knockdown of both BgS6K isoforms induces an increase in the length of the last nymphal instar, together with a reduction in the mRNA levels of a number of enzymes of the Juvenile Hormone biosynthetic pathway in the corpora allata, vitellogenin mRNA levels in the fat body and basal oocyte length. Thus, BgS6K is important for nymphal development and is necessary for the full induction of Juvenile Hormone synthesis and vitellogenin production in adult females.

A viral kinase mimics S6 kinase to enhance cell proliferation

Viruses depend upon the host cell for manufacturing components of progeny virions. To mitigate the inextricable dependence on host cell protein synthesis, viruses can modulate protein synthesis through a variety of mechanisms. We report that the viral protein kinase (vPK) encoded by open reading frame 36 (ORF36) of Kaposi's sarcoma-associated herpesvirus (KSHV) enhances protein synthesis by mimicking the function of the cellular protein S6 kinase (S6KB1). Similar to S6KB1, vPK phosphorylates the ribosomal S6 protein and up-regulates global protein synthesis. vPK also augments cellular proliferation and anchorage-independent growth. Furthermore, we report that both vPK and S6KB1 phosphorylate the enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 2 (PFKFB2) and that both kinases promote endothelial capillary tubule formation.

Exercise and dietary change ameliorate high fat diet induced obesity and insulin resistance via mTOR signaling pathway.

[Purpose]The purpose of this study was to investigate the effect of exercise and dietary change on obesity and insulin resistance and mTOR signaling protein levels in skeletal muscles of obese rats.[Methods]Sixty male Sprague-Dawley rats were divided into CO (Normal diet) and HF (High Fat diet) groups in order to induce obesity for 15 weeks. The rats were then subdivided into CO, COT (CO + Training), HF, HFT (HF + Training), HFND (Dietary change), and HFNDT (HFND + Training) groups (10 rats / group). The training groups underwent moderate-intensity treadmill exercise for 8 weeks, after which soleus muscles were excised and analyzed. Data was statistically analyzed by independent t-test and One-way ANOVA tests with a 0.05 significance level.[Results]Fasting blood glucose, plasma insulin, and HOMA-IR in the HF group were significantly higher, as compared with other groups (p <.05). Protein levels of insulin receptor subunit-1 (IRS-1), IRS-2, and p-Akt were significantly higher in the HFT, HFND, and HFNDT groups, as compared with HF group. In addition, the protein levels of the mammalian target of rapamycin complex 1 (mTORC1) and ribosomal S6 protein kinase 1 were significantly decreased by exercise and dietary change (p <.05). However, mTORC2 and phosphoinositide 3-kinase were significantly increased (p <.05).[Conclusion]In summary, despite the negative impact of continuous high fat intake, regular exercise and dietary change showed a positive effect on insulin resistance and mTOR signaling protein levels.

Leucine alleviates dexamethasone-induced suppression of muscle protein synthesis via synergy involvement of mTOR and AMPK pathways.

Glucocorticoids (GCs) are negative muscle protein regulators that contribute to the whole-body catabolic state during stress. Mammalian target of rapamycin (mTOR)-signalling pathway, which acts as a central regulator of protein metabolism, can be activated by branched-chain amino acids (BCAA). In the present study, the effect of leucine on the suppression of protein synthesis induced by GCs and the pathway involved were investigated. Invitro experiments were conducted using cultured C2C12 myoblasts to study the effect of GCs on protein synthesis, and the involvement of mTOR pathway was investigated as well. After exposure to dexamethasone (DEX, 100μmol/l) for 24h, protein synthesis in muscle cells was significantly suppressed (P<0.05), the phosphorylations of mTOR, ribosomal protein S6 protein kinase 1 (p70s6k1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) were significantly reduced (P<0.05). Leucine supplementation (5mmol/l, 10mmol/l and 15mmol/l) for 1h alleviated the suppression of protein synthesis induced by DEX (P<0.05) and was accompanied with the increased phosphorylation of mTOR and decreased phosphorylation of AMPK (P<0.05). Branched-chain amino transferase 2 (BCAT2) mRNA level was not influenced by DEX (P>0.05) but was increased by leucine supplementation at a dose of 5mmol/l (P<0.05).

