Phosphotidylinositol 3-kinase Research Articles

Nrf2 as a potential target for Parkinson's disease therapy.

Parkinson's disease (PD) is a complex neurodegenerative disorder featuring both motor and nonmotor symptoms associated with a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Conventionally, PD treatment options have focused on dopamine replacement and provide only symptomatic relief. However, disease-modifying therapies are still unavailable. Mechanistically, genetic and environmental factors can produce oxidative stress which has been implicated as a core contributor to the initiation and progression of PD through the degeneration of dopaminergic neurons. Importantly, nuclear factor erythroid 2-related factor 2 (Nrf2) is essential for maintaining redox homeostasis by binding to the antioxidant response element which exists in the promoter regions of most genes coding for antioxidant enzymes. Furthermore, protein kinase C, mitogen-activated protein kinases, and phosphotidylinositol 3-kinase have been implicated in the regulation of Nrf2 activity during PD. Here, we review the evidence supporting the regulation of Nrf2 through Keap1-dependent and Keap1-independent mechanisms. We also address that targeting Nrf2 may provide a therapeutic option to mitigate oxidative stress-associated PD. Finally, we discuss currently known classes of small molecule activators of Nrf2, including Nrf2-activating compounds in PD.

Neurotensin: a neuropeptide induced by hCG in the human and rat ovary during the periovulatory period†.

Neurotensin (NTS) is a tridecapeptide that was first characterized as a neurotransmitter in neuronal cells. The present study examined ovarian NTS expression across the periovulatory period in the human and the rat. Women were recruited into this study and monitored by transvaginal ultrasound. The dominant follicle was surgically excised prior to the luteinizing hormone (LH) surge (preovulatory phase) or women were given 250μg human chorionic gonadotropin (hCG) and dominant follicles collected 12-18h after hCG (early ovulatory), 18-34h (late ovulatory), and 44-70h (postovulatory). NTS mRNA was massively induced during the early and late ovulatory stage in granulosa cells (GCs) (15000 fold) and theca cells (700 fold). In the rat, hCG also induced Nts mRNA expression in intact ovaries and isolated GCs. In cultured granulosa-luteal cells (GLCs) from IVF patients, NTS expression was induced 6h after hCG treatment, whereas in cultured rat GCs, NTS increased 4h after hCG treatment. Cells treated with hCG signaling pathway inhibitors revealed that NTS expression is partially regulated in the human and rat GC by the epidermal-like growth factor pathway. Human GLC, and rat GCs also showed that Nts was regulated by the protein kinase A (PKA) pathway along with input from the phosphotidylinositol 3- kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. The predominat NTS receptor present in human and rat GCs was SORT1, whereas NTSR1 and NTSR2 expression was very low. Based on NTS actions in other systems, we speculate that NTS may regulate crucial aspects of ovulation such as vascular permeability, inflammation, and cell migration.

Insulin receptor substrate-1 inhibits high-fat diet-induced obesity by browning of white adipose tissue through miR-503.

Genetic variation of insulin receptor substrate 1 (IRS-1) was found to modulate the insulin resistance of adipose tissues, but the underlying mechanism was not clear. To investigate how the IRS-1 was involved in the browning of white adipose tissue through miRNA, we identified a mutated Irs-1 (Irs-1-/- ) mice model and found that this mice had a reduced subcutaneous WAT (sWAT) and increased brown adipose tissue (BAT) in the interscapular region. So we isolated the bone marrow stromal cells and analyzed differentially expressed miRNAs and adipogenesis-related genes with miRNA arrays and PCR arrays. Irs-1-/- mice showed decreased miR-503 expression, but increased expression of its target, bone morphogenetic protein receptor type 1a (BMPR1a). Overexpression of miR-503 in preadipocytes downregulated BMPR1a and impaired adipogenic activity through the phosphotidylinositol 3-kinase (PI3K/Akt) pathway, while the inhibitor had the opposite effect. In both Irs-1-/- and cold-induced models, sWAT exhibited BAT features, and showed tissue-specific increased BMPR1a expression, PI3K expression, and Akt phosphorylation. Thus, our results showed that IRS-1 regulated brown preadipocyte differentiation and induced browning in sWAT through the miR-503-BMPR1a pathway, which played important roles in high-fat diet-induced obesity.

