Phosphatidylinositide 3-kinase Pathway Research Articles

Inhibition of PI3K by copanlisib exerts potent antitumor effects on Merkel cell carcinoma cell lines and mouse xenografts

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer with steadily increasing incidence and poor prognosis. Despite recent success with immunotherapy, 50% of patients still succumb to their diseases. To date, there is no Food and Drug Administration-approved targeted therapy for advanced MCC. Aberrant activation of phosphatidylinositide-3-kinase (PI3K)/AKT/mTOR pathway is frequently detected in MCC, making it an attractive therapeutic target. We previously found PI3K pathway activation in human MCC cell lines and tumors and demonstrated complete clinical response in a Stage IV MCC patient treated with PI3K inhibitor idelalisib. Here, we found that both PI3K-α and -δ isoforms are abundantly expressed in our MCC cell lines and clinical samples; we therefore examined antitumor efficacy across a panel of five PI3K inhibitors with distinctive isoform-specificities, including idelalisib (PI3K-δ), copanlisib (PI3K-α/δ), duvelisib (PI3K-γ/δ), alpelisib (PI3K-α), and AZD8186 (PI3K-β/δ). Of these, copanlisib exerts the most potent antitumor effects, markedly inhibiting cell proliferation, survival, and tumor growth by suppressing PI3K/mTOR/Akt activities in mouse models generated from MCC cell xenografts and patient-derived tumor xenografts. These results provide compelling preclinical evidence for application of copanlisib in advanced MCC with aberrant PI3K activation for which immunotherapy is insufficient, or patients who are unsuitable for immunotherapy.

Therapeutic hypothermia reduced brain damage on rats after cardiopulmonary resuscitation by activating III-type PI3K pathway to increase autophagy

To investigate the effect of III-type phosphatidylinositide 3 kinase (PI3K) pathway adjusting autophagy on brain damage mechanism after cardiopulmonary resuscitation (CPR) and hypothermia treatment. The asphyxia induce cardiac arrest-CPR model was reproduced on healthy male Sprague-Dawley (SD) rats. Sixty rats after restoration of spontaneous circulation (ROSC) were randomly divided into normothermia group, therapeutic hypothermia group and PI3K inhibitor 3-methyl adenine (3-MA) pretreatment group, differentiated by 24 hours and 48 hours after ROSC. Each group had 10 rats at each time point. The anal temperature in the normothermia group was maintained at (37.0±0.2) centigrade, and the rats in the hypothermia group were given cooling treatment immediately after ROSC, and the target rectal temperature was 32-34 centigrade. In the 3-MA pretreatment group, 10 mmol/L 3-MA 5 μL was injected into the ventricle 20 minutes before asphyxia, and other groups were given the same amount of normal saline. Ten rats without CPR were included in Sham group only received anesthesia and catheterization. The rats were sacrificed at 24 hours and 48 hours after ROSC respectively, and the brain tissues were harvested, the brain water content (BWC) was measured by dry-wet weight method. Western Blot was used to determine the autophagy related proteins Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3), apoptosis related proteins Bcl-2 and caspase-3, and the III-type PI3K pathway proteins Vps34 and Atg14. Ultrastructural changes in brain tissue were observed with transmission electron microscope. Neurological deficit scores (NDS) was obtained in each group at 48 hours after ROSC. Compared with Sham group, the cortex at 24 hours after ROSC in normothermic group showed obvious edema, apoptosis and autophagy began to appear under transmission electron microscope, and the expressions of autophagy, apoptosis and III- type PI3K-related proteins in brain tissue were significantly increased in a time-dependent manner, and the neurological function at 48 hours after ROSC was significantly damaged. After hypothermia intervention, brain edema of rats was significantly reduced, no obvious apoptosis was found, but autophagy was increased, the expressions of autophagy-related proteins Vps34, Atg14 and III-type PI3K-related proteins Beclin-1 and LC3 at 48 hours after ROSC were further higher than those of normothermic group (Vps34/GAPDH: 0.25±0.03 vs. 0.15±0.04, Atg14/GAPDH: 0.12±0.03 vs. 0.05±0.04, Beclin-1/GAPDH: 0.060±0.002 vs. 0.018±0.002, LC3 -II/GAPDH: 0.160±0.010 vs. 0.050±0.010, all P < 0.05), the expressions of apoptosis related proteins Bcl-2 and caspase-3 were significantly lowered (Bcl-2/GAPDH: 0.05±0.03 vs. 0.20±0.04, caspase-3/GAPDH: 0.050±0.002 vs. 0.140±0.015, both P < 0.05), neurological function was significantly improved (NDS: 157±85 vs. 343±198, P < 0.05). Pretreatment with 3-MA inhibited the protective effect of hypothermia on brain tissues. The expressions of Vps34, Atg14, Beclin-1 and LC3 in brain tissues at 48 hours after ROSC in 3-MA pretreatment group was significantly lower than those in the hypothermia group (Vps34/GAPDH: 0.18±0.03 vs. 0.25±0.03, Atg14/GAPDH: 0.07±0.04 vs. 0.12±0.03, Beclin-1/GAPDH: 0.015±0.003 vs. 0.060±0.002, LC3-II/GAPDH: 0.045±0.030 vs. 0.160±0.010, all P < 0.05), the expressions of Bcl-2 and caspase-3 were significantly increased (Bcl-2/GAPDH: 0.15±0.04 vs. 0.05±0.03, caspase-3/GAPDH: 0.120±0.015 vs. 0.050±0.002, both P < 0.05), and NDS score was significantly increased (341±208 vs. 157±85, P < 0.05). Hypothermia treatment reduced brain edema and ameliorated brain function after CPR, which might be related to increase autophagy and inhibit apoptosis adjustment by activating III-type PI3K pathway.