Inhibition of mammalian target of rapamycin activation in the rostral anterior cingulate cortex attenuates pain-related aversion in rats

Pain is a complex experience that comprises both sensory and affective dimensions. Mammalian target of rapamycin (mTOR) plays an important role in the modulation of neuronal plasticity associated with the pathogenesis of pain sensation. However, the role of mTOR in pain affect is unclear. Using a formalin-induced conditioned place avoidance (F-CPA) test, the current study investigated the effects of the mTOR specific inhibitor rapamycin on noxious stimulation induced aversion in the rostral anterior cingulate cortex (rACC). Intraplantar injection of 5% formalin was associated with significant activation of mTOR, as well as p70 ribosomal S6 protein (p70S6K), its downstream effector, in the rACC. The inhibition of mTOR activation with rapamycin disrupted pain-related aversion; however, this inhibition did not affect formalin-induced spontaneous nociceptive behaviors in rats. These findings demonstrated for the first time that mTOR and its downstream pathway in the rACC contribute to the induction of pain-related negative emotion.

Quantitative Proteome Analysis of Leishmania donovani under Spermidine Starvation.

We have earlier reported antileishmanial activity of hypericin by spermidine starvation. In the current report, we have used label free proteome quantitation approach to identify differentially modulated proteins after hypericin treatment. A total of 141 proteins were found to be differentially regulated with ANOVA P value less than 0.05 in hypericin treated Leishmania promastigotes. Differentially modulated proteins have been broadly classified under nine major categories. Increase in ribosomal protein S7 protein suggests the repression of translation. Inhibition of proteins related to ubiquitin proteasome system, RNA binding protein and translation initiation factor also suggests altered translation. We have also observed increased expression of Hsp 90, Hsp 83–1 and stress inducible protein 1. Significant decreased level of cyclophilin was observed. These stress related protein could be cellular response of the parasite towards hypericin induced cellular stress. Also, defective metabolism, biosynthesis and replication of nucleic acids, flagellar movement and signalling of the parasite were observed as indicated by altered expression of proteins involved in these pathways. The data was analyzed rigorously to get further insight into hypericin induced parasitic death.

092 Merkel cell polyomavirus small T antigen co-expressed with Atoh1 drives development of intraepidermal MCC

092 Merkel cell polyomavirus small T antigen co-expressed with Atoh1 drives development of intraepidermal MCC ME Verhaegen, D Mangelberger, PW Harms, D Wilbert, J Meireles, T Saunders and AA Dlugosz 1 Dermatology, University of Michigan, Ann Arbor, MI, 2 Pathology, University of Michigan, Ann Arbor, MI and 3 Internal Medicine, University of Michigan, Ann Arbor, MI Merkel cell carcinoma (MCC) is a rare and deadly neuroendocrine skin tumor that frequently expresses viral transforming antigens, sTAg and tLTAg, encoded by Merkel cell polyomavirus (MCPyV). While the cell of origin is unknown, MCCs express markers detected in normal Merkel cells, including Keratin (K) 20, K8, and synaptophysin. Most MCCs and MCC cell lines also express ATOH1, a bHLH protein that is required for Merkel cell specification, and when expressed ectopically, drives de novo development of Merkel cells from epidermis. To test the potential of MCPyV viral oncoproteins to drive MCC development, we co-expressed MCPyV TAgs and Atoh1 in epidermis of transgenic mice. Combined expression of sTAg and Atoh1 in skin of multiple independent founders induced striking alterations that mimicked the appearance of human intraepidermal MCC, including numerous foci of small blue cells with nuclear hyperchromasia, mitotic activity and epidermal clefting. These tumor-like collections of cells expressed typical Merkel cell and MCC markers, including synaptophysin, K8 and K20. In contrast to normal Merkel cells, however, K20 was present in a paranuclear dot-like pattern which is pathognomonic of MCC. Moreover, many tumor cells expressed both K8 and the proliferation maker Ki67, which is characteristic of MCC tumor cells but not Merkel cells, which are post-mitotic. Addition of tLTAg did not appreciably alter the MCC-like phenotype produced by co-expression of sTAg and Atoh1. Thus, expression of MCPyV sTAg in Atoh1expressing epidermal cells of pre-term mouse embryos leads to widespread development of MCC-like tumors, strongly supporting the concept that sTAg is the primary oncogenic driver of MCC when expressed in the appropriate progenitor population. 093 Cell fate determination in the initiation of high-risk skin carcinoma WK Ryan, J Fernandez and EC Torchia Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO There are few effective treatment options for high-risk Squamous Cell Carcinomas (SCCs). Our goal in this study was to define the early signaling pathways in “initiated” Keratinocyte (KC) progenitors that contribute to the progression of high-risk tumors in a well-characterized mouse SCC model. Here, the activation of a Gain-Of-Function (GOF) p53 mutant resulted in tumors with centrosome amplification, dysregulation of the mitotic Kinase Aurora-A (Aur-A), genomic DNA instability, and enhanced metastasis, implicating abnormal mitotic regulation in the progression to high-risk SCCs. We initially observed a marked S-phase increase in both GOF and Loss-Of-Function (LOF) p53-KCs after a short course of the tumor promoter, TPA. However, histological presentation remained similar. The genetic deletion of Aur-A (Aur-A) in GOF or LOF p53 skin led to dramatic morphological changes associated with diffused hyperplasia, cellular pleomorphism, cell karyomegaly, prominent nucleoli, and multinucleation. These effects were more pronounced in Aur-A; GOF p53 skin, but surprisingly, there was minimal induction of DNA damage or cell death. By RNA seq, subtle differences between GOF and LOF p53 skin were observed. However, co-deletion of Aur-A markedly altered gene expression profiles. Analysis by the IPA software predicted the activation of the PI3K/mTOR pathway in AurA;GOF p53 skin. By immunoblotting, phospho/ total S6 protein ratios were increased in GOF vs LOF p53 skin, but markedly elevated after Aur-A deletion, whereas Bcl-xl protein levels remained unchanged. We confirmed p-S6 levels by IF and noted its predominantly suprabasal expression in untreated controls that extended to the basal layer in Aur-A skin. Lastly, we sorted KCs from TPA treated skin and perform single cell RNA sequencing. Pathway analysis revealed the enrichment of mTOR pathway signatures in GOF p53 KCs, suggesting that the involvement of the pathway in GOF p53 skin was cell-autonomous to KCs. Our data indicate that the PI3K/mTOR pathway may have role in the initiation of high-risk SCCs.