Neferine inhibits epidermal growth factor-induced proliferation and migration of retinal pigment epithelial cells through downregulating p38 MAPK and PI3K/AKT signalling

Purpose: Proliferative vitreoretinopathy (PVR) occurs in approximately 5–10% of patients after retinal detachment surgery. Neferine is a bis-benzylisoquinoline alkaloid found in the green seed embryos (Nelumbo nucifera) of the lotus flower and has various properties, such as being antithrombotic, antioxidant, neuroprotective, anticancerous, and anti-inflammatory. Although the effects of neferine on the proliferation and migration of cancer cells have been partially shown, their possible role and the mechanism of action on PVR remain unclear.Materials and methods: To mimic a PVR model in vitro, retinal pigment epithelial (RPE) cells were exposed to epidermal growth factor (EGF) and treated with various concentrations of neferine. Cell viability was determined by MTT test. Cell-cycle phase distribution and cell migration were examined by image-based cytometry and wound healing test, respectively. Messenger RNA (mRNA) and protein expression were determined by RT-qPCR and Western blotting, respectively.Results: Stimulation of the cells with EGF significantly increased the rate of proliferation, whilst treatment with low concentrations of neferine-reduced proliferation to a level equal to that seen in untreated cells. Neferine significantly downregulated EGF-increased cell viability, and survivin mRNA expression was depressed to the basal level. In addition, neferine treatment contributed to cell proliferation loss by upregulating p21 and p27 expression leading to cycle arrest at the G1 phase. The treatment significantly inhibited cell migration by upregulating the expression of epithelial markers, such as E-cadherin and occludin, and decreased MMP2, MMP9, α-SMA, and vimentin. Neferine treatment markedly reduced phosphotidyl inositol 3-kinase (PI3K), AKT, p-p38 mitogen-activated protein kinase (MAPK), and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) protein expression.Conclusion: It can be considered that neferine may be a potential candidate molecule in the treatment of PVR by inhibiting cell proliferation and the migration of EGF-induced RPE cells through the modulation of various transcriptional activities.

Rationale for the advancement of PI3K pathway inhibitors for personalized chordoma therapy.

Chordomas are rare and serious tumors with few effective treatments outside of aggressive surgery and radiation. Targeted therapies may present a more effective option for a subset of patients with lesions possessing certain genetic biomarkers. A small molecule inhibitor library was tested in patient-derived UM-Chor1 cells to identify targeted therapies with potential efficacy. Targeted exome sequencing of UM-Chor1 and UM-Chor2 cells was performed to investigate genetic aberrations in relevant pathways. Chordoma cell lines were treated with inhibitors of the phosphotidylinositol 3-kinase (PI3K), epidermal growth factor receptor (EGFR), and cyclin dependent kinase (CDK) pathways, and responses were determined using resazurin cell viability assays, Annexin V apoptosis assays, and western blotting. Pan-PI3K inhibitor BKM120 was also tested in five chordoma xenograft models. Unbiased small molecule profiling nominated PI3K-AKT-mTOR pathway inhibitors as a promising therapy in chordoma, and genetic analyses of UM-Chor1 and UM-Chor2 cell lines revealed aberrations in PTEN, EGFR, and CDKN2A. Treatment of UM-Chor1 and UM-Chor2 with targeted PI3K, EGFR, and CDK inhibitors inhibited growth and proliferation and induced apoptosis more robustly than imatinib, a currently used chordoma therapy. Furthermore, BKM120 significantly inhibited tumor growth in a subset of the xenograft models tested. Targeted therapies, especially those inhibiting PI3K, display promising effects in multiple chordoma cell line and xenograft models. Nevertheless, the limited effects of PI3K, EGFR, and CDK targeting agents in other models reveal the presence of resistance mechanisms, which motivates future research to both identify biomarkers of response and develop combination therapies.

