Phosphoinositide 3-kinase Signaling Cascade Research Articles

Curcumin's Dose-Dependent Attenuation of Gastric Cancer Cell Progression Via the PI3K Pathway Blockade.

Background: Gastric cancer stands as a primary cause of cancer-related deaths worldwide, making the discovery of new therapeutic agents essential for enhancing treatment outcomes. Curcumin, a polyphenolic compound found in turmeric (Curcuma longa), has demonstrated potential in multiple cancer types due to its anti-cancer characteristics. This research aimed to examine the impact of curcumin on gastric cancer cell growth, migration, and invasion, as well as its influence on the phosphoinositide 3-kinase (PI3K) signaling cascade. Methods: Gastric cancer cell lines were exposed to varying curcumin concentrations, followed by assessments of cell viability, migration, and invasion. Furthermore, gene and protein expression levels associated with the PI3K signaling cascade were evaluated using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Results: The findings revealed a dose-dependent decrease in cell viability, migration, and invasion in gastric cancer cells treated with curcumin. Additionally, curcumin administration led to the downregulation of key genes and proteins within the PI3K signaling process, such as PI3K, Akt, and mTOR. Conclusion: These findings propose that curcumin may exercise its anti-cancer effects on gastric cancer cells, partly by suppressing the PI3K signaling pathway. This study's outcomes support curcumin's potential as a therapeutic agent for gastric cancer and encourage further exploration of its underlying molecular mechanisms and in vivo effectiveness.

Antcin K Inhibits TNF-α, IL-1β and IL-8 Expression in Synovial Fibroblasts and Ameliorates Cartilage Degradation: Implications for the Treatment of Rheumatoid Arthritis

Extracts from Taiwan’s traditional medicinal mushroom, Antrodia cinnamomea, exhibit anti-inflammatory activities in cellular and preclinical studies. However, this paper is the first to report that Antcin K, a triterpenoid isolated from A. cinnamomea, inhibits proinflammatory cytokine production in human rheumatoid synovial fibroblasts (RASFs), which are major players in rheumatoid arthritis (RA) disease. In our analysis of the mechanism of action, Antcin K inhibited the expression of three cytokines (tumor necrosis factor alpha [TNF-α], interleukin 1 beta [IL-1β] and IL-8) in human RASFs; cytokines that are crucial to RA synovial inflammation. Notably, incubation of RASFs with Antcin K reduced the phosphorylation of the focal adhesion kinase (FAK), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT) and nuclear factor-κB (NF-κB) signaling cascades, all of which promote cytokine production in RA. Intraperitoneal injections of Antcin K (10 mg/kg or 30 mg/kg) attenuated paw swelling, cartilage degradation and bone erosion, and decreased serum levels of TNF-α, IL-1β, IL-8 in collagen-induced arthritis (CIA) mice; in further experiments, IL-6 levels were similarly reduced. The inhibitory effects of Antcin K upon TNF-α, IL-1β and IL-8 expression in human RASFs was achieved through the downregulation of the FAK, PI3K, AKT and NF-κB signaling cascades. Our data support clinical investigations using Antcin K in RA disease.

Emergence of mTOR mutation as an acquired resistance mechanism to AKT inhibition, and subsequent response to mTORC1/2 inhibition

Acquired resistance to molecular targeted therapy is a significant challenge of the precision medicine era. The ability to understand these mechanisms of resistance may improve patient selection and allow for the development of rationally designed next-line or combination treatment strategies and improved patient outcomes. AKT is a critical effector of the phosphoinositide 3-kinase signaling cascade, one of the most commonly activated pathways in human cancer. Deregulation of signaling pathways, such as RAF/MEK/ERK are previously described mechanisms of resistance to AKT/PI3K inhibitors. Mutations in the mTOR gene, however, are exceedingly rare. We present a case of acquired mTOR resistance, following targeted AKT inhibition, and subsequent response to mTOR1/2 inhibitor in a patient with metastatic endometrial cancer, the first documented response to ATP-competitive mTOR inhibition in this setting. This case supports mTOR mutation as a mechanism of resistance, and underscores the importance of tumor molecular profiling, exemplifying precision medicine in action.