Thioredoxin Confers Intrinsic Resistance to Cytostatic Drugs in Human Glioma Cells.

Thioredoxin (Trx) overexpression is known to be a cause of chemotherapy resistance in various tumor entities. However, Trx effects on resistance are complex and depend strictly on tissue type. In the present study, we analyzed the impact of the Trx system on intrinsic chemoresistance of human glioblastoma multiforme (GBM) cells to cytostatic drugs. Resistance of GBM cell lines and primary cells to drugs and signaling inhibitors was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Impact of Trx inhibition on apoptosis was investigated by proteome profiling of a subset of proteins and annexin V apoptosis assays. Trx-interacting protein (TXNIP) was overexpressed by transfection and protein expression was determined by immunoblotting. Pharmacological inhibition of Trx by 1-methyl-2-imidazolyl-disulfide (PX-12) reduced viability of three GBM cell lines, induced expression of active caspase-3, and reduced phosphorylation of AKT-kinase and expression of β-catenin. Sensitivity to cisplatin could be restored by both PX-12 and recombinant expression of the upstream Trx inhibitor TXNIP, respectively. In addition, PX-12 also sensitized primary human GBM cells to temozolomide. Combined inhibition of Trx and the phosphatidylinositide 3-kinase (PI3K) pathway resulted in massive cell death. We conclude that the Trx system and the PI3K pathway act as a sequential cascade and could potentially present a new drug target.

The phosphatidylinositide 3-kinase (PI3K) signaling pathway is a determinant of zileuton response in adults with asthma

Variable responsiveness to zileuton, a leukotriene antagonist used to treat asthma, may be due in part to genetic variation. While individual SNPs were previously associated with zileuton-related lung function changes, specific quantitative trait loci (QTLs) and biological pathways that may contribute have not been identified. In this study, we investigated the hypothesis that genetic variation within biological pathways is associated with zileuton response. We performed an integrative QTL mapping and pathway enrichment study to investigate data from a GWAS of zileuton response, in addition to mRNA expression profiles and leukotriene production data from lymphoblastoid cell lines (LCLs) (derived from asthmatics) that were treated with zileuton or ethanol (control). We identified 1060 QTLs jointly associated with zileuton-related differential LTB4 production in LCLs and lung function change in patients taking zileuton, of which eight QTLs were also significantly associated with persistent LTB4 production in LCLs following zileuton treatment (i.e., ‘poor’ responders). Four nominally significant trans-eQTLs were predicted to regulate three candidate genes (SELL, MTF2, and GAL), the expression of which was significantly reduced in LCLs following zileuton treatment. Gene and pathway enrichment analyses of QTL associations identified multiple genes and pathways, predominantly related to phosphatidyl inositol signaling via PI3K. We validated the PI3K pathway activation status in a subset of LCLs demonstrating variable zileuton-related LTB4 production, and show that in contrast to LCLs that responded to zileuton, the PI3K pathway was activated in poor responder LCLs. Collectively, these findings demonstrate a role for the PIK3 pathway and its targets as important determinants of differential responsiveness to zileuton.

S1PR1 drives a feedforward signalling loop to regulate BATF3 and the transcriptional programme of Hodgkin lymphoma cells

The Hodgkin/Reed–Sternberg cells of classical Hodgkin lymphoma (HL) are characterised by the aberrant activation of multiple signalling pathways. Here we show that a subset of HL displays altered expression of sphingosine-1-phosphate (S1P) receptors (S1PR)s. S1P activates phosphatidylinositide 3-kinase (PI3-K) in these cells that is mediated by the increased expression of S1PR1 and the decreased expression of S1PR2. We also showed that genes regulated by the PI3-K signalling pathway in HL cell lines significantly overlap with the transcriptional programme of primary HRS cells. Genes upregulated by the PI3-K pathway included the basic leucine zipper transcription factor, ATF-like 3 (BATF3), which is normally associated with the development of dendritic cells. Immunohistochemistry confirmed that BATF3 was expressed in HRS cells of most HL cases. In contrast, in normal lymphoid tissues, BATF3 expression was confined to a small fraction of CD30-positive immunoblasts. Knockdown of BATF3 in HL cell lines revealed that BATF3 contributed to the transcriptional programme of primary HRS cells, including the upregulation of S1PR1. Our data suggest that disruption of this potentially oncogenic feedforward S1P signalling loop could provide novel therapeutic opportunities for patients with HL.

Regulation of the Phosphatidylinositide 3-Kinase Pathway by the Lipid Phosphatase PTEN.