The effect of Prosopis farcta extract on the expression of some key genes of the glycolysis pathway and the genes involved in insulin signaling in HepG2 cells

ObjectiveHyperglycemia in patients with Diabetic Mellitus (DM) results from the demolition of islet β cells in the pancreas as well as failure in insulin action or poor response to insulin secretion and there are no further methods to treat diabetes apart from insulin injection and oral hypoglycemic medicines. Thus, the main objective of the current research investigates hydro-alcoholic extracts of Prosopis farcta on impact on the Pyruvate kinase (Pk), Phospho fructokinase (PFK), Glucokinase (GK) and signaling insulin Phosphotidylinositol 3-Kinase (PI3K), Insulin Receptor Substrate (IRS1), Glucose Transporter type2 (GLUT2) in HepG2 cells. MethodBy various concentrations (0–10 mg/mL) of the extract (leaf-root – fruit) for 24 and 48 h, HepG2 cells were cultured and treated in this experimental research. MTT test evaluated cell proliferation by ELISA Reader measured the optical density of the colored solution at 570 nm wavelengths. Real-time-PCR method defined the fold changes of genes expression against β-actin. Data were analyzed by ANOVA. Variations with P-value < 0.05 were considered significant. ResultsOur results showed cells were treated with various concentrations of extract (leaf-root – fruit) for 24 and 48 h had markedly declined cell viability in HepG2 cell line as compared to controls in a time and dose-dependent methods, an evident deviation is seen between different concentrations of the extracts. Real Time-PCR analysis showed that the expression of glucokinase, phosphofructo and pyruvate kinase were down regulated and expression of IRS1-PI3K-GLUT2 genes were up regulated in compare to control cells (P < 0.05). ConclusionIt appears the anti-diabetic impact of the Prosopis farcta plant not to be over the glycolysis path. This acts mechanisms via enhancing the gene expression in the insulin signaling pathway. The plant, thus, may serve to enhance the insulin secretion function through triggering insulin signaling paths and, in turn, inhibiting increased blood glucose impact on diabetic patients. It is, therefore, expected that the various extracts of such plant may have an impact on diabetic patients, type-2 diabetes in particular.

STK33 Promotes the Growth and Progression of Human Pancreatic Neuroendocrine Tumour via Activation of the PI3K/AKT/mTOR Pathway

Background: Serine/threonine kinase 33 (STK33) has been reported to play an important role in cancer cell proliferation. We investigated the role of STK33 in pancreatic neuroendocrine tumour (PNET) and the underlying mechanisms. Methods: PNET specimens and adjacent non-tumorous pancreatic tissues from 84 patients who underwent curative surgery for PNET were stained by immunochemistry for STK33. The relationships among STK33 expression, clinicopathological parameters and clinical prognosis were statistically analysed. MTT, scratching, Transwell and apoptosis assays were employed to detect the effects of STK33 knockdown (siSTK33) or STK33 overexpression (pSTK33) on major oncogenic properties of cells of 2 PNET cell lines (BON and QGP-1), and real-time PCR and western blot were used to examine the expression of relevant genes. Results: Relative to its expression in normal pancreatic tissue, STK33 was overexpressed in PNET specimens. Furthermore, STK33 expression was significantly associated with World Health Organization classification (p < 0.001), American Joint Committee on Cancer stage (p < 0.001), lymph node metastasis (p < 0.001), tumour size (p = 0.022), sex (p = 0.003), perineural invasion (p < 0.001) and shorter disease-free survival of patients with PNET (p < 0.001). Enforced STK33 expression promoted PNET cell proliferation, migration and invasion and tumour growth and inhibited cell apoptosis, whereas STK33 depletion exerted the opposite effects. Mechanistic studies revealed that STK33 promoted growth and progression of PNET via activation of the phosphotidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. Conclusions: STK33 plays important roles in the tumour growth and progression of PNET via activation of the PI3K/AKT/mTOR pathway and has potential as a therapeutic target to improve PNET treatment.

Anticancer effects of limonene and phosphatidylinositol 3- Kinase (pi3k) inhibitor combination in colon cancer cell lines

Aberrant activation of phosphotidylinositol 3-kinase (PI3K) pathway effectors has been observed in many cancers including colorectal cancer. As cancers develop due to multiple genetic alterations it is seldom possible to treat them with monotherapy. So, combination therapy has been the standard care in cancer treatment. Therefore, the anticancer effects of the combination of limonene which is a natural monoterpene and NVP-BEZ235, a dual inhibitor of PI3K/mammalian target of rapamycin (mTOR) pathway were investigated in colorectal cancer cell lines. In vitro anticancer assays such as cell viability, cell cycle analysis, apoptosis, caspases estimation and Western blot analysis were performed to determine the combination effects on cells. Results showed that both drugs exhibited dose and time dependent cytotoxicity. Drug combination significantly decreased the phosphorylation of p-70S6K, p-AKTSer473andp-AKTThr308. The inhibition of p-70S6K phosphorylation was more sensitive than p-AKTSer473. Disruption of the known S6K1-IRS-1 negative feedback loop may possibly explain the reversal of p-AKT after 48h treatment with either of the drugs alone. However, the combination of limonene and BEZ may help to alleviate the negative feedback activation of PI3K signaling. This combination also induced G1 phase arrest in the cells and increased the expression of Bcl-2 family mediated apoptosis markers. In conclusion, the results indicate that limonene and BEZ combination demonstrated anticancer effects and are mediated by down regulation of PI3K/mTOR signaling and induction of apoptosis by mitochondrial death pathway.