Ephrin A1 promotes proliferation of bovine endometrial cells with abundant expression of proliferating cell nuclear antigen and cyclin D1 changing the cell population at each stage of the cell cycle.

Ephrin A1 has a role in a variety of biological events, including cell proliferation, differentiation, migration, and angiogenesis. Ephrin A1 expression is abundant in trophoblasts and endometrial cells during the implantation period; however, its intracellular activities have not yet been reported in bovine endometrial (BEND) epithelial cells. The aim of this study was to identify the functional role of ephrin A1 in BEND cells, which have served as a good model system for investigating the regulation of signal transduction following treatment with interferon-τ (IFNT) in vitro. Supplementation of ephrin A1 to BEND cells increased cell proliferation and increased levels of proliferating cell nuclear antigen and cyclin D1 protein in BEND cell nuclei. To investigate intracellular mechanisms regulated by ephrin A1, we performed Western blot analysis focused on mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling, which are significantly involved in the successful maintenance of pregnancy. Ephrin A1 dose-dependently increased phosphorylation of extracellular signal-regulated kinases (ERK)1/2, c-Jun N-terminal kinases (JNK), P38, protein kinase B (AKT), P70S6K, S6, and cyclin D1, and the activated proteins were suppressed by pharmacological inhibitors including wortmannin (a PI3K inhibitor), U0126 (an ERK1/2 inhibitor), and SP600125 (a JNK inhibitor). Among ephrin A1 receptors, abundant expression of EPHA2 and EPHA4 messenger RNA was detected in BEND cells by reverse transcription polymerase chain reaction analysis. Furthermore, tunicamycin-induced endoplasmic reticulum (ER) stress was inactivated by ephrin A1 treatment of BEND cells. Our findings suggest that ephrin A1 promotes the development of BEND cells and likely enhances uterine capacity and maintenance of pregnancy by activating MAPK and PI3K signaling cascades and by restoring ER stress.

Phosphoinositide 3-Kinase Signaling Pathway in Pancreatic Ductal Adenocarcinoma Progression, Pathogenesis, and Therapeutics.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by its sudden manifestation, rapid progression, poor prognosis, and limited therapeutic options. Genetic alterations in key signaling pathways found in early pancreatic lesions are pivotal for the development and progression of pancreatic intraepithelial neoplastic lesions into invasive carcinomas. More than 90% of PDAC tumors harbor driver mutations in K-Ras that activate various downstream effector-signaling pathways, including the phosphoinositide-3-kinase (PI3K) pathway. The PI3K pathway also responds to stimuli from various growth factor receptors present on the cancer cell surface that, in turn, modulate downstream signaling cascades. Thus, the inositide signaling acts as a central node in the complex cellular signaling networks to impact cancer cell growth, motility, metabolism, and survival. Also, recent publications highlight the importance of PI3K signaling in stromal cells, whereby PI3K signaling modifies the tumor microenvironment to dictate disease outcome. The high incidence of mutations in the PI3K signaling cascade, accompanied by activation of parallel signaling pathways, makes PI3K a promising candidate for drug therapy. In this review, we describe the role of PI3K signaling in pancreatic cancer development and progression. We also discuss the crosstalk between PI3K and other major cellular signaling cascades, and potential therapeutic opportunities for targeting pancreatic ductal adenocarcinoma.

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

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

Advances in the Development of Class I Phosphoinositide 3-Kinase (PI3K) Inhibitors.

The PI3K signaling cascade is the key moderator of cell proliferation, survival, motility, and apoptosis. Class I PI3K proteins are well characterized and linked to thrombosis (PI3Kβ), rheumatoid arthritis (PI3Kδ), and cancer (PI3Kα). In this review, we explore the latest progress in the design and development of selective Class I PI3K inhibitors from the perspective of drug design and structure activity relationships.

