Levels Of Activated Akt Research Articles

Increased androgen receptor levels and signaling in ovarian cancer cells by VEPH1 associated with suppression of SMAD3 and AKT activation

Studies indicate androgens contribute to initiation or progression of epithelial ovarian cancer through poorly understood mechanisms. We provide evidence that the androgen receptor (AR) interacts in a ligand-independent manner with the putative armadillo repeat domain of ventricular zone expressed PH domain-containing 1 (VEPH1). This interaction was increased by mutation of the two nuclear receptor-interacting LxxLL motifs present within the VEPH1 armadillo repeat domain. Androgen treatment did not result in nuclear co-localization of VEPH1 with AR, suggesting that VEPH1 does not function as a nuclear co-regulatory protein. VEPH1 expression decreased SMAD3 and activated AKT levels in ovarian cancer cell lines and increased AR activity and protein levels, consistent with an impact on receptor stability. Treatment of cells with dihydrotestosterone (DHT) increased AR protein levels measured 24 h after treatment, an effect augmented in VEPH1-transfected cells, and inhibited by knock-down of endogenous VEPH1. SMAD3 overexpression decreased AR protein levels and prevented the VEPH1-dependent increase in AR; however, silencing of SMAD3 paradoxically also decreased AR levels. DHT treatment led to a rapid and sustained decrease in phosphorylated AKT (pAKT) levels that was enhanced by VEPH1 expression. Inhibition of PI3K resulted in increased AR protein levels. These studies indicate that VEPH1 acts to enhance AR activity in ovarian cancer cells by decreasing SMAD3 and pAKT levels, resulting in increased levels of AR protein.

Akt Signaling in Macrophage Polarization, Survival, and Atherosclerosis.

The PI3K/Akt pathway plays a crucial role in the survival, proliferation, and migration of macrophages, which may impact the development of atherosclerosis. Changes in Akt isoforms or modulation of the Akt activity levels in macrophages significantly affect their polarization phenotype and consequently atherosclerosis in mice. Moreover, the activity levels of Akt signaling determine the viability of monocytes/macrophages and their resistance to pro-apoptotic stimuli in atherosclerotic lesions. Therefore, elimination of pro-apoptotic factors as well as factors that antagonize or suppress Akt signaling in macrophages increases cell viability, protecting them from apoptosis, and this markedly accelerates atherosclerosis in mice. In contrast, inhibition of Akt signaling by the ablation of Rictor in myeloid cells, which disrupts mTORC2 assembly, significantly decreases the viability and proliferation of blood monocytes and macrophages with the suppression of atherosclerosis. In addition, monocytes and macrophages exhibit a threshold effect for Akt protein levels in their ability to survive. Ablation of two Akt isoforms, preserving only a single Akt isoform in myeloid cells, markedly compromises monocyte and macrophage viability, inducing monocytopenia and diminishing early atherosclerosis. These recent advances in our understanding of Akt signaling in macrophages in atherosclerosis may have significant relevance in the burgeoning field of cardio-oncology, where PI3K/Akt inhibitors being tested in cancer patients can have significant cardiovascular and metabolic ramifications.

The cholesterol transporter ABCG1 modulates macrophage polarization in human monocyte-derived macrophages