Dissecting the role of Phosphotidylinositol 3-Kinase (PI3K) δ in the germinal center reaction through a mouse model of hyperactivated Pik3cd

Abstract The catalytic p110δ subunit of PI3K drives AKT pathways orchestrating activation and differentiation of T and B cells. Patients with gain of function mutations in Pik3cd (encoding for p110δ) exhibit a primary immunodeficiency characterized by lymphopenia, lymphadenopathy, recurrent and chronic infections, and occasionally lymphoma. They have reduced circulating naïve and increased effector T cell numbers, fewer class switched memory B cells and inefficient responses to vaccination. Nonetheless, in patient peripheral blood we find increased frequencies of T follicular helper (Tfh) cells. We have generated a mouse model expressing constitutively active p110δ that recapitulates many features of the human disease. Pik3cdMut mice, both in steady state condition and after immunization, display increased Tfh and germinal center (GC) B cell numbers in secondary lymphoid organs. Moreover, naïve mutant transgenic T cells (OT-II) preferentially acquire Tfh cell phenotype when adoptively transferred to WT mice, indicative of a T cell intrinsic defect. Nonetheless, despite increased Tfh and GC B cells, antigen specific B cell responses are reduced, particularly antigen-specific IgG. In contrast, we find the appearance of autoantibodies in Pik3cdMut mice. We further find that mutant OT-2 cells differentiate to Tfh cells independently from ICOS-ICOSL interactions. In line with this, mutant T cells show higher phosphorylation and inactivation of FoxO1, potent inhibitor of Tfh cell differentiation, that is phosphorylated and inhibited by AKT activation downstream of ICOS. Our results demonstrate that hyperactivated PI3Kδ leads to increased generation of Tfh cells and germinal centers, yet with defective in vivo function.

Association between high expression of phosphorylated Akt and mammalian target of rapamycin and improved survival in salivary gland adenoid cystic carcinoma.

Previous genomic studies revealed phosphotidylinositol-3-kinase (PI3K)/Akt pathway mutation in human salivary gland adenoid cystic carcinoma (ACC). No validation of its prognostic value has been reported. P-Akt, pan-Akt, phosphorylated-mammalian target of rapamycin (p-mTOR), PI3K, and insulin-like growth factor-1 receptor beta (IGF-1Rβ) were detected on 120 salivary gland ACC/adjacent salivary gland pairs immunohistochemically and were correlated with clinicopathological data. Expression of cytoplasmic and nuclear p-Akt, cytoplasmic p-mTOR, nuclear pan-Akt, and nuclear IGF-1Rβ were higher in ACC than in adjacent salivary glands. P-Akt, p-mTOR, PI3K, and IGF-1Rβ expression were correlated with one another in both cytoplasm and nucleus. Low p-mTOR expression in both subcellular compartments was associated with locoregional recurrence, poor disease-free survival (DFS), and overall survival (OS). Low nuclear p-Akt (Ser473) and p-mTOR expression were independent predictors for poor OS and DFS, respectively. High level of Akt/mTOR activation in ACC is correlated with a significantly improved survival. P-mTOR and nuclear p-Akt are prognostic biomarkers of salivary gland ACC. © 2017 Wiley Periodicals, Inc. Head Neck 39: 1145-1154, 2017.

Combination of phosphotidylinositol-3-kinase targeting with cetuximab and irradiation: A preclinical study on an orthotopic xenograft model of head and neck cancer.