PIK3CA mutations can initiate pancreatic tumorigenesis and are targetable with PI3K inhibitors.

Aberrations in the phosphoinositide 3-kinase (PI3K) signaling pathway have a key role in the pathogenesis of numerous cancers by altering cell growth, metabolism, proliferation and apoptosis. Interest in targeting the PI3K signaling cascade continues, as new agents are being clinically evaluated. PIK3CA mutations result in a constitutively active PI3K and are present in a subset of pancreatic cancers. Here we examine mutant PIK3CA-mediated pancreatic tumorigenesis and the response of PIK3CA mutant pancreatic cancers to dual PI3K/mammalian target of rapamycin (mTOR) inhibition. Two murine models were generated expressing a constitutively active PI3K within the pancreas. An increase in acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasms (PanINs) was identified. In one model these lesions were detected as early as 10 days of age. Invasive pancreatic ductal adenocarcinoma developed in these mice as early as 20 days of age. These cancers were highly sensitive to treatment with dual PI3K/mTOR inhibition. In the second model, PanINs and invasive cancer develop with a greater latency owing to a lesser degree of PI3K pathway activation in this murine model. In addition to PI3K pathway activation, increased ERK1/2 signaling is common in human pancreatic cancers. Phosphorylation of ERK1/2 was also investigated in these models. Phosphorylation of ERK1/2 is demonstrated in the pre-neoplastic lesions and invasive cancers. This activation of ERK1/2 is diminished with dual PI3K/mTOR inhibition. In summary, PIK3CA mutations can initiate pancreatic tumorigenesis and these cancers are particularly sensitive to dual PI3K/mTOR inhibition. Future studies of PI3K pathway inhibitors for patients with PIK3CA mutant pancreatic cancers are warranted.

Phosphoinositide-3-kinase p110δ isoform promotes influenza pathogenicity (MUC5P.858)

Abstract We have found that mice deficient in a tissue specific isoform of the signaling enzyme phosphoinositide-3 kinase (PI3K) p110δ are protected from PR8 influenza virus infection and have reduced weight loss and lung viral loads post-infection. Lung inflammation and immune cell infiltration is also decreased after viral challenge. In addition, p110δ KO mice had fewer influenza virus infected alveolar epithelial cells when infected with a GFP-flu virus and GFP expression was measured in lung epithelial cells. Cytokine measurements in bronchoalveolar lavages suggest p110δ KO mice exhibit increased IFN-gamma and MIG post PR8 infection. Slightly increased numbers of NK cells are detected in the lungs of p110δ KO mice as early as day 3 post-infection however activation was decreased within this population as compared to controls, suggesting they are not the primary source of resistance to influenza. In order to determine which cell type was causing the decreased pathogenicity we used two different tissue specific knockout mouse lines. Mice with a myeloid specific KO of p110δ showed similar weight loss and viral loads to C57BL/6 controls, suggesting alveolar macrophages are not primarily responsible for the observed protection in p110δ KO mice. However, mice with alveolar epithelial cell (AEC) specific deletion of p110δ were protected from influenza virus challenge. These data suggest PI3K signaling cascade is important for influenza virus protection within AEC.

Initiation of human astrovirus type 1 infection was blocked by inhibitors of phosphoinositide 3-kinase

BackgroundUpon initial contact with a virus, host cells activate a series of cellular signaling cascades that facilitate viral entry and viral propagation within the cell. Little is known about how the human astrovirus (HAstV) exploits signaling cascades to establish an infection in host cells. Recent studies showed that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is important for HAstV infection, though the involvement of other signaling cascades remains unclear.MethodsA panel of kinase blockers was used to search for cellular signaling pathways important for HAstV1 infection. To determine their impact on the infectious process, we examined viral gene expression, RNA replication, and viral RNA and capsid protein release from host cells.ResultsInhibitors of phosphoinositide 3-kinase (PI3K) activation interfered with the infection, independent of their effect on ERK 1/2 activation. Activation of the PI3K signaling cascade occurred at an early phase of the infection, judging from the timeframe of Akt phosphorylation. PI3K inhibition at early times, but not at later times, blocked viral gene expression. However, inhibiting the downstream targets of PI3K activation, Akt and Rac1, did not block infection. Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production.ConclusionsOur results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1. PI3K participates in the early stage of infection, possibly during the viral entry process. Our results also reveal the role of PKA in viral production.