Abstract The ATP-binding Cassette Transporter G1 (ABCG1) promotes cholesterol efflux from cells and regulates intracellular cholesterol homeostasis. Recent studies have shown that in mice in the absence of ABCG1, macrophages shift from a tumor-promoting M2 phenotype to a tumor-fighting M1 phenotype within the tumor and suppress bladder cancer growth in vivo. Two important aspects of this effect are still unknown: (1) whether ABCG1-would also impact polarization of human macrophages, and (2) the underlying molecular mechanism of this effect. Here, we show that in human monocyte-derived macrophages the inhibition of ABCG1 by siRNA significantly increased the expression of the M1 markers IDO1, CXCL10, CD64 and TNF after LPS/IFNγ (M1) stimulation. In contrast, after IL-4 (M2) stimulation the expression of the M2 markers MRC1, TGM2 and CD163 was significantly reduced. These data indicate that ABCG1-deficiency promotes the polarization of macrophage to an M1 phenotype in human macrophages. To address the molecular mechanism, we analysed cell signalling in bone marrow-derived macrophages from Abcg1−/− mice. These macrophages displayed reduced levels of Akt activation after stimulation with LPS/IFNγ or IL4. In addition, while Abcg1−/− macrophages stimulated with LPS/IFNγ produced more TNF compared to WT macrophages, after treatment with an Akt activator the TNF-production of Abcg1−/− and WT macrophages was comparable. These data suggest that the M1 bias of Abcg1−/− macrophages is mediated through the Akt signalling pathway. In summary, these findings deepen our mechanistic understanding of the M1/M2 switch in macrophages and suggest that ABCG1 could be a potential new target to modulate macrophage polarization for cancer immunotherapy.

Microvesicles from human adipose stem cells promote wound healing by optimizing cellular functions via AKT and ERK signaling pathways

BackgroundHuman adipose stem cells (ASCs) have emerged as a promising treatment paradigm for skin wounds. Recent works demonstrate that the therapeutic effect of stem cells is partially mediated by extracellular vesicles, which comprise exosomes and microvesicles. In this study, we investigate the regenerative effects of isolated microvesicles from ASCs and evaluate the mechanisms how ASC microvesicles promote wound healing.MethodsAdipose stem cell-derived microvesicles (ASC-MVs) were isolated by differential ultracentrifugation, stained by PKH26, and characterized by electron microscopy and dynamic light scattering (DLS). We examined ASC-MV effects on proliferation, migration, and angiogenesis of keratinocytes, fibroblasts, and endothelial cells both in vitro and in vivo. Next, we explored the underlying mechanisms by gene expression analysis and the activation levels of AKT and ERK signaling pathways in all three kinds of cells after ASC-MV stimulation. We then assessed the effect of ASC-MVs on collagen deposition, neovascularization, and re-epithelialization in an in vivo skin injury model.ResultsASC-MVs could be readily internalized by human umbilical vein endothelial cells (HUVECs), HaCAT, and fibroblasts and significantly promoted the proliferation, migration, and angiogenesis of these cells both in vitro and in vivo. The gene expression of proliferative markers (cyclin D1, cyclin D2, cyclin A1, cyclin A2) and growth factors (VEGFA, PDGFA, EGF, FGF2) was significantly upregulated after ASC-MV treatment. Importantly, ASC-MVs stimulated the activation of AKT and ERK signaling pathways in those cells. The local injection of ASC-MVs at wound sites significantly increased the re-epithelialization, collagen deposition, and neovascularization and led to accelerated wound closure.ConclusionsOur data suggest that ASC-MVs can stimulate HUVEC, HaCAT, and fibroblast functions. ASC-MV therapy significantly accelerates wound healing, and the benefits of ASC-MVs may due to the involvement of AKT and ERK signaling pathways. This illustrates the therapeutic potential of ASC-MVs which may become a novel treatment paradigm for cutaneous wound healing.

PI3K blockage synergizes with PLK1 inhibition preventing endoreduplication and enhancing apoptosis in anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is among the most lethal malignancies. The mitotic kinase PLK1 is overexpressed in the majority of ATCs and PLK1 inhibitors have shown preclinical efficacy. However, they also cause mitotic slippage and endoreduplication, leading to the generation of tetraploid, genetically unstable cell populations.We hypothesized that PI3K activity may facilitate mitotic slippage upon PLK1 inhibition, and thus tested the effect of combining PLK1 and PI3K inhibitors in ATC models, in vitro and in vivo. Treatment with BI6727 and BKM120 resulted in a significant synergistic effect in ATC cells, independent of the levels of AKT activity. Combination of the two drugs enhanced growth suppression at doses for which the single drugs showed no effect, and led to a massive reduction of the tetraploid cells population. Furthermore, combined treatment in PI3Khigh cell lines showed a significant induction of apoptosis.Finally, combined inhibition of PI3K and PLK1 was extremely effective in vivo, in an immunocompetent allograft model of ATC.Our results demonstrate a clear therapeutic potential of combining PLK1 and PI3K inhibitors in anaplastic thyroid tumors.