The purpose of this study was to investigate the effects of combining the phosphotidylinositol-3-kinase (PI3K) inhibitor buparlisib (BKM)120 with the anti-epidermal growth factor receptor (EGFR) agent cetuximab and radiotherapy (RT) on an orthotopic model of head and neck squamous cell carcinoma (HNSCC). We evaluated the antitumor efficacy of BKM120, cetuximab, and RT, administered alone or in combination, using the human PIK3CA-mutated HNSCC cell line, CAL33, injected into the floor of the mouth of nude mice. Compared with control, the BKM120-cetuximab and the BKM120-cetuximab-RT combinations led to the highest tumor inhibition (p < .001). The highest inhibitory effect of treatments on cell proliferation, mitogen-activated protein kinase (MAPK) and PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathways were found with the BKM120-cetuximab association. The association of BKM120 and cetuximab with RT inhibited RT-induced activation of the MAPK pathway. These results can serve as a preclinical rationale for innovative treatments combining PI3K inhibition with anti-EGFR therapies and irradiation in patients with HNSCC. © 2016 Wiley Periodicals, Inc. Head Neck 39: 151-159, 2017.

A Randomized, Phase II Trial of Cetuximab With or Without PX-866, an Irreversible Oral Phosphatidylinositol 3-Kinase Inhibitor, in Patients With Metastatic Colorectal Carcinoma

BackgroundThe phosphotidylinositol-3 kinase (PI3K)/serine-threonine kinase/mammalian target of rapamycin signaling pathway is frequently altered in colorectal cancer (CRC). PX-866 is an oral, irreversible, pan-isoform inhibitor of PI3K. This randomized phase II study evaluated cetuximab with or without PX-866 in patients with metastatic, anti-epidermal growth factor receptor-naive, KRAS codon 12 and 13 wild-type CRC. Patients and MethodsPatients with metastatic CRC who had received both oxaliplatin and irinotecan were randomized (1:1) to cetuximab (400 mg/m2 loading then 250 mg/m2 weekly) with or without PX-866 (8 mg orally daily; arms A and B, respectively). The primary endpoint was progression-free survival (PFS). Secondary endpoints included objective response rate, overall survival (OS), toxicity, and correlation of relevant biomarkers with efficacy outcomes. ResultsA total of 85 patients were enrolled. The median PFS was 59 days versus 104 days for arms A (cetuximab + PX-866) and B (cetuximab alone), respectively (P = .77). OS between the 2 arms (266 vs. 333 days for arm A vs. B) were similar (P = .83). Overall toxicity, including treatment-related toxicity, was higher in arm A compared with arm B, especially in terms of all-grade nausea (66% vs. 37%), vomiting (50% vs. 29%), diarrhea (64% vs. 18%), and rash (66% vs. 37%). Grade 3 diarrhea occurred in 19% of patients in Arm A and 0% in Arm B. PIK3CA mutations and PTEN loss by immunohistochemistry were infrequently seen. ConclusionThe addition of PX-866 to cetuximab did not improve PFS, objective response rate, or OS in patients with metastatic CRC. The combination arm had greater toxicity and may have been harmful in this study.

Human epidermal growth factor receptor 3 in head and neck squamous cell carcinomas.

Human epidermal growth factor receptor 3 (HER3) is a member of the human epidermal growth factor receptor (HER) family. The main characteristic of HER3 is that it does not possess tyrosine kinase activity, unlike other HERs. The role of HER3 in tumorigenesis has now been recognized, particularly in head and neck squamous cell carcinomas (HNSCCs). Despite conflicting studies, HER3 was found to be overexpressed in HNSCC samples, and correlates with disease progression and poor survival, especially when it is coexpressed with other HERs. HER3 is a significant factor in HNSCC treatment resistance. Indeed, HER3 is a major mechanism described for cetuximab resistance because of modification of epidermal growth factor receptor (EGFR) internalization and by phosphotidylinositol-3-kinase (PI3K)/AKT signaling pathway activation. HER3 also affects resistance to tyrosine kinase inhibitors (TKIs) and thereby promotes treatment escape and radiotherapy resistance by activation of the survival signaling pathway. To counteract this, pharmacologic inhibitors of HER3 are currently in development and could significantly improve HNSCC treatment. © 2016 Wiley Periodicals, Inc. Head Neck 38: E2412-E2418, 2016.