Depletion of histone deacetylase 3 antagonizes PI3K-mediated overgrowth of Drosophila organs through the acetylation of histone H4 at lysine 16

Core histone modifications play an important role in chromatin remodeling and transcriptional regulation. Histone acetylation is one of the best-studied gene modifications and has been shown to be involved in numerous important biological processes. Herein, we demonstrated that the depletion of histone deacetylase 3 (Hdac3) in Drosophila melanogaster resulted in a reduction in body size. Further genetic studies showed that Hdac3 counteracted the organ overgrowth induced by overexpression of insulin receptor (InR), phosphoinositide 3-kinase (PI3K) or S6 kinase (S6K), and the growth regulation by Hdac3 was mediated through the deacetylation of histone H4 at lysine 16 (H4K16). Consistently, the alterations of H4K16 acetylation (H4K16ac) induced by the overexpression or depletion of males-absent-on-the-first (MOF), a histone acetyltransferase that specifically targets H4K16, resulted in changes in body size. Furthermore, we found that H4K16ac was modulated by PI3K signaling cascades. The activation of the PI3K pathway caused a reduction in H4K16ac, whereas the inactivation of the PI3K pathway resulted in an increase in H4K16ac. The increase in H4K16ac by the depletion of Hdac3 counteracted the PI3K-induced tissue overgrowth and PI3K-mediated alterations in the transcription profile. Overall, our studies indicated that Hdac3 served as an important regulator of the PI3K pathway and revealed a novel link between histone acetylation and growth control.

Apo- and holo-lactoferrin stimulate proliferation of mouse crypt cells but through different cellular signaling pathways

Lactoferrin (Lf), an iron-binding glycoprotein in human milk, plays an important role in intestinal epithelial cell proliferation, but the underlying molecular mechanisms remain unknown. Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinases (ERK) and the phosphoinositide 3-kinase (PI3K) cascades are chief pathways controlling cell proliferation. We hypothesized that Lf stimulates proliferation of intestinal epithelial cells by activating ERK and PI3K signaling cascades and that lactoferrin receptor (LfR) is involved in these processes. Confocal microscopy showed that LfR is expressed and localized at the plasma membrane of mouse crypt cells isolated from 7 to 10 days old pups, specifically binding both iron-free Lf (apo-Lf) and iron-saturated Lf (holo-Lf). Using the BrdU assay, we observed that apo-Lf had a stronger stimulatory effect than holo-Lf on proliferation of crypt cells. Both function-blocking antibody against LfR and MEK inhibitor (U0126) resulted in inhibitory effects on apo-Lf-induced cell proliferation, whereas PI3K inhibitor (LY294002) significantly decreased both apo- and holo-Lf-induced proliferation. Although both apo- and holo-Lf up-regulated transcription of cyclin D1, an effector of ERK1/2 and PI3K/Akt signaling cascades, only apo-Lf initiated ERK1/2 signaling and both apo- and holo-Lf were capable of activating the PI3K/Akt signaling pathway. Function-blocking antibody against LfR and LfR siRNA inhibited apo-Lf induced activation of the ERK1/2 signaling pathway, suggesting that LfR is involved in apo-Lf induced activation of the ERK1/2, but not the PI3K signaling pathway. In conclusion, apo-Lf and holo-Lf stimulate intestinal proliferation but through different signaling pathways.