Featured Article: Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T, et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 1998;95:29–39.4 PTEN is a lipid phosphatase whose action targets phosphatidylinositol 3,4,5-trisphosphate (PIP3),5 a component of the lipid cellular membrane. Its role as a tumor suppressor was defined in 1998 in the article by Stambolic et al. highlighted here (1), which reported the extensive effects of the disruption of PTEN (phosphatase and tensin homolog) in mice. By the late 1990s, a quest for the major tumor suppressor gene within the frequently deleted region of chromosome 10 found in advanced cancers culminated in the discovery of PTEN, named based on amino acid sequence homology with the protein tyrosine phosphatases and tensin (1). The observations reported in the 1998 article contributed to the understanding of the cellular functions of PTEN and the severity of the consequences associated with PTEN loss. Homozygous …

Development and characterization of a novel long-acting recombinant follicle stimulating hormone agonist by fusing Fc to an FSH-β subunit

Does a novel long-acting recombinant human FSH, KN015, a heterodimer composed of FSHα and FSHβ-Fc/Fc, offer a potential FSH alternative? KN015 had in vitro activity and superior in vivo bioactivity than recombinant human FSH (rhFSH), suggesting KN015 could serve as a potential FSH agonist for clinical therapy. rhFSH has very short half-life so that repeat injections are needed, resulting in discomfort and inconvenience for patients. The longest-acting rhFSH available in clinics is corifollitropin alpha (FSH-CTP), but its half-life is not long enough to sustain the whole therapy period, and additional injections of rhFSH are needed. Plasmids containing FSHα, FSHβ-Fc and Fc cDNA were transfected into Chinese hamster ovary (CHO) cells for KN015 production. The pharmacokinetics of KN015 was investigated in 6-week-old SD rats (n = 6/group) and healthy Cynomolgus monkeys in two different dose groups (n = 2/group). A series of experiments were designed for in vitro and in vivo characterization of the bioactivity of KN015 relative to rhFSH. The purity and molecular weight of KN015 were determined by reducing and non-reducing SDS-PAGE. To measure KN015 half-life, sera were collected at increasing time points and the remaining FSH concentration was measured by enzyme-linked immunosorbent assay. To assess the bioactivity of KN015 versus rhFSH in vitro, firstly cAMP production was assessed in CHO cells expressing FSH receptor (FSHR) with the treatment of Fc/Fc, rhFSH or KN015 at eight different doses (0.03, 0.09, 0.28, 0.83, 2.5, 7.5, 22.5, 67.5 nM), and secondly cumulus oocyte complexes (COCs; n = 20/group) of ICR mice (primed-PMSG 44 h before sacrificed) were collected and cultured in medium containing 1.25 pM Fc/Fc, rhFSH or KN015 at 37°C and then germinal vesicle breakdown (GVBD) and COC expansion were observed at 4 and 16 h, respectively. The in vivo activity of KN015 was compared with rhFSH by ovary weight gain and ovulation assays. In the former, ovary weight gains in 21-day-old female SD rats, after a single subcutaneous injection of KN015, were compared with those after several injections of rhFSH over a range of doses (n = 8/group). Sera were harvested for estradiol (E2) analysis, and the ovaries were processed for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL), RT-PCR and western blot. In the latter, 26-day-old female SD rats (n = 8/group) were injected with different doses of KN015 or rhFSH, and were sacrificed at 24 h after an injection of hCG (20 IU/rat). Moreover, the molecular responses stimulated by KN015 or rhFSH in the ovary were also analyzed through detecting expression of the FSH target genes (Cyp19a1, Fshr and Lhcgr) and phosphatidylinositide 3-kinase (PI3K) pathway activation. KN015 has a molecular weight of 82 kD and its half-life is 84 h in SD rats (10-fold longer than that of rhFSH) and 215 h in Cynomolgus monkeys. The EC50 value of the cAMP induction in CHO cells (KN015 versus rhFSH, 1.84 versus 0.87 nM), COC expansion and oocyte maturation assays showed KN015 had approximately half of rhFSH's activity in vitro. A single dose of KN015 (1.5 pmol/rat, 166.1 ± 19.7 mg, P < 0.01) stimulated significantly larger ovary weight gain than several injections of rhFSH (1.5 pmol/rat, 59.3 ± 28.1 mg, P < 0.01). The serum E2 level in the KN015 group was significantly higher than that in rhFSH group. The number of oocytes obtained by ovulation induction was comparable with or higher in the KN015 group than in the rhFSH group. KN015 was more effective than rhFSH in inducing FSH target genes (Cyp19a1, Fshr, Lhcgr) or activating the PI3K pathway in vivo. Moreover, a single injection of KN015 promoted granulosa cell proliferation and prevented follicle atresia to the same extent as several injections of rhFSH. All assays in this study were operated only in animals and clinical trials are needed to confirm they can be extrapolated to humans. KN015 is a valuable alternative to FSH and may have great potential for therapeutic applications. This study was supported by National Basic Research Program of China (2011|CB944504, 2012CB944403) and National Natural Science Foundation of China (81172473, 31371449). The authors have no conflicts of interest to declare.