Regulation of AKT Activity by Inhibition of the Pleckstrin Homology Domain-PtdIns(3,4,5)P3 Interaction Using Flavonoids.

The serine-threonine kinase AKT plays a pivotal role in tumor progression and is frequently overactivated in cancer cells; this protein is therefore a critical therapeutic target for cancer intervention. We aimed to identify small molecule inhibitors of the pleckstrin homology (PH) domain of AKT to disrupt binding of phosphatidylinositol-3,4,5-trisphosphate (PIP3), thereby downregulating AKT activity. Liposome pulldown assays coupled with fluorescence spectrometry were used to screen flavonoids for inhibition of the AKT PH-PIP3 interaction. Western blotting was used to determine the effects of the inhibitors on AKT activation in cancer cells, and in silico docking was used for structural analysis and optimization of inhibitor structure. Several flavonoids showing up to 50% inhibition of the AKT PH-PIP3 interaction decreased the level of AKT activation at the cellular level. In addition, the modified flavonoid showed increased inhibitory effects and the approach would be applied to develop anticancer drug candidates. In this study, we provide a rationale for targeting the lipid-binding domain of AKT, rather than the catalytic kinase domain, in anticancer drug development.

Reducing the Levels of Akt Activation by PDK1 Knock-in Mutation Protects Neuronal Cultures against Synthetic Amyloid-Beta Peptides.

The Akt kinase has been widely assumed for years as a key downstream effector of the PI3K signaling pathway in promoting neuronal survival. This notion was however challenged by the finding that neuronal survival responses were still preserved in mice with reduced Akt activity. Moreover, here we show that the Akt signaling is elevated in the aged brain of two different mice models of Alzheimer Disease. We manipulate the rate of Akt stimulation by employing knock-in mice expressing a mutant form of PDK1 (phosphoinositide-dependent protein kinase 1) with reduced, but not abolished, ability to activate Akt. We found increased membrane localization and activity of the TACE/ADAM17 α-secretase in the brain of the PDK1 mutant mice with concomitant TNFR1 processing, which provided neurons with resistance against TNFα-induced neurotoxicity. Opposite to the Alzheimer Disease transgenic mice, the PDK1 knock-in mice exhibited an age-dependent attenuation of the unfolding protein response, which protected the mutant neurons against endoplasmic reticulum stressors. Moreover, these two mechanisms cooperatively provide the mutant neurons with resistance against amyloid-beta oligomers, and might singularly also contribute to protect these mice against amyloid-beta pathology.

Hydrogen Sulfide Donor NaHS Improves Metabolism and Reduces Muscle Atrophy in Type 2 Diabetes: Implication for Understanding Sarcopenic Pathophysiology

Sarcopenia, a loss of muscle mass and functionality, constitutes a major contributor to disability in diabetes. Hydrogen sulfide (H2S) dynamics and muscle mass regulatory signaling were studied in GK rats, a model for type 2 diabetes (T2D). GK rats exhibited a number of features that are consistent with sarcopenia and T2D including loss of muscle mass and strength, in addition to glucose intolerance, insulin resistance, and impaired β-cell responsiveness to glucose. Mechanistically, activation levels of Akt, a key modulator of protein balance, were decreased in T2D. Consequently, we confirmed reduced activity of mTOR signaling components and higher expression of atrophy-related markers typified by FoxO1/atrogin-1/MuRF1 and myostatin-Smad2/3 signaling during the course of diabetes. We observed in GK rat reduced antioxidant capacity (↓GSH/GSSG) and increased expression and activity of NADPH oxidase in connection with augmented rate of oxidation of lipids, proteins, and DNA. H2S bioavailability and the expression of key enzymes involved in its synthesis were suppressed as a function of diabetes. Interestingly, GK rats receiving NaHS displayed increased muscle Akt/mTOR signaling and decreased expression of myostatin and the FoxO1/MuRF1/atrogin-dependent pathway. Moreover, diabetes-induced heightened state of oxidative stress was also ameliorated in response to NaHS therapy. Overall, the current data support the notion that a relationship exists between sarcopenia, heightened state of oxidative stress, and H2S deficiency at least in the context of diabetes. Moreover, treatment with a potent H2S donor at an early stage of diabetes is likely to mitigate the development of sarcopenia/frailty and predictably reduces its devastating sequelae of amputation.