A stimulatory TSH receptor antibody enhances adipogenesis via phosphoinositide 3-kinase activation in orbital preadipocytes from patients with Graves' ophthalmopathy

Graves' ophthalmopathy (GO) is characterized by expanded volume of the orbital tissues associated with elevated serum levels of TSH receptor (TSHR) autoantibodies. Because previous studies have demonstrated evidence of adipogenesis within the GO orbit, we sought to determine whether M22, a human monoclonal antibody directed against TSHR, enhances adipogenesis in orbital fibroblasts from patients with GO and, if so, to identify signaling mechanisms involved. GO orbital fibroblast cultures (n=10) were treated for 10 days with bovine TSH (1 or 10·0 U/l) or M22 (1 or 10 ng/ml) in serum-free adipocyte differentiation medium. Some cultures also received a phosphoinositide 3-kinase (PI3K) inhibitor or an inhibitor of cAMP production. In other experiments, confluent cultures (n=8) were treated for between 1 and 30 min with TSH (0·1–10·0 U/l) or M22 (0·1–100 ng/ml) with measurement of cAMP production or levels of phosphorylated AKT (pAKT). We found levels of adiponectin, leptin, and TSHR mRNA to be increased in GO cultures treated for 10 days with either M22 (2·6 mean fold ±0·7; P=0·03) or TSH (13·2±5·8-fold, P=0·048). In other studies, M22 and TSH stimulated cAMP production and pAKT levels in GO cells. Inhibition of PI3K activity during 10 days in culture decreased the levels of M22-stimulated mRNA encoding adiponectin (67±12%; P=0·021), as well as adiponectin and CCAAT/enhancer-binding protein α protein levels. In conclusion, M22 is a pro-adipogenic factor in GO orbital preadipocytes. This antibody appears to act via the PI3K signaling cascade, suggesting that inhibition of PI3K signaling may represent a potential novel therapeutic approach in GO.

LFA-1-dependent Ca2+ Entry following Suboptimal T Cell Receptor Triggering Proceeds without Mobilization of Intracellular Ca2+

A surge in cytosolic calcium ion concentration by entry of extracellular Ca2+ is a hallmark of T cell activation. According to store-operated Ca2+ entry mechanism, the Ca2+ entry is preceded by activation of phospholipase C-gamma1 (PLC-gamma1) and the consequent mobilization of intracellular Ca2+. Using membrane vesicles expressing the mouse class I major histocompatibility complex, i.e. Ld plus costimulatory ligands, i.e. B7-1 and intercellular adhesion molecule-1 along with 2C T cell receptor transgenic T cells, we investigated the roles of CD28 and LFA-1 (lymphocyte function-associated antigen-1) in the activation of PLC-gamma1 and Ca2+ signaling. Both CD28 and LFA-1 made significant and comparable contributions to the activation of PLC-gamma1 as gauged by the level of its phosphorylation at tyrosine 783. In contrast, their roles in Ca2+ signaling were quite distinct so that LFA-1/intercellular adhesion molecule-1 interaction exerted a determining role, whereas CD28/B7-1 interaction played only a minimal role. In particular, when the T cells were activated by suboptimal T cell receptor stimulation, LFA-1 played an indispensable role in the Ca2+ signaling. Further experiments using Ca2+-free medium demonstrated that the entry of extracellular Ca2+ was not always accompanied by mobilization of intracellular Ca2+. Thus, intracellular Ca2+ mobilization was hardly detected under the condition that LFA-1 played the indispensable role in the entry of extracellular Ca2+, while a distinct level of intracellular Ca2+ mobilization was readily detected under the condition that LFA-1 played only the supporting role. These results ensure the unique role of LFA-1 in T cell Ca2+ signaling and reveal that LFA-1-dependent Ca2+ entry proceeds via a mechanism separate from store-operated Ca2+ entry.

Synthetic lethality when combining phosphoinositide 3-kinase alpha and beta catalytic subunit-directed RNAi and estrogen deprivation for estrogen receptor positive breast cancer: implications for clinical trial design with pharmacological inhibitors.