Inhibition of Akt Signaling Alleviates MDS/MPN Driven By KrasD12 or Nf1 Loss

Juvenile and chronic myelomonocytic leukemias (JMML and CMML) are aggressive myeloid malignancies categorized as myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). Chemotherapy has little benefit for MDS/MPN patients, and new therapies are needed. We have used mouse models investigate the potential of signal transduction inhibitors in MDS/MPN, as JMML and CMML are associated with mutations in NRAS, KRAS, PTPN11, CBL, or NF1 that activate Ras signaling. Conditional Mx1-Cre, KrasLSL-D12 (designated KrasD12) mice develop an aggressive and fully penetrant MDS/MPN characterized by leukocytosis, splenomegaly, anemia, and death by 10-16 weeks of age. Mx1-Cre, Nf1flox/- mice (hereafter Nf1Δ/-) undergo conditional loss of Nf1. These mice also develop MDS/MPN, but the disease is more indolent. We and others have investigated inhibition of effector networks downstream of Ras, such as the Raf/MEK/ERK (MAPK) and phosphotidylinositol-3 kinase (PI3K)/Akt pathways. We previously showed that the MEK inhibitor PD0325901 induced sustained hematologic improvement in both KrasD12 and Nf1Δ/- mice. We also have reported that the class I PI3K inhibitor GDC-0941 improves hematologic function and prolongs survival in KrasD12 mice. However, GDC-0941 and other PI3K inhibitors attenuate both PI3K/Akt and Raf/MEK/ERK pathways due to effects of PI3K upstream of Ras. Therefore, the benefit from GDC-0941 could have been due to its modulation of Raf/MEK/ERK signaling. Here, we specifically test the importance of Akt signaling in MDS/MPN in KrasD12 and Nf1 mouse models using the allosteric inhibitor MK-2206. This compound binds to the interface of the PH and kinase domains of Akt1, Akt2, and Akt3, and does not inhibit any of 250 other kinases at 1 µM. MK-2206 induced substantial improvement in both KrasD12 and Nf1Δ/- mice. Mice treated with MK-2206 had pronounced reduction in leukocytosis, reticulocytosis and splenomegaly, increased hemoglobin concentration, and prolonged survival. MK-2206 had no hematologic effects in control WT mice, indicating some selectivity against aberrant hematopoiesis. Importantly, MK-2206 inhibited Akt but not Raf/MEK/ERK or Jak/STAT signaling. This demonstrates that canonical PI3K/Akt signaling plays an important role in Ras-driven MDS/MPN. Furthermore, combined inhibition of MEK and Akt with PD0325901+MK-2206 yielded a greater improvement in splenomegaly than either agent alone in both KrasD12 and Nf1Δ/- models. Akt has multiple effectors relevant to hematopoiesis and leukemia. Of these, mTOR is of particular interest for targeted cancer therapy. Therefore, we tested the response of KrasD12 mice to rapamycin, a partial inhibitor of mTOR with preferential activity against the mTORC1 complex. KrasD12 mice demonstrated variable responses to rapamycin, with approximately half undergoing a complete and durable hematologic response and the remainder having no response. Together, these studies further implicate PI3K/Akt signaling as a pathogenic effector downstream of Ras in MDS/MPN and support the idea that inhibitors targeting this pathway may have a role in treatment of JMML or CMML. DisclosuresNo relevant conflicts of interest to declare.

CHANGING OF RGS TRANSCRIPTS LEVELS BY LOW-DOSE-RATE IONIZING RADIATION IN MOUSE TESTIS

Deleterious effects of high dose radiation exposure with high-dose-rate are unarguable, but they are still controversial in low-dose-rate. The regulator of G-protein signaling (RGS) is a negative regulator of G proteincoupled receptor (GPCR) signaling. In addition, it is reported that irradiation stress led to GPCR-mediated mitogen-activated protein kinase (MAPK) and phosphotidylinositol 3-kinase (PI3-k) signaling. The RGS mRNA expression profiles by whole body radiation with low-dose-rate has not yet been explored. In the present study, we, therefore, examined which RGS was modulated by the whole body radiation with low-dose-rate (3.49 mGy·h -1 ). Among 16 RGS expression tested, RGS6, RGS13 and RGS16 mRNA were down-regulated by low-dose-rate irradiation. This is the first report that whole body radiation with low-dose-rate can modulate the different RGS expression levels. These results are expected to reveal the potential target and/or the biomarker proteins associated with male testis toxicity induced by low-dose-rate irradiation, which might contribute to understanding the mechanism beyond the testis toxicity.