Abstract PR01: Inhibitory role of phosphatidylinositol-3,4-bisphosphate in triple-negative breast cancers

Abstract Triple-negative breast cancer (lacking expression of estrogen receptor, progesterone receptor and amplification of HER2/Neu) remains one of the most aggressive subtypes, affects the youngest patients and yet still lacks an effective targeted therapy. Novel insights into the molecular mechanisms that drive these cancers are imperative to guide development and application of such targeted therapies. Data from The Cancer Genome Atlas and other sources have suggested that phospho-Akt (pAkt) levels are significantly higher in triple-negative tumors compared to either hormone receptor positive tumors (express estrogen and/or progesterone receptor) or Her2/Neu amplified tumors. This has led to the hypothesis that these tumors are dependent on phosphatidylinositide-3-kinase (PI3K) and that PI3K pathway inhibitors may be effective therapeutically in triple-negative tumors. Both PI3K-α and β are ubiquitously expressed and therefore may be important for the oncogenesis of solid tumors, including those of the breast. INPP4B catalyzes the removal of the 4′ phosphate of phosphatidylinositol-(3,4)-bisphosphate creating phosphatidylinositol-(3)-phosphate. There has been debate concerning whether phosphatidylinositol-(3,4)-bisphosphate can contribute to activation of Akt and other downstream effectors, in addition to phosphatidylinositol-(3,4,5)-trisphosphate, the canonical signaling second messenger effector downstream of PI3K. If this were true, INPP4B would have a negative effect on signaling. Utilizing a panel of cell lines derived from triple-negative breast tumors that are null for PTEN, we used both siRNA-mediated silencing and stable transfectants expressing either Flag-INPP4B or a mutant of Flag-INPP4B(Cys842Ala) lacking phosphatase activity to manipulate levels of phosphatidylinositol-(3,4,5)-trisphosphate and phosphatidylinositol-(3,4)-bisphosphate. We then employed a panel of PI3K pan- and isoform-selective inhibitors to investigate how changing the levels of phosphatidylinositol phosphate species effected inhibitor sensitivity. We have found that silencing INPP4B in several triple-negative breast cancer cell lines sensitizes cells to PI3Kα-isoform selective inhibitors and, to an even greater degree, PI3Kβ-isoform selective inhibitors. This is evidenced by both a decrease in pAkt levels, as detected by Western blot, as well as a decrease in GI50, as detected by proliferation assays following siRNA treatment. Conversely, introduction of an extra copy of INPP4B into the same cell lines results in a decrease in sensitization to both PI3Kα- and PI3Kβ-isoform selective inhibitors, in this case to a greater degree with the PI3Kα-isoform selective inhibitor than the PI3Kβ-isoform selective inhibitor. This is especially evident by a marked dampening of the decrease in pAkt levels that is seen in response to the inhibitors in both the parental cell line and stable cell lines expressing a catalytically dead mutant of INPP4B. Thus, this is an effect requiring the phosphatase activity of INPP4B. Unexpectedly, our data support a model in which phosphatidylinositol-(3,4)-bisphosphate is in fact inhibitory toward PI3K, at least under conditions of strong signaling, as in triple negative breast cancers. When levels of this inhibitor accumulate following siRNA-mediated knockdown of INPP4B, cells become sensitized to PI3K pathway inhibitors. Conversely, when levels are depleted following overexpression of INPP4B, cells become resistant to PI3K pathway inhibitors. This may indicate a novel feedback inhibition by a downstream substrate within the PI3K pathway under conditions of excessive signaling. This work has important implications for the future of targeted therapy by PI3K pathway inhibitors. Furthermore, it may reveal a unique indication for PI3Kβ-isoform selective inhibitors in triple-negative breast cancers that have lost both PTEN and INPP4B. This abstract is also being presented as Poster A21. Citation Format: Darien E. Reed, Kevan M. Shokat. Inhibitory role of phosphatidylinositol-3,4-bisphosphate in triple-negative breast cancers. [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 PR01.

Inhibitory effect of Puerariae radix flavones on platelet-derived growth factor-BB-induced proliferation of vascular smooth muscle cells via PI3K and ERK pathways.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) results in intimal thickening of the aorta, which may lead to arteriosclerosis. Therefore, VSMC antiproliferative agents may be efficient in the prevention and treatment of arteriosclerosis. Puerariae radix (PR) is the dried root of Pueraria lobata Ohwi or Pueraria thomsonii Benth. Flavones are the main components of PR and have been shown to have a protective effect on vascular disorders in traditional Chinese medicine treatments. However, the underlying molecular mechanism remains unclear. The aim of the present study was to explore the effect of PR flavone (PRF) on platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation. PDGF-BB (25 ng/ml) and different doses of PRF (10, 50, 100 and 200 ng/ml) were used to treat VSMCs. The results revealed that PRF notably inhibited the PDGF-BB-induced VSMC proliferation and induced a cell cycle arrest at growth 1 phase of the cell cycle. In addition, cell cycle-associated proteins, including cyclin D1, proliferating cell nuclear antigen and cyclin-dependent kinase 4, were found to be downregulated. Furthermore, PRF inhibited the PDGF-BB-stimulated downregulation of VSMC markers, including α-smooth muscle actin, desmin and smoothelin. PDGF-BB upregulated the phosphorylation levels of phosphatidylinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK), which are associated with cell proliferation; however, these were decreased following PRF treatment. These observations indicated that PRF had a suppressive effect on PDGF-BB-induced VSMC proliferation by inhibiting PI3K and ERK pathways.