Nose-to-brain delivery of insulin enhanced by a nanogel carrier

Recent evidences suggest that insulin delivery to the brain can be an important pharmacological therapy for some neurodegenerative pathologies, including Alzheimer disease (AD). Due to the presence of the Blood Brain Barrier, a suitable carrier and an appropriate route of administration are required to increase the efficacy and safety of the treatment. Here, poly(N-vinyl pyrrolidone)-based nanogels (NG), synthetized by e-beam irradiation, alone and with covalently attached insulin (NG-In) were characterized for biocompatibility and brain delivery features in a mouse model. Preliminarily, the biodistribution of the “empty” nanocarrier after intraperitoneal (i.p.) injection was investigated by using a fluorescent-labeled NG. By fluorescence spectroscopy, SEM and dynamic light scattering analyses we established that urine clearance occurs in 24h. Histological liver and kidneys inspections indicated that no morphological alterations of tissues occurred and no immunological response was activated after NG injection. Furthermore, after administration of the insulin-conjugated nanogels (NG-In) through the intranasal route (i.n.) no alteration or immunogenic response of the nasal mucosa was observed, suggesting that the formulation is well tolerated in mouse. Moreover, an enhancement of NG-In delivery to the different brain areas and of its biological activity, measured as Akt activation levels, with reference to free insulin administration was demonstrated. Taken together, these results indicate that the synthesized NG-In enhances brain insulin delivery upon i.n. administration and strongly encourage its further evaluation as therapeutic agent against some neurodegenerative diseases.

Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration

BackgroundOsteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC) recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca2+-containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO4) on MSC migration. In addition, to evaluate the influence of CaSO4 on MSC differentiation and the potential molecular mechanisms involved.MethodsA circular calvarial bone defect (5 mm diameter) was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO4 concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO4 effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO4 treatment was also evaluated by qPCR.ResultsCaSO4 increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO4 significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO4-containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO4 and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO4 effects on MSC migration.ConclusionsSpecific CaSO4 concentrations induce bone regeneration of calvarial defects in part by acting on the host’s undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO4 regulates BMP-2-induced MSC migration by differentially activating the PI3K/AKT pathway. Altogether, these results suggest that CaSO4 scaffolds could have potential applications for bone regeneration.

RGS12 Is a Novel Tumor-Suppressor Gene in African American Prostate Cancer That Represses AKT and MNX1 Expression.

African American (AA) men exhibit a relatively high incidence and mortality due to prostate cancer even after adjustment for socioeconomic factors, but the biological basis for this disparity is unclear. Here, we identify a novel region on chromosome 4p16.3 that is lost selectively in AA prostate cancer. The negative regulator of G-protein signaling RGS12 was defined as the target of 4p16.3 deletions, although it has not been implicated previously as a tumor-suppressor gene. RGS12 transcript levels were relatively reduced in AA prostate cancer, and prostate cancer cell lines showed decreased RGS12 expression relative to benign prostate epithelial cells. Notably, RGS12 exhibited potent tumor-suppressor activity in prostate cancer and prostate epithelial cell lines in vitro and in vivo We found that RGS12 expression correlated negatively with the oncogene MNX1 and regulated its expression in vitro and in vivo Further, MNX1 was regulated by AKT activity, and RGS12 expression decreased total and activated AKT levels. Our findings identify RGS12 as a candidate tumor-suppressor gene in AA prostate cancer, which acts by decreasing expression of AKT and MNX1, establishing a novel oncogenic axis in this disparate disease setting. Cancer Res; 77(16); 4247-57. ©2017 AACR.