Abstract Abstract #3063 Background: Endocrine therapy with estrogen deprivation (ED) induces cell cycle arrest in sensitive estrogen receptor positive (ER+) breast cancers, but typically not apoptosis, thus relapse is common despite ED due to residual surviving tumor cells. The phosphoinositide 3-kinase (PI3K) pathway, which promotes cell survival, is hyperactivated in ER+ breast cancer due to mutations of genes in the PI3K signaling cascade, most commonly PIK3CA. We therefore investigated the role of p110α and p110β, the gene products of PIK3CA and PIK3CB, in promoting cell survival in ER+ breast cancer.
 Materials and Methods: RNA interference (RNAi) was used to selectively inhibit p110α and p110β expression in the ER+, PIK3CA mutant MCF-7 and T47D cell lines; the ER+, PIK3CA wild-type but PIK3CB gene amplified HCC712 cell line; and the ER-, PIK3CA and PIK3CB wild-type MDA-MB-231 cell line. Western blotting was used to confirm p110α and p110β knockdown efficiencies and to determine the effects of specific knockdowns on PI3K pathway signaling. Cell line growth was measured using a resazurin reduction assay. Apoptosis was quantified using TUNEL/Hoechst staining. Gene copy number for PIK3CB was assessed by FISH in cell lines and tumor samples.
 Results: PIK3CA RNAi inhibited growth and activated apoptosis in all tested ER+ cell lines under ED, whereas the addition of estrogen reversed PIK3CA RNAi-induced apoptosis. PIK3CB RNAi inhibited proliferation and promoted apoptosis in the absence of estrogen only in the PIK3CB-gene amplified HCC712 cell line. In combination, PIK3CA and PIK3CB gene knockdowns produced a synergistic apoptotic effect on T47D and HCC712 cell lines, but not in MCF-7 cells which contain low levels of p110β protein. In contrast PIK3CA and PIK3CB RNAi did not promote apoptosis in the ER- MDA-MB-231 cell line. Finally, since PIK3CB gene amplification has not been described previously we conducted a screen by FISH and array CGH and detected low-level PIK3CB copy number gain or amplification in ∼5% of clinical samples from a series of breast cancer patients representing diverse clinical phenotypes (ER+, ER-, ERBB2+).
 Conclusions: Inhibition of the PI3K pathway through suppression of PI3K catalytic subunit expression produces a synthetic lethal effect in the absence of estrogen specifically in ER+ breast cancer cells. These data establish a preclinical rationale for combining endocrine therapy with PI3K pharmacological inhibitors in ER+ breast cancer, but suggest that both PIK3CA and PIK3CB are therapeutic targets and gene mutation or amplification in both catalytic subunits may have to be taken into account when identiying a sensitive population for PI3 kinase-directed therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3063.

Delayed preconditioning with NS1619 protects cultured cortical neurons against both apoptotic and necrotic cell deaths

NS1619, a potent activator of the large conductance Ca2+ activated potassium (BKCa) channel, has been demonstrated to induce preconditioning in the heart. The aim of our study was to test the delayed preconditioning effect of NS1619 in rat cortical neuronal cultures against oxygen‐glucose deprivation, H2O2, or glutamate excitotoxicity. We also investigated its actions on reactive oxygen species (ROS) generation, and on mitochondrial and plasma membrane potentials. In addition, we tested the activation of the phosphoinositide 3‐kinase (PI3K) signaling pathway, and the effect of NS1619 on caspase‐3/7. NS1619 dose‐dependently protected the cells against the toxic insults, and the protection was blocked by a superoxide dismutase mimetic and a PI3K antagonist, but not by BKCa channel inhibitors. Furthermore, NS1619 increased ROS generation, depolarized isolated mitochondria, hyperpolarized the neuronal cell membrane, and activated the PI3K signaling cascade. Finally, NS1619 inhibited the activation of capase‐3/7. In summary, NS1619 is a potent inducer of delayed neuronal preconditioning. However, the neuroprotective effect seems to be independent of cell membrane and mitochondrial BKCa channels. Rather it is the consequence of ROS generation, activation of the PI3K pathway, and inhibition of caspase activation. Supported by the NIH Grants HL‐030260, HL‐065380, and HL‐077731.