Exposure of breast cancer cells to a subcytotoxic dose of apigenin causes growth inhibition, oxidative stress, and hypophosphorylation of Akt

Epidemiological studies show that fruit- and vegetable-rich diets are associated with a reduced risk of developing certain forms of cancer, including breast cancer. In this study we demonstrate that a subcytotoxic concentration of apigenin, which is a flavone found at high concentrations in parsley, onions, grapefruit, oranges, and chamomile tea, inhibited DNA synthesis in a panel of human breast cancer cell lines (MDA-MB-231, MBA-MB-468, MCF-7, SK-BR-3). Decreased proliferation of MDA-MB-468 cells in the presence of apigenin was associated with G2/M phase cell cycle arrest and the production of reactive oxygen species. Apigenin-treated MDA-MB-468 cells also showed reduced phosphorylation of Akt (protein kinase B), which is an essential effector serine/threonine kinase in the phosphatidylinositide 3-kinase pathway that promotes tumor growth and progression. However, exposure to the antioxidant reduced glutathione failed to reverse apigenin-mediated inhibition of Akt phosphorylation and cell proliferation, indicating that these effects were not due to oxidative stress. Taken together, these findings suggest that low-dose apigenin has the potential to slow or prevent breast cancer progression.

Signals Controlling Un‐Differentiated States in Embryonic Stem and Cancer Cells: Role of the Phosphatidylinositol 3′ Kinase Pathway

The capacity of embryonic stem (ES) cells to differentiate into cell lineages comprising the three germ layers makes them powerful tools for studying mammalian early embryonic development in vitro. The human body consists of approximately 210 different somatic cell types, the majority of which have limited proliferative capacity. However, both stem cells and cancer cells bypass this replicative barrier and undergo symmetric division indefinitely when cultured under defined conditions. Several signal transduction pathways play important roles in regulating stem cell development, and aberrant expression of components of these pathways is linked to cancer. Among signaling systems, the critical role of leukemia inhibitory factor (LIF) coupled to the Jak/STAT3 (signal transduction and activation of transcription-3) pathway in maintaining stem cell self-renewal has been extensively reviewed. This pathway additionally plays multiple roles in tumorigenesis. Likewise, the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway has been determined to play an important role in both stem cell maintenance and tumor development. This pathway is often induced in cancer with frequent mutational activation of the catalytic subunit of PI3K or loss of a primary PI3K antagonist, phosphatase and tensin homolog deleted on chromosome ten (PTEN). This review focusses on roles of the PI3K signal transduction pathway components, with emphasis on functions in stem cell maintenance and cancer. Since the PI3K pathway impinges on and collaborates with other signaling pathways in regulating stem cell development and/or cancer, aspects of the canonical Wnt, Ras/mitogen-activated protein kinase (MAPK), and TGF-β signaling pathways are also discussed. J. Cell. Physiol. 229: 1312–1322, 2014.

PIK3CA Activating Mutations in Facial Infiltrating Lipomatosis

Facial infiltrating lipomatosis is a nonheritable disorder characterized by hemifacial soft-tissue and skeletal overgrowth, precocious dental development, macrodontia, hemimacroglossia, and mucosal neuromas. The authors tested the hypothesis that this condition is caused by a somatic mutation in the phosphatidylinositide-3 kinase (PI3K) signaling pathway, which has been indicted in other anomalies with overgrowth. The authors extracted DNA from abnormal tissue in six individuals, generated sequencing libraries, enriched the libraries for 26 genes involved in the PI3K pathway, and designed and applied a sequential filtering strategy to analyze the sequence data for mosaic mutations. Unfiltered sequence data contained variant reads affecting ~12 percent of basepairs in the targeted genes. Filtering reduced the fraction of targeted basepairs containing variant reads to ~0.008 percent, allowing the authors to identify causal missense mutations in PIK3CA (p.E453K, p.E542K, p.H1047R, or p.H1047L) in each affected tissue sample. Affected tissue from individuals with facial infiltrating lipomatosis contains PIK3CA mutations that have previously been reported in cancers and in affected tissue from other nonheritable, overgrowth disorders, including congenital lipomatous overgrowth, vascular, epidermal, and skeletal anomalies syndrome, Klippel-Trenaunay syndrome, hemimegalencephaly, fibroadipose overgrowth, and macrodactyly. Because PIK3CA encodes a catalytic subunit of PI3K, and in vitro studies have shown that the overgrowth-associated mutations increase this enzyme's activity, PI3K inhibitors currently in clinical trials for patients with cancer may have a therapeutic role in patients with facial infiltrating lipomatosis. The strategy used to identify somatic mutations in patients with facial infiltrating lipomatosis is applicable to other somatic mosaic disorders that have allelic heterogeneity.