Age- and Tissue-Dependent Modulation of IGF-1/PI3K/Akt Protein Expression by Dietary Restriction in Mice.

Insulin-like growth factor-1 (IGF-1), a key mediator of growth hormone action in mammals, plays an important role in the regulation of cellular and tissue functions. In the present investigation, the normal age-dependent and effect of dietary restriction on the expression levels of serum IGF-1, phosphotidylinositol 3-kinase (PI3K), and protein kinase B (PKB/Akt) in the liver, kidney and skeletal muscles of 1-, 9-, and 21-month-old female mice were ascertained using ELISA and Western blot analysis. Results showed that serum IGF-1 peaks at 9-month-old and decreases in older age. Dietary restriction (DR) decreases IGF-1 in both age groups. In liver, PI3K and relative amount of phosphorylated Akt (pAkt/Akt) showed an age-dependent correlated expression level as IGF-1 protein. In kidney and skeletal muscles, PI3K levels showed no changes with age. However, pAkt/Akt ratio increased in 9-month-old mice and decreased in 21-month-old mice. In liver, DR increased the level of PI3K and pAkt/Akt ratio in 9-month-old mice, but no significant change was seen in 21-month-old mice as compared to ad libitum-fed group. In kidney and skeletal muscles, DR mice showed an increase in PI3K level in both age groups while the pAkt/Akt ratio showed no change in 9-month-old mice, but exhibited a decrease in 21-month-old mice. These results indicate that there is a tissue-specific regulation of IGF-1/PI3K/Akt during normal aging and upon DR to help maintain the metabolic status of these tissues at those phases of animal's lifespan.

Abstract A25: Effect of Rab25 on Akt1 phosphorylation

Abstract Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease and despite therapeutic improvements and reconstructive modalities, the overall survival rate remains relatively poor. Although, tobacco and alcohol consumption are recognized as critical risk factors, genetic and epigenetics factors and viral infections also contribute to the progression of HNSCC. Several studies have assessed the status of known oncogenes and tumor suppressor genes to identify novel genomic events in HNSCC. Rab25 belongs to the Rab family of small GTPases. Rab25 has been described in conflicting previous studies as a promoter or suppressor of cancer. More recently, Zhang et al. reported that overexpression of Rab25 contributes to metastasis through activation of the Akt/GSK-3β/Snail pathway signaling in bladder cancer, which act as molecular switches that belong to the Ras protein superfamily and regulate vesicle trafficking in eucaryotic cells. The phosphotidylinositol 3-Kinase (PI3K) signaling pathway plays a critical role in cellular growth, proliferation and survival. Signaling through this pathway is commonly upregulated during tumorigenesis. Akt/PKB is a Ser/Thr kinase and the central member of the PI3K/Akt signaling pathway. It is activated by phosphorylation and subsequently phosphorylates diverse growth –controlling effectors and regulates the synthesis, stability and subcellular localization of cell cycle regulators. In this study, we investigated the phosphorylation of Akt1 and whether it occurs under the effect of Rab25 expression in HNSCC. For this purpose we analyzed the Rab25 protein by Western Blot in 57 HNSCC tumors and corresponding non-cancerous tissue samples. To detect the effect of the Rab25 protein on Akt1 activation we also analyzed the amount of total Akt1 and phosphorylated Akt1 in the same tissue samples. Our results indicate that Rab25 is down-regulated in 56.2% of the HNSCC tumor samples compared to their normal counterparts. On the other hand, the change in Akt1 phosphorylation was in the same direction with Rab25 protein expression (p: 0.04) Citation Format: Nur Buyru, Didem Seven, Soydan Dogan, Hikmet Koseoglu, Emin Karaman. Effect of Rab25 on Akt1 phosphorylation. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A25.