Delayed neuronal preconditioning by NS1619 is independent of calcium activated potassium channels

1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619), a potent activator of the large conductance Ca2+ activated potassium (BK(Ca)) channel, has been demonstrated to induce preconditioning (PC) in the heart. The aim of our study was to test the delayed PC effect of NS1619 in rat cortical neuronal cultures against oxygen-glucose deprivation, H2O2, or glutamate excitotoxicity. We also investigated its actions on reactive oxygen species (ROS) generation, and on mitochondrial and plasma membrane potentials. Furthermore, we tested the activation of the phosphoinositide 3-kinase (PI3K) signaling pathway, and the effect of NS1619 on caspase-3/7. NS1619 dose-dependently protected the cells against the toxic insults, and the protection was completely blocked by a superoxide dismutase mimetic and a PI3K antagonist, but not by BK(Ca) channel inhibitors. Application of NS1619 increased ROS generation, depolarized isolated mitochondria, hyperpolarized the neuronal cell membrane, and activated the PI3K signaling cascade. However, only the effect on the cell membrane potential was antagonized by BK(Ca) channel blockers. NS1619 inhibited the activation of capase-3/7. In summary, NS1619 is a potent inducer of delayed neuronal PC. However, the neuroprotective effect seems to be independent of cell membrane and mitochondrial BK(Ca) channels. Rather it is the consequence of ROS generation, activation of the PI3K pathway, and inhibition of caspase activation.

PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase.

Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K.

MTOR as a positive regulator of tumor cell responses to hypoxia.

Rapamycin is a clinically approved immunosuppressive agent that has recently shown promising antitumor activities in human patients. In contrast to many conventional chemotherapeutic agents, rapamycin displays a remarkably high level of selectivity for certain types of tumors. The pharmacological activities of rapamycin are attributable to the functional inhibition of a single target protein, termed the mammalian target of rapamycin (mTOR). Because mTOR is widely expressed in both normal and transformed cells, variations in mTOR expression levels are likely not a primary determinant of tumor sensitivity to rapamycin. However, recent studies highlighted an intriguing link between cancer cell sensitivity to rapamycin and deregulated signaling through the phosphoinositide (PI) 3-kinase pathway. These findings have prompted a search for cancer-related responses that are jointly regulated by the PI 3-kinase signaling cascade and mTOR. The oxygen-regulated transcription factor, hypoxia-induced factor (HIF)-1, has emerged as a candidate target for both of these two highly interactive signaling proteins. Here we review evidence that mTOR functions as a positive regulator of HIF-1-dependent responses to hypoxic stress in human cancer cells.

Cellular Function of Phosphoinositide 3-Kinases: Implications for Development, Immunity, Homeostasis, and Cancer

The phosphoinositide 3-kinase (PI3K) family of enzymes is recruited upon growth factor receptor activation and produces 3' phosphoinositide lipids. The lipid products of PI3K act as second messengers by binding to and activating diverse cellular target proteins. These events constitute the start of a complex signaling cascade, which ultimately results in the mediation of cellular activities such as proliferation, differentiation, chemotaxis, survival, trafficking, and glucose homeostasis. Therefore, PI3Ks play a central role in many cellular functions. The factors that determine which cellular function is mediated are complex and may be partly attributed to the diversity that exists at each level of the PI3K signaling cascade, such as the type of stimulus, the isoform of PI3K, or the nature of the second messenger lipids. Numerous studies have helped to elucidate some of the key factors that determine cell fate in the context of PI3K signaling. For example, the past two years has seen the publication of many transgenic and knockout mouse studies where either PI3K or its signaling components are deregulated. These models have helped to build a picture of the role of PI3K in physiology and indeed there have been a number of surprises. This review uses such models as a framework to build a profile of PI3K function within both the cell and the organism and focuses, in particular, on the role of PI3K in cell regulation, immunity, and development. The evidence for the role of deregulated PI3K signaling in diseases such as cancer and diabetes is reviewed.