Suppression of a cancer stem-like phenotype mediated by alpha-lipoic acid in human lung cancer cells through down-regulation of β-catenin and Oct-4.

Cancer stem cells (CSCs) that possess the ability of self-renewal and multi-potency have been shown to drive tumor progression and metastasis. The majority of recent studies has focused on potential molecules targeting CSCs so as to develop novel strategies for efficient cancer treatment or protection. Here, we show how alpha-lipoic acid (LA), an endogenous mitochondrial anti-oxidant, affects the CSC-like phenotypes of human non-small cell lung cancer-derived H23, H292 and H460 cells. CSC-like phenotypes were verified by anchorage-independent growth, three-dimensional (3D) spheroid formation and the expression of CSC markers. Enriched CSC populations were used to confirm the effects of LA. Protein ubiquitination and degradation were assessed using immunoprecipitation. We found that treatment with LA reduced the CSC-like phenotype, as indicated by a decreased expression of known CSC markers (CD133, CD44, ALDH1A1, Oct-4 and Nanog) in H460 cells. In addition, we found that LA reduced the CSC-related abilities of anchorage-independent growth and 3D spheroid formation, and suppressed factors related to epithelial-mesenchymal transition, such as E-cadherin, Vimentin, Slug and Snail. Mechanistically, we found that LA suppresses CSC through depletion of the cellular stemness proteins β-catenin and Oct-4 via decreasing the level of active (phosphorylated) Akt. This resulted in the induction of GSK3β-dependent β-catenin ubiquitin-proteasomal degradation and a decrease in the stabilized (phosphorylated) form of Oct-4. The effects of LA on the CSC-like phenotypes were confirmed in CSC enriched H460, H292 and H23non-small cell lung cancer-derived cells. Our data are indicative for a novel regulatory role and underlying mechanism of LA in the negative regulation of a CSC-like phenotype in non-small cell lung cancer-derived cells.

Abstract 3162: HRAS G12V predicts for innate resistance to PI3Kα inhibition in head and neck squamous cell cancer

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with an incidence of ~600 000 new cases per year and a 50% mortality rate for advanced disease. The mutational landscape of HNSCC has been recently elucidated, introducing the possibility for targeted therapeutic approaches. PIK3CA—which encodes the α-catalytic subunit of PI3K (PI3Kα)—is the most frequently altered actionable target in HNSCC, however it is not presently clear which patients benefit most from PI3Kα-inhibition. BYL719 is a leading PI3Kα inhibitor in clinical development for HNSCC. We previously examined the responses of a large panel of genetically-characterized HNSCC cell lines to BYL719 and identified activating HRAS G12V mutations as strong predictors for BYL719 resistance. Here we examine if this mutation is able to individually modulate BYL719 response through overexpression and knockdown studies. Methods: To determine if HRAS G12V was able to modulate BYL719 sensitivity, we knocked down HRAS in HRAS G12V and HRAS wild-type (WT) cell lines using RNA interference, and then treated cells over 10-point dose ranges with BYL719. Sensitivity was determined by calculating IC50 (half-maximal inhibitory concentration) values at 72 hours using PrestoBlue®. Constructs expressing wildtype (WT) HRAS or HRAS G12V were transfected into BYL719-sensitive cells for overexpression studies and cells were selected using G418 Sulfate for 1 month. Proliferation and BYL719 sensitivity (IC50 values) were measured as described. Immunoblotting was used to examine activity of the PI3K-AKT axis with and without BYL719 (1μM) for 24 hours. Results & Conclusions: HRAS knockdown sensitized HRAS G12V lines to BYL719 (lower IC50), but did not affect the response of WT HRAS cells. WT HRAS and HRAS G12V overexpression significantly increased cellular proliferation and promoted BYL719 resistance. In HRAS G12V cell lines, we observed high levels of active phosphorylated AKT (S473/T308), even in the presence of BYL719. Collectively these findings highlight the predictive role of HRAS G12V for innate BYL719 resistance and contribute to our understanding of which patients may respond best on BYL719 therapy. Citation Format: Kara M. Ruicci, Ren Sun, Morgan Black, Nicole Pinto, John Yoo, Kevin Fung, Danielle Macneil, Lauire Aillies, Joseph S. Mymryk, Paul C. Boutros, John W. Barrett, Anthony C. Nichols. HRAS G12V predicts for innate resistance to PI3Kα inhibition in head and neck squamous cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3162. doi:10.1158/1538-7445.AM2017-3162