PIK3CA, HRAS and PTEN in human papillomavirus positive oropharyngeal squamous cell carcinoma

BackgroundRecent genomic evidence suggests frequent phosphatidylinositide 3-kinase (PI3K) pathway activation in human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma. Mutations/amplification of the gene encoding p110α catalytic subunit of phosphoinositide 3-kinase (PIK3CA), loss of phosphatase and tensin homolog (PTEN) and HRAS mutations are known to activate PI3K pathway.Methods and resultsPIK3CA mutations were identified by Sanger sequencing in 23 of 75 (31%) HPV-positive oropharyngeal carcinomas, including exon 9 (p.E545K [n = 10] and p.E542K [n = 5]) or exon 20 (p.H1047Y, n = 2) mutations. Five rare and one novel (p.R537Q) PIK3CA mutations were identified. HRAS mutation (p.Q61L) was detected in 1 of 62 tested cases. PIK3CA amplification by fluorescence in situ hybridization (FISH) was identified in 4 cases (4/21, 20%), while PTEN loss was seen in 7 (7/21, 33%) cases (chromosome 10 monosomy [n = 4], homozygous deletion [n = 3]).ConclusionsOverall, genetic alterations that likely lead to PI3K pathway activation were identified in 34 of 75 cases (45%) and did not correlate with disease specific survival. These findings offer a molecular rationale for therapeutic targeting of PI3K pathway in patients with HPV-positive oropharyngeal carcinoma.

PI3K inhibitor GDC-0941 enhances apoptotic effects of BH-3 mimetic ABT-737 in AML cells in the hypoxic bone marrow microenvironment

Both phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling and antiapoptotic Bcl-2 family members are critical for survival of acute myeloid leukemia (AML) cells. Here, we demonstrate the antileukemic effects of simultaneous inhibition of PI3K by the selective class I PI3K inhibitor GDC-0941 and of Bcl-2 family members by the BH3 mimetic ABT-737 in the context of the bone marrow microenvironment, where hypoxia and interactions with bone marrow stromal cells promote AML cell survival and chemoresistance. The combination of GDC-0941 and ABT-737 profoundly downregulated antiapoptotic Mcl-1 expression levels, activated BAX, and induced mitochondrial apoptosis in AML cells co-cultured with bone marrow stromal cells under hypoxic conditions. Hypoxia caused degradation of Mcl-1 and rendered Mcl-1-overexpressing OCI-AML3 cells sensitive to ABT-737. Our findings suggest that pharmacologic PI3K inhibition by GDC-0941 enhances ABT-737-induced leukemia cell death even under the protective conditions afforded by the bone marrow microenvironment. Combined blockade of PI3K and Bcl-2 pathways down-regulates anti-apoptotic Mcl-1 expression PI3K and Bcl-2 induced Mcl-1 down-regulation activates BAX PI3K and Bcl-2 blockage induces apoptosis in AML under hypoxic BM microenvironment.

Inhibiting the phosphatidylinositide 3-kinase pathway blocks radiation-induced metastasis associated with Rho-GTPase and Hypoxia-inducible factor-1 activity

Background and purposeUndifferentiated follicular and anaplastic thyroid tumours often respond poorly to radiotherapy and show increased metastatic potential. We evaluated radiation-induced effects on metastasis in thyroid carcinoma cells and tumours, mechanistically focusing on phosphatidylinositide 3-kinase (PI3K) and associated pathways. Material and methodsMigration was analysed in follicular (FTC133) and anaplastic (8505c) cells following radiotherapy (0–6 Gray) with concomitant pharmacological (GDC-0941) or genetic inhibition of PI3K. Hypoxia-inducible factor-1 (HIF-1)-activity was measured using luciferase reporter assays and was inhibited using a dominant-negative variant. Activation and subcellular localisation of target proteins were assessed via Western blot and immunofluorescence. In vivo studies used FTC133 xenografts with metastatic lung dissemination assessed ex vivo. ResultsRadiation induced migration in a HIF-dependent manner in FTC133 cells but decreased migration in 8505c’s. Post-radiation HIF-activity correlated with migratory phenotype. PI3K-targeting inhibited migration under basal and irradiated conditions through inhibition of HIF-1α, Rho-GTPase expression/activity and localisation whilst having little effect on src/FAK. In vivo, radiation induced PI3K, HIF, Rho-GTPases and src but only PI3K, HIF and Rho-GTPases were inhibited by GDC-0941. Co-treatment with GDC-0941 and radiation significantly reduced metastatic dissemination versus radiotherapy alone. ConclusionsRadiation modifies metastatic characteristics of thyroid carcinoma cells, which can be successfully inhibited by targeting PI3K using GDC-0941 in vitro and in vivo.

Molecular changes in the phosphatidylinositide 3‐kinase (PI3K) pathway are common in gastric cancer

The phosphatidylinositide 3-kinase (PI3K) pathway is an important signalling pathway that is frequently activated in cancer cells. This has led to the emergence of PI3K inhibitors as potential new treatment modalities for many cancers. We have investigated the frequency of molecular changes in the PI3K pathway in gastric cancer. A series of sixty one human gastric cancer specimens and nine human gastric cancer cell lines were screened for PIK3CA mutations and copy number gain by direct sequencing and multiplex ligation-dependent probe amplification (MLPA), respectively. PTEN protein levels were assessed by immunohistochemistry. Alterations in the PI3K pathway were found in 33 of 61 (54%) gastric tumours. PIK3CA mutation and copy number gain were detected in 3 (4.9%) and 8 (13.1%), respectively, of 61 gastric cancer samples while PTEN loss was detected in 24 (39%) of the tumours. Two tumours had both PTEN loss and PIK3CA copy number gain. There were no significant associations between these PI3K pathway changes and the clinical features of the tumours. Alterations in the PI3K pathway are frequent in gastric tumours implicating this pathway as a legitimate therapeutic target in gastric cancer.

