Catalytic Subunit Of Phosphoinositide 3-kinase Research Articles

MiR-30a Suppresses Cell Migration and Invasion Through Downregulation of PIK3CD in Colorectal Carcinoma

Background/Aims: MicroRNAs (miRNAs) play key roles in tumor metastasis. The aim of this study was to determine the regulation and function of miR-30a in colorectal carcinoma (CRC) metastasis. Methods: The expression of miR-30a was detected in CRC cell lines and samples by qRT-PCR. The anti-metastatic effect of miR-30a was determined by both in vitro and in vivo assays. A luciferase reporter assay was performed to determine target association between miR-30a and phosphoinositide 3-kinase catalytic subunit delta (PIK3CD). Results: miR-30a was significantly downregulated in highly metastatic CRC cell lines and metastatic tissues. Overexpression of miR-30a suppressed CRC cell migration and invasion in vitro and liver metastasis in vivo, whereas miR-30a deletion dramatically promoted cell migration and invasion. Further studies revealed that PIK3CD is a direct target of miR-30a as miR-30a bounds directly to the 3'-UTR of PIK3CD, subsequently reducing its expression. Similar to the restoring miR-30a expression, PIK3CD downregulation inhibited cell migration and invasion, whereas PIK3CD overexpression rescued the suppressive effect of miR-30a. Moreover, significant downregulation of miR-30a in metastatic CRC tissues was found to be inversely correlated with PIK3CD expression. Mechanistic studies revealed that miR-30a down-regulated the expression of key components of the Akt/mTOR pathway, whereas PIK3CD overexpression reversed this negative effect. Conclusion: Our findings indicate that miR-30a might function as a metastasis suppressor in CRC. miR-30a may be a potential therapeutic target to block CRC metastasis.

MiR-124 suppresses cell proliferation in hepatocellular carcinoma by targeting PIK3CA

MicroRNAs (miRNAs) have crucial roles in the development and progression of human cancers, including hepatocellular carcinoma (HCC). Recent studies have shown that microRNA-124 (miR-124) was downregulated in HCC; however, the underlying mechanisms by which miR-124 suppresses tumorigenesis in HCC are largely unknown. In this study, we report that phosphoinositide 3-kinase catalytic subunit alpha (PIK3CA) is a novel target of miR-124 in HepG2 cells. Overexpression of miR-124 resulted in decreased expression of PIK3CA at both mRNA and protein levels. We found that miR-124 overexpression markedly suppressed cell proliferation by inducing G1-phase cell-cycle arrest in vitro. Consistent with the restoring miR-124 expression, PIK3CA knockdown suppressed cell proliferation, whereas overexpression of PIK3CA abolished the suppressive effect of miR-124. Mechanistic studies showed that miR-124-mediated reduction of PIK3CA resulted in suppression of PI3K/Akt pathway. The expressions of Akt and mTOR, key components of the PI3K/Akt pathway, were all downregulated. Moreover, we found overexpressed miR-124 effectively repressed tumor growth in xenograft animal experiments. Taken together, our results demonstrate that miR-124 functions as a growth-suppressive miRNA and plays an important role in inhibiting the tumorigenesis through targeting PIK3CA.

Integrative genomic analysis implicates gain of PIK3CA at 3q26 and MYC at 8q24 in chronic lymphocytic leukemia.

The disease course of chronic lymphocytic leukemia (CLL) varies significantly within cytogenetic groups. We hypothesized that high-resolution genomic analysis of CLL would identify additional recurrent abnormalities associated with short time-to-first therapy (TTFT). We undertook high-resolution genomic analysis of 161 prospectively enrolled CLLs using Affymetrix 6.0 SNP arrays, and integrated analysis of this data set with gene expression profiles. Copy number analysis (CNA) of nonprogressive CLL reveals a stable genotype, with a median of only 1 somatic CNA per sample. Progressive CLL with 13q deletion was associated with additional somatic CNAs, and a greater number of CNAs was predictive of TTFT. We identified other recurrent CNAs associated with short TTFT: 8q24 amplification focused on the cancer susceptibility locus near MYC in 3.7%; 3q26 amplifications focused on PIK3CA in 5.6%; and 8p deletions in 5% of patients. Sequencing of MYC further identified somatic mutations in two CLLs. We determined which catalytic subunits of phosphoinositide 3-kinase (PI3K) were in active complex with the p85 regulatory subunit and showed enrichment for the α subunit in three CLLs carrying PIK3CA amplification. Our findings implicate amplifications of 3q26 focused on PIK3CA and 8q24 focused on MYC in CLL.