Abstract 1357: The small GTPase Arf6 potentiates melanoma metastasis by activating Akt

Abstract Arf6 is a member of the Ras-superfamily of small GTPases and controls membrane trafficking and cytoskeletal remodeling, functioning mainly in endocytosis pathways at the cell periphery. Arf6 is activated by various extracellular signals and oncogenic events and has been shown to promote cell migration and pro-invasive phenotype in human cancer cells. Small molecule inhibition of ARF6 reduces spontaneous metastasis in xenograft models of human cutaneous melanoma, suggesting that ARF6 is necessary for disease progression. Using a genetically-engineered mouse model of BRAF-mutant melanoma, we determined whether activation of Arf6 is sufficient to induce spontaneous metastasis in vivo. For melanocyte-specific primary tumor induction, Cre recombinase was delivered via local injection of RCAS virus into DCT-TVA;Cdkn2alox/lox;BRAFV600E mice. In the experimental mice, a constitutively active mutant form of Arf6 (Q67L) was virally delivered with Cre. In this study, we observed a significant increase in spontaneous metastatic disease burden in Arf6 Q67L mice. Likewise, tail vein injection of melanoma cell lines derived from Arf6 Q67L tumors consistently show a diffuse pattern of pulmonary metastasis compared to controls that show rare, microscopic metastasis. Recently, it has been demonstrated that activation of Akt, but not Pten loss, leads to an aggressive phenotype in melanoma that includes the acquisition of brain metastases. Immunohistochemical analysis of phospho-Akt revealed that Arf6 Q67L is sufficient to induce Akt activation in tumors. In addition, ARF6 is necessary for AKT activation in human melanoma lines. We did not observe brain metastasis in DCT-TVA;Cdkn2alox/lox;BRAFV600E + Arf6 Q67L mice. When we added Ptenlox/lox allele to this genetic background, however, we observed microscopic brain metastases at a low frequency, suggesting that the combination of Pten loss and Arf6 activation reaches a threshold level of Akt activation that is sufficient to cause brain metastasis. Taken together our data indicate that activation of Arf6 is sufficient to potentiate melanoma metastasis, consistent with the proinvasive cellular phenotype attributed to Arf6. In addition, our data suggests a novel signaling mechanism by which Arf6, a small GTPase involved in trafficking, is somehow involved in Akt activation and that this step may be important for the acquisition of metastatic capacity. Citation Format: Lehi Acosta, Aaron Rogers, Jae H. Yoo, Shannon J. Odelberg, Dean Y. Li, Sheri Holmen, Allie H. Grossmann. The small GTPase Arf6 potentiates melanoma metastasis by activating Akt [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1357. doi:10.1158/1538-7445.AM2017-1357

Abstract 1068: Driver pathway blockage synergizes with PLK1 inhibition in anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies, with a median survival of less than 6 months from the time of diagnosis. Molecular changes that characterize ATC have been recently well defined and involve most often p53 mutations, and activation of PI3K, RAS and BRAF. PLK1, an essential mitotic regulator, has been found overexpressed in ATC. Previous data from our lab have shown that PLK1 inhibitors are effective in ATC cell lines. However PLK1 inhibition often results in escape from growth arrest through mitotic slippage. This leads to the generation of polyploid, genetically unstable, cell populations. We have tested the effect of combining PLK1 and PI3K inhibitors in ATC cell lines. Combined treatment with the PLK1 inhibitor BI6727 and the PI3K inhibitor BKM120 resulted in a significant synergistic effect in ATC cell lines with high levels of AKT activity, but not in those with undetectable pAKT. Combination of the two drugs enhanced the G2/M arrest at doses in which the single drugs showed no effect. In addition, it led to a massive reduction of the tetraploid cells population. Furthermore, combined treatment with BI6727 and BKM120 in PI3Khigh cell lines showed a significant induction of apoptosis in a time- and dose-dependent manner. Combined inhibition of PI3K and PLK1 was extremely effective in inhibiting tumor growth in vivo, in an immunocompetent allograft model of ATC. Our results show that combination of PLK1 and PI3K inhibitors is an effective treatment for ATC cells with high levels of PI3K signaling. This combination results in cell cycle arrest, inhibition of of tetraploid cell generation, and induction of apoptosis, suggesting a clear therapeutic potential. Citation Format: Daniela De Martino, Emrullah Yilmaz, Arturo Orlacchio, Antonio Di Cristofano. Driver pathway blockage synergizes with PLK1 inhibition in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1068. doi:10.1158/1538-7445.AM2017-1068