MTOR Inhibition Elicits a Dramatic Response in PI3K-Dependent Colon Cancers

The phosphatidylinositide-3-kinase (PI3K) signaling pathway is critical for multiple cellular functions including metabolism, proliferation, angiogenesis, and apoptosis, and is the most commonly altered pathway in human cancers. Recently, we developed a novel mouse model of colon cancer in which tumors are initiated by a dominant active PI3K (FC PIK3ca*). The cancers in these mice are moderately differentiated invasive mucinous adenocarcinomas of the proximal colon that develop by 50 days of age. Interestingly, these cancers form without a benign intermediary or aberrant WNT signaling, indicating a non-canonical mechanism of tumorigenesis. Since these tumors are dependent upon the PI3K pathway, we investigated the potential for tumor response by the targeting of this pathway with rapamycin, an mTOR inhibitor. A cohort of FC PIK3ca* mice were treated with rapamycin at a dose of 6 mg/kg/day or placebo for 14 days. FDG dual hybrid PET/CT imaging demonstrated a dramatic tumor response in the rapamycin arm and this was confirmed on necropsy. The tumor tissue remaining after treatment with rapamycin demonstrated increased pERK1/2 or persistent phosphorylated ribosomal protein S6 (pS6), indicating potential resistance mechanisms. This unique model will further our understanding of human disease and facilitate the development of therapeutics through pharmacologic screening and biomarker identification.

Abstract 362: [18F]-FLT Positron Emission Tomography can be used to image the response of sensitive tumours to PI3-Kinase inhibition with the novel agent GDC-0941.

Abstract Emerging targeted therapeutics are characterised by high efficacy rates in selected patient sub-populations as well as potentially high running costs. This calls for companion diagnostic tools that contribute to the better management of such therapies, in terms of patient selection and monitoring therapeutic response. The Phosphatidylinositide 3-kinase pathway is deregulated in a range of cancers, and several targeted inhibitors are entering the clinic. This study aimed to investigate whether the PET tracer [18F]-FLT is suitable to mark the effect of the novel PI-3K inhibitor GDC-0941 which has entered Phase II clinical trial. Methods Nude mice bearing U87 and HCT116 xenografts were imaged at baseline with [18F]-FLT (10-15MBq per mouse produced using a GE Tracerlab). GDC-0941 or vehicle was then administered at a concentration of 50mg/kg twice daily by gavage and animals were rescanned at acute (∼18h) and chronic (∼186h) timepoints post-therapy. Standard uptake value (SUV)s for tumour uptake were calculated, and tissue was analysed at sacrifice for phospho-AKT. To generate normalised uptake values (NUV)s the SUV [tumour] was divided by the SUV for heart. NUV figures are given for the 100-105min post-injection time interval. Results Growth of U87 xenografts was significantly inhibited by GDC-0941 treatment throughout the course of the study (average growth rate of treated = –1.0±9.3 mm3, control = 69.2±22.1mm3 per day). Growth of HCT116 xenografts was not significantly different between groups (average growth rate of treated = 34.6±15.9 mm3, control =54.8±11.1 mm3 per day). Tumour uptake of [18F]-FLT was significantly reduced in treated animals bearing U87 xenografts at the acute timepoint compared to baseline (NUVmax 2.17 ± 0.38 vs 1.59 ± 0.29, p&amp;lt;0.01), whereas uptake in HCT116 tumours was unchanged. Analysis of the downstream marker phospho-AKT showed that this was decreased in U87, but not HCT116, tumours. Conclusion [18F]-FLT is a strong candidate for the non-invasive measurement of GDC-0941 action. In addition to FLT, we are assessing the performance of a range of other imaging agents, including proprietary tracers, that can potentially bring value in monitoring of PI3-kinase targeting therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 362. doi:1538-7445.AM2012-362

Insulin-like growth factor-1 induces the phosphorylation of PRAS40 via the PI3K/Akt signaling pathway in PC12 cells

Insulin-like growth factor-1 (IGF-1) is a polypeptide tropic factor that plays an important role in the survival and differentiation of both neuronal and non-neuronal cells. Numerous studies have demonstrated that IGF-1 promotes neuronal cell survival via the PI3K/Akt signaling pathway. Proline-rich Akt substrate of 40kDa (PRAS40) is a recently discovered downstream target of Akt. However, the relationship between IGF-1 and PRAS40 is not known. In this study, we characterized the phosphorylation of PRAS40 induced by IGF-1 in PC12 cells and explored the signaling pathway responsible for the effect of IGF-1. IGF-1 induced the phosphorylation of Akt at Thr473 and PRAS40 at Thr246 in PC12 cells. The phosphorylation of Akt and PRAS40 induced by IGF-1 (100ng/ml) was inhibited by the phosphatidylinositide 3-kinase (PI3K) specific inhibitor LY294002 (50μM), while no inhibitory effect was observed for a MAPK kinase pathway specific inhibitor PD98059 nor a p38 MAPK inhibitor PD169316, suggesting that the phosphorylation of PRAS40 induced by IGF-1 is mediated by the PI3K pathway in PC12 cells and primary cultured neurons. In further support this hypothesis, an Akt kinase specific inhibitor, Akt inhibitor VIII, attenuated IGF-1-induced phosphorylation of PRAS40 at the concentration that blocked the phosphorylation of Akt induced by IGF-1. Taken together, these data demonstrate that IGF-1 stimulates the phosphorylation of PRAS40 at Thr246 in neuronal cells and the effect of IGF-1 is mediated, at least in part, by the PI3K/Akt signaling pathway.