Serine 302 Phosphorylation of Mouse Insulin Receptor Substrate 1 (IRS1) Is Dispensable for Normal Insulin Signaling and Feedback Regulation by Hepatic S6 Kinase

Constitutive activation of the mammalian target of rapamycin complex 1 and S6 kinase (mTORC1→ S6K) attenuates insulin-stimulated Akt activity in certain tumors in part through "feedback" phosphorylation of the upstream insulin receptor substrate 1 (IRS1). However, the significance of this mechanism for regulating insulin sensitivity in normal tissue remains unclear. We investigated the function of Ser-302 in mouse IRS1, the major site of its phosphorylation by S6K in vitro, through genetic knock-in of a serine-to-alanine mutation (A302). Although insulin rapidly stimulated feedback phosphorylation of Ser-302 in mouse liver and muscle, homozygous A302 mice (A/A) and their knock-in controls (S/S) exhibited similar glucose homeostasis and muscle insulin signaling. Furthermore, both A302 and control primary hepatocytes from which Irs2 was deleted showed marked inhibition of insulin-stimulated IRS1 tyrosine phosphorylation and PI3K binding after emetine treatment to raise intracellular amino acids and activate mTORC1 → S6K signaling. To specifically activate mTORC1 in mouse tissue, we deleted hepatic Tsc1 using Cre adenovirus. Although it moderately decreased IRS1/PI3K association and Akt phosphorylation in liver, Tsc1 deletion failed to cause glucose intolerance or promote hyperinsulinemia in mixed background A/A or S/S mice. Moreover, Tsc1 deletion failed to stimulate phospho-Ser-302 or other putative S6K sites within IRS1, whereas ribosomal S6 protein was constitutively phosphorylated. Following acute Tsc1 deletion from hepatocytes, Akt phosphorylation, but not IRS1/PI3K association, was rapidly restored by treatment with the mTORC1 inhibitor rapamycin. Thus, within the hepatic compartment, mTORC1 → S6K signaling regulates Akt largely through IRS-independent means with little effect upon physiologic insulin sensitivity.

Stimulatory Effects of Coumestrol on Embryonic and Fetal Development Through AKT and ERK1/2 MAPK Signal Transduction.