AKT and its related molecular feature in aged mice skin.

Previous studies suggest that Akt signaling promotes tissue regeneration and decreased Akt activities are found in aged tissues. However, this study finds that the expression and activation levels of Akt in the mice skin increased with age. Additionally, the expression levels of Pten, p16, p21 and p53 also elevated with increased age. Immuno-fluorescence analysis showed that Akt phosphorylation found in the epidermal cells (with increased levels of NF-κB activation) were also found. In vivo inhibition of AKT activity result in reduced NF-κB activation. Our results suggest that increasing Akt/ NF-κB is a crucial mediator of skin aging, which can increase the susceptibility of cell transformation.

Ras, Erk, and PI3K signaling pathways in the central selection of B cells

Abstract B cell selection mechanisms in the bone marrow control the nature of the primary B cell repertoire, generating diverse specificities while reducing autoreactivity. Our studies aim to characterize the mechanisms that drive the positive selection of immature B cells in the bone marrow. It has been shown that the B cell receptor (BCR) is able to signal in the absence of ligand (tonic signal) and this signal is required for B cell development. We hypothesize that alterations in tonic signaling could cause aberrant positive selection of autoreactive immature B cells. We have previously shown that low levels of active Ras, Erk, and Akt correlate with levels of BCR tonic signal and that constitutive activation of Ras allows for differentiation of autoreactive immature B cells in vitro. We are currently testing the individual contributions of the Erk and PI3K pathways in this selection process. By using retroviral transduction of primary bone marrow B cells in vitro, we show that Erk activation can overcome a lack of tonic signaling to induce differentiation of nonautoreactive or slightly autoreactive B cells. We have also found that the Dual Specificity Phosphatase 5 (DUSP5), a novel phosphatase, may play a role in Erk regulation and the selection of immature B cells. In order to study these pathways in vivo we generated mouse models in which constitutively active forms of MEK or PI3K are specifically expressed in autoreactive B cells to determine if activation of these molecules is sufficient to bypass central tolerance. Preliminary data suggests that in vivo activation of PI3K can drive development of autoreactive B cells and allow some escape to the periphery.

VEPH1 expression decreases vascularisation in ovarian cancer xenografts and inhibits VEGFA and IL8 expression through inhibition of AKT activation

Background:VEPH1 is amplified in several cancers including ovarian but its impact on tumour progression is unknown. Previous work has shown that VEPH1 inhibits TGFβ signalling while its Drosophila ortholog increases tissue growth, raising the possibility that VEPH1 could impact tumour growth or progression.Methods:A CRISPR approach was used to disrupt VEPH1 expression in ovarian cancer ES-2 cells, while VEPH1-negative SKOV3 cells were stably transfected with VEPH1 cDNA. The impact of altered VEPH1 expression was assessed using in vitro and in vivo assays and mechanistic studies were performed in vitro.Results:VEPH1 expression in SKOV3 cells resulted in a reduced tumour growth rate associated with increased necrotic area, and decreased microvessel density and VEGF-A levels relative to tumours formed by mock-transfected cells. VEPH1 expression also decreased VEGFA and IL8 expression in SKOV3 cells and was associated with decreased activated AKT levels. These effects were not observed in ES-2 cells, which bear a BRAFV600E activating mutation that leads to constitutively increased IL8 and VEGFA expression.Conclusions:VEPH1 expression in SKOV3 ovarian cancer cells inhibits AKT activation to decrease VEGFA and IL8 expression, which leads to decreased tumour vascularisation and progression.