Phosphatidylinositide-3-Kinase Inhibitors: Addressing Questions of Isoform Selectivity and Pharmacodynamic/Predictive Biomarkers in Early Clinical Trials

In Journal of Clinical Oncology, Bendell et al present a clinical phase I dose-escalation study of the orally bioavailable phosphatidylinositide-3-kinase (PI3K) inhibitor NVP-BKM120. Over the last 2 years, preliminary conference reports have described the initial phase I clinical experience for several PI3K inhibitors with different selectivity profiles. The article from Bendell et al is the first full peer-reviewed publication reporting the phase I clinical results with a pan–class I selective PI3K inhibitor lacking activity against mammalian target of rapamycin (mTOR). Briefly, the authors report that NVP-BKM120 is well tolerated with an acceptable dosedependent safety profile. Toxicities reported were predominantly rash, hyperglycaemia, and mood alterations, which may reflect ontarget effects of PI3K inhibition in normal tissues. Dose-dependent molecular pharmacodynamic biomarker modulation was also reported, and there was evidence of biologic activity, including a single partial response and a number of patients with prolonged stable disease of at least 8 months duration. In this Understanding the Pathway article, we discuss the findings in the context of our present knowledge of pathogenic PI3K signaling in cancer, and we highlight topical issues in the field, particularly addressing currently unresolved questions concerning the optimal isoform selectivity of PI3K inhibitors and use of proof-of-mechanism and predictive biomarkers. PI3Ks are a family of mainly lipid kinases that regulate an intracellular signal transduction pathway—or, more accurately, a signaling network—which in turn controls many features of cell behavior, including growth, survival, motility, metabolism, and additional specialized functions (Fig 1). Four distinct PI3K subfamilies exist— commonly referred to as classes I, II, III and IV—categorized by their substrate specificities, primary structures, modes of regulation, and domain content. Of these, it is the class I isoforms (p110 , , , ) and class IV PI3K-related protein kinase mTOR that have become the most intensively examined PI3K family targets for small-molecule therapeutics. The key mechanistic rationale for this stems from the observation of frequent genetic and epigenetic activations of the PI3K pathway in cancer by a range of different molecular mechanisms (Fig 1). These findings strongly suggest that activation of the PI3K pathway is a critical step in oncogenesis, a view supported by multiple lines of experimental evidence. Since the identification of the fungal natural product wortmannin as a PI3K inhibitor, high-throughput screening of compound collections coupled to medicinal chemistry optimization, often supported by structure-based design, has led to the discovery and development of a plethora of chemically diverse inhibitors that possess a range of overlapping or distinct PI3K subtype selectivity profiles. Many of these are now progressing into early clinical trials. Proof-of-mechanism pharmacodynamic biomarkers used to demonstrate target engagement and pathway modulation are critical for the implementation of the pharmacologic audit trail, enabling rational optimization of dose and schedule of administration. The dose-dependent hyperglycemia reported for NVP-BKM120 is an example of a class effect consistent with PI3K pathway inhibition. Inhibition of PI3K abrogates the actions of insulin, mainly mediated by the PI3K p110 isoform, resulting in increased blood glucose and a compensatory release of insulin and C-peptide. Increased blood glucose has commonly been used as a pharmacodynamic marker of PI3K pathway inhibition; however, Bendell et al suggest that C-peptide levels may provide a better, indirect biomarker. Reduced [F]fluorodeoxyglucose levels in tumors detected by positron emission tomography also indicated a metabolic response, resulting at least in part from a direct effect of PI3K inhibition on glucose uptake. It is important to establish the degree and duration of PI3K pathway inhibition necessary to achieve durable antitumor effects. Molecular pharmacodynamic biomarkers of signaling output such as phosphorylation of ribosomal protein S6 (RPS6), PRAS40, 4E binding protein 1, and AKT (Fig 1) have been used as indicators of PI3K pathway inhibition in both tumor and surrogate tissues. We previously showed that concentrations of PI3K inhibitors required to block phosphorylation of downstream pathway proteins such as RPS6 are approximately 10-fold higher than those needed for inhibition of the more proximal AKT phosphorylation (Fig 1). Here, Bendell et al detected a 40% to 85% decrease of RPS6 phosphorylation in skin biopsies; there were no equivalent RPS6 biomarker data from tumors, so it is difficult to assess how this degree of inhibition in skin would relate to effects in malignant tissue. However, previous studies with another pan–class I PI3K inhibitor—GDC-0941—showed that a JOURNAL OF CLINICAL ONCOLOGY U N D E R S T A N D I N G T H E P A T H W A Y VOLUME 30 NUMBER 3 JANUARY 2