Successful establishment of pregnancy is required for fetal-maternal interactions regulating implantation, embryonic development and placentation. A uterine environment with insufficient growth factors and nutrients increases the incidence of intrauterine growth restriction (IUGR) leading to an impaired uterine environment. In the present study, we demonstrated the effects of the phytoestrogen coumestrol on conceptus development in the pig that is regarded as an excellent biomedical animal model for research on IUGR. Results of this study indicated that coumestrol induced migration of porcine trophectoderm (pTr) cells in a concentration-dependent manner. In response to coumestrol, the phosphorylation of AKT, P70S6K, S6, ERK1/2 MAPK, and P90RSK proteins were activated in pTr cells and ERK1/2 MAPK and P90RSK phosphorylation was prolonged for a longer period than for the other proteins. To identify the signal transduction pathway induced by coumestrol, pharmacological inhibitors U0126 (an ERK1/2 inhibitor) and LY294002 (a PI3K inhibitor) were used to pretreat pTr cells. The results showed that coumestrol-induced phosphorylation of ERK1/2 MAPK and P90RSK was blocked by U0126. In addition, the increased phosphorylation in response to coumestrol was completely inhibited following pre-treatment incubation of pTr cells in the presence of LY294002 and U0126. Furthermore, these two inhibitors suppressed the ability of coumestrol to induce migration of pTr cells. Collectively, these findings suggest that coumestrol affects embryonic development through activation of the PI3K/AKT and ERK1/2 MAPK cell signal transduction pathways and improvement in the uterine environment through coumestrol supplementation may provide beneficial effects of enhancing embryonic and fetal survival and development. J. Cell. Physiol. 231: 2733-2740, 2016. © 2016 Wiley Periodicals, Inc.

Impact of rapamycin on status epilepticus induced hippocampal pathology and weight gain

Growing evidence implicates the dentate gyrus in temporal lobe epilepsy (TLE). Dentate granule cells limit the amount of excitatory signaling through the hippocampus and exhibit striking neuroplastic changes that may impair this function during epileptogenesis. Furthermore, aberrant integration of newly-generated granule cells underlies the majority of dentate restructuring. Recently, attention has focused on the mammalian target of rapamycin (mTOR) signaling pathway as a potential mediator of epileptogenic change. Systemic administration of the mTOR inhibitor rapamycin has promising therapeutic potential, as it has been shown to reduce seizure frequency and seizure severity in rodent models. Here, we tested whether mTOR signaling facilitates abnormal development of granule cells during epileptogenesis. We also examined dentate inflammation and mossy cell death in the dentate hilus. To determine if mTOR activation is necessary for abnormal granule cell development, transgenic mice that harbored fluorescently-labeled adult-born granule cells were treated with rapamycin following pilocarpine-induced status epilepticus. Systemic rapamycin effectively blocked phosphorylation of S6 protein (a readout of mTOR activity) and reduced granule cell mossy fiber axon sprouting. However, the accumulation of ectopic granule cells and granule cells with aberrant basal dendrites was not significantly reduced. Mossy cell death and reactive astrocytosis were also unaffected. These data suggest that anti-epileptogenic effects of mTOR inhibition may be mediated by mechanisms other than inhibition of these common dentate pathologies. Consistent with this conclusion, rapamycin prevented pathological weight gain in epileptic mice, suggesting that rapamycin might act on central circuits or even peripheral tissues controlling weight gain in epilepsy.

Sulforaphane improves dysregulated metabolic profile and inhibits leptin-induced VSMC proliferation: Implications toward suppression of neointima formation after arterial injury in western diet-fed obese mice

Sulforaphane (SFN), a dietary phase-2 enzyme inducer that mitigates cellular oxidative stress through nuclear factor erythroid 2-related factor 2 (Nrf2) activation, is known to exhibit beneficial effects in the vessel wall. For instance, it inhibits vascular smooth muscle cell (VSMC) proliferation, a major event in atherosclerosis and restenosis after angioplasty. In particular, SFN attenuates the mitogenic and pro-inflammatory actions of platelet-derived growth factor (PDGF) and tumor necrosis factor-α (TNFα), respectively, in VSMCs. Nevertheless, the vasoprotective role of SFN has not been examined in the setting of obesity characterized by hyperleptinemia and insulin resistance. Using the mouse model of western diet-induced obesity, the present study demonstrates for the first time that subcutaneous delivery of SFN (0.5mg/Kg/day) for~3weeks significantly attenuates neointima formation in the injured femoral artery [↓ (decrease) neointima/media ratio by~60%; n=5–8]. This was associated with significant improvements in metabolic parameters, including ↓ weight gain by~52%, ↓ plasma leptin by~42%, ↓ plasma insulin by~63%, insulin resistance [↓ homeostasis model assessment of insulin resistance (HOMA-IR) index by~73%], glucose tolerance (↓ AUCGTT by~24%), and plasma lipid profile (e.g., ↓ triglycerides). Under in vitro conditions, SFN significantly decreased leptin-induced VSMC proliferation by~23% (n=5) with associated diminutions in leptin-induced cyclin D1 expression and the phosphorylation of p70S6kinase and ribosomal S6 protein (n=3–4). The present findings reveal that, in addition to improving systemic metabolic parameters, SFN inhibits leptin-induced VSMC proliferative signaling that may contribute in part to the suppression of injury-induced neointima formation in diet-induced obesity.