Adipose tissue p50α/p55α abundance may drive acute high fat diet induced insulin resistance

Insulin resistance (IR), a metabolic feature of obesity, drives a number of diseases. Recently, the focus of obesity research has been adipocyte‐immune cell crosstalk and its influence on systemic insulin sensitivity (IS). Currently, obesity induced IR is thought to arise secondary to inflammation due to infiltration of adipose tissue (AT) with pro‐inflammatory immune cells. Obesity is a progressive disease, and metabolic alterations in AT following acute nutrient excess, prior to development of obesity, are not well understood. Phosphotidylinositol 3‐kinase (PI3K) has been proposed as the integration point between immune cell infiltration and adipocyte insulin action. Global knockdown of pik3r1 (gene encoding regulatory subunits (p85α/p55α/p50α) of PI3K) resulted in enhanced adipocyte and systemic IS, with parallel decrease in AT macrophage infiltration despite marked obesity. Here we demonstrate that acute (3d) high fat diet (HFD) significantly increases bodyweight due to a significant increase in food intake in wild type mice. Body composition analysis by NMR indicates a significant increase in percent body fat with a concomitant decrease in lean mass. Energy expenditure measured by indirect calorimetry was also significantly decreased after 3d HFD. Importantly, both systemic IS measured by oral glucose challenge and insulin‐stimulated adipocyte 2‐deoxyglucose uptake was significantly reduced. No difference was found in adipocyte chemokine or cytokine secretion. We propose that reduction in adipocyte IS and subsequent systemic IS, is due to an increase in AT p50α/p55α abundance and occurs prior to initiation of a pro‐inflammatory response. Identifying primary steps in development of IR may guide treatments for obesity related metabolic diseases.NIH DK095926 (CEM)

A randomized, phase II trial of cetuximab with or without PX-866, an irreversible oral phosphatidylinositol 3-kinase inhibitor, in patients with relapsed or metastatic head and neck squamous cell cancer

The phosphotidylinositol-3 kinase (PI3K)/serine-threonine kinase/mammalian target of rapamycin signaling pathway is frequently altered in head and neck squamous cell cancer (HNSCC). PX-866 is an oral, irreversible, pan-isoform inhibitor of PI3K. A phase I trial demonstrated tolerability of this combination. This randomized phase II study evaluated PX-866 combined with cetuximab in patients with advanced, refractory HNSCC. Patients with recurrent or metastatic HNSCC who had received at least one and no more than two prior systemic treatment regimens were randomized (1 : 1) to cetuximab with or without PX-866 (8 mg p.o. daily; arms A and B, respectively). The primary end point was progression-free survival (PFS). Secondary end points included objective response rate (ORR), overall survival (OS), toxicity, and correlation of key biomarkers with efficacy outcomes. Eighty-three patients were enrolled. There was a similar response rate between arms (10% versus 7%). Of patients for whom tissue was assessable, 57% were human papillomavirus (HPV) positive. Median PFS was 80 days in both arms and there was no difference in OS between the two arms (211 versus 256 days). Overall toxicity was higher in arm A compared with arm B, especially in terms of nausea (53% versus 23%), vomiting (45% versus 15%), fatigue (43% versus 23%), diarrhea (40% versus 21%), and hypokalemia (25% versus 10%). Grade 3 or higher adverse events were infrequent, but more common in the combination arm although without a specific pattern. PIK3CA mutations were observed in 17% of the cases assessed, and PTEN loss was infrequently observed. The addition of PX-866 to cetuximab did not improve PFS, RR, or OS in patients with advanced, refractory HNSCC enrolled without molecular preselection. In this contemporary cohort, HPV-positive patients comprised the majority, and neither HPV-positive nor HPV-negative patients derived clinical benefit for the addition of cetuximab plus PX-866.

Radioresistance in head and neck squamous cell carcinoma: Biological bases and therapeutic implications.

Head and neck squamous cell carcinoma (HNSCC) is strongly associated with alcohol and tobacco consumption. Lately, the incidence of human papillomavirus (HPV)-related tumors has shown a significant increase, and HPV-related tumors show distinctive features if compared with the HPV-negative counterpart. Locally advanced HNSCC can be treated with concomitant chemoradiotherapy, but early recurrences sometimes occur. Relapses are often related to an intrinsic radioresistance of the tumors. Alterations in intracellular pathways, primarily involved in cell proliferation, apoptosis, and DNA repair, can lead to radioresistance. Preclinical and clinical evidence highlighted that 3 main pathways, including the epidermal growth factor receptor (EGFR), the phosphotidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), and the p53 signaling cascades, play a crucial role in radioresistance development. A future approach may consist in the association of radiotherapy (RT) and selective inhibition of the key pathways involved in radioresistance. Phase I, II, and III clinical trials are currently testing these novel treatment strategies.