CCDC88A, a prognostic factor for human pancreatic cancers, promotes the motility and invasiveness of pancreatic cancer cells.

BackgroundCoiled-Coil Domain Containing 88A (CCDC88A) was identified as a substrate of the serine/threonine kinase Akt that is capable of binding to the actin cytoskeleton. The aim of this study was to investigate the potential role of CCDC88A in the migration and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells.MethodsImmunohistochemistry was performed to determine whether high CCDC88A expression in human PDAC tissues is correlated with poor prognosis. Immunoprecipitation, immunoblotting and immunocytochemistry were performed to determine the intracellular distribution of CCDC88A, and its association with the serine/threonine kinase Akt and actin-filaments in PDAC cells. Phosphoprotein array analysis was performed to determine CCDC88A-associated intracellular signaling pathways. Finally, immunofluorescence analyses and Matrigel invasion assays were performed to examine the effects of CCDC88A on the formation of cell protrusions and PDAC cell invasion.ResultsExpression of CCDC88A in PDAC tissue was significantly correlated with overall survival. CCDC88A was co-localized with peripheral actin structures in cell protrusions of migrating PDAC cells. Knockdown of CCDC88A inhibited the migration and invasiveness of PDAC cells through a decrease in cell protrusions. Although CCDC88A has been previously reported to be a binding partner and substrate of Akt, the level of active Akt was not associated with the translocation of CCDC88A towards cell protrusions. CCDC88A-dependent promotion of cell migration and invasiveness was not modulated by Akt signaling. Knockdown of CCDC88A decreased phosphorylated Src and ERK1/2 and increased phosphorylated AMPK1 in PDAC cells. Knockdown of AMPK1 inhibited the migration and invasiveness of PDAC cells. The combined data suggest that CCDC88A may be a useful marker for predicting the outcome of patients with PDAC and that CCDC88A can promote PDAC cell migration and invasion through a signaling pathway that involves phosphorylation of Src and ERK1/2 and/or dephosphorylation of AMPK1.ConclusionsCCDC88A was accumulated in cell protrusions, contributed to the formation of membrane protrusions, and increased the migration and invasiveness of PDAC cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0466-0) contains supplementary material, which is available to authorized users.

The relevance of PTEN-AKT in relation to NOTCH1-directed treatment strategies in T-cell acute lymphoblastic leukemia.

The tumor suppressor phosphatase and tensin homolog (PTEN) negatively regulates phosphatidylinositol 3-kinase (PI3K)-AKT signaling and is often inactivated by mutations (including deletions) in a variety of cancer types, including T-cell acute lymphoblastic leukemia. Here we review mutation-associated mechanisms that inactivate PTEN together with other molecular mechanisms that activate AKT and contribute to T-cell leukemogenesis. In addition, we discuss how Pten mutations in mouse models affect the efficacy of gamma-secretase inhibitors to block NOTCH1 signaling through activation of AKT. Based on these models and on observations in primary diagnostic samples from patients with T-cell acute lymphoblastic leukemia, we speculate that PTEN-deficient cells employ an intrinsic homeostatic mechanism in which PI3K-AKT signaling is dampened over time. As a result of this reduced PI3K-AKT signaling, the level of AKT activation may be insufficient to compensate for NOTCH1 inhibition, resulting in responsiveness to gamma-secretase inhibitors. On the other hand, de novo acquired PTEN-inactivating events in NOTCH1-dependent leukemia could result in temporary, strong activation of PI3K-AKT signaling, increased glycolysis and glutaminolysis, and consequently gamma-secretase inhibitor resistance. Due to the central role of PTEN-AKT signaling and in the resistance to NOTCH1 inhibition, AKT inhibitors may be a promising addition to current treatment protocols for T-cell acute lymphoblastic leukemia.