Cooperative Activation of PI3K by Ras and Rho Family Small GTPases

Phosphoinositide 3-kinases (PI3Ks) and Ras and Rho family small GTPases are key regulators of cell polarization, motility, and chemotaxis. They influence each other's activities by direct and indirect feedback processes that are only partially understood. Here, we show that 21 small GTPase homologs activate PI3K. Using a microscopy-based binding assay, we show that K-Ras, H-Ras, and five homologous Ras family small GTPases function upstream of PI3K by directly binding the PI3K catalytic subunit, p110. In contrast, several Rho family small GTPases activated PI3K by an indirect cooperative positive feedback that required a combination of Rac, CDC42, and RhoG small GTPase activities. Thus, a distributed network of Ras and Rho family small GTPases induces and reinforces PI3K activity, explaining past challenges to elucidate the specific relevance of different small GTPases in regulating PI3K and controlling cell polarization and chemotaxis.

PI 3-kinase and disease

Phosphoinositide 3-Kinase (PI3K) is a central enzyme in a signaling pathway that mediates cellular responses to growth factors. This enzyme phosphorylates the 3 position of phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the plasma membrane. A number of signaling proteins, including the Ser/Thr protein kinases, AKT and PDK1, contain pleckstrin homology domains that bind specifically to PIP3. Thus, the generation of PIP3 at the plasma membrane in response to activation of PI3K by growth factors results in the initiation of downstream Ser/Thr phosphorylation cascades that control a variety of cellular responses. The signaling pathway downstream of PI3K is highly conserved from worms and flies to humans and genetic analysis of the pathway has revealed a conserved role in regulating glucose metabolism and cell growth. Based on deletion of genes encoding the catalytic or regulatory subunits of PI3K in the mouse, PI3K mediates insulin dependent regulation of glucose metabolism, and defects in activation of this pathway result in insulin resistance. In contrast, mutational events that lead to hyperactivation of the PI3K pathway result in cancers. Activating mutations in PIK3CA, encoding the p110alpha catalytic subunit of PI3K or inactivating mutations in PTEN, a phosphoinositide 3-phosphatases that reverses the effects of PI3K, are among the most common events in solid tumors. We have generated mouse models in which a mutated form of the PIK3CA gene is expressed in a tissue specific and reversibly inducible manner. These mice develop cancers that are dependent on continuous expression of the mutant PIK3CA gene. The PIK3CA driven tumors are FDG-PET positive and turning off PI 3-Kinase with PI3K inhibitors that are in human clinical trials results in an acute decline in FDG-PET signal that precedes tumor shrinkage. These results suggest that the ability of PI3K to stimulate high rates of glucose uptake and metabolism may be critical for the survival of PIK3CA mutant tumors. The role of PI3K inhibitors for treating cancers in mouse models and in human trials will be discussed.

MicroRNA-7 inhibits tumor growth and metastasis by targeting the phosphoinositide 3-kinase/Akt pathway in hepatocellular carcinoma

MicroRNAs (miRNAs) are known to be involved in carcinogenesis and tumor progression in hepatocellular carcinoma (HCC). Recently, microRNA-7 (miR-7) has been proven to play a substantial role in glioblastoma and breast cancer, but its functions in the context of HCC remain unknown. Here, we demonstrate that miR-7 inhibits HCC cell growth and metastasis in vitro and in vivo. We first screened and identified a novel miR-7 target, phosphoinositide 3-kinase catalytic subunit delta (PIK3CD). Overexpression of miR-7 would specifically and markedly down-regulate its expression. miR-7-overexpressing subclones showed significant cell growth inhibition by G(0) /G(1) -phase cell-cycle arrest and significant impairment of cell migration in vitro. To identify the mechanisms, we investigated the phosphoinositide 3-kinase (PI3K)/Akt pathway and found that Akt, mammalian target of rapamycin (mTOR), and p70S6K were down-regulated, whereas 4EBP1 was up-regulated in miR-7-overexpressing subclones. We also identified two novel, putative miR-7 target genes, mTOR and p70S6K, which further suggests that miR-7 may be a key regulator of the PI3K/Akt pathway. In xenograft animal experiments, we found that overexpressed miR-7 effectively repressed tumor growth (3.5-fold decrease in mean tumor volume; n = 5) and abolished extrahepatic migration from liver to lung in a nude mouse model of metastasis (n = 5). The number of visible nodules on the lung surface was reduced by 32-fold. A correlation between miR-7 and PIK3CD expression was also confirmed in clinical samples of HCC. These findings indicate that miR-7 functions as a tumor suppressor and plays a substantial role in inhibiting the tumorigenesis and reversing the metastasis of HCC through the PI3K/Akt/mTOR-signaling pathway in vitro and in vivo. By targeting PIK3CD, mTOR, and p70S6K, miR-7 efficiently regulates the PI3K/Akt pathway. Given these results, miR-7 may be a potential therapeutic or diagnostic/prognostic target for treating HCC.

Down-regulation of p110β expression increases chemosensitivity of colon cancer cell lines to oxaliplatin

This study examined the synergetic effect of class IA Phosphoinositide 3-kinases catalytic subunit p110β knockdown in conjunction with oxaliplatin treatment on colon cancer cells. Down-regulation of p110β by siRNA interference and oxaliplatin treatment were applied in colon cancer cell lines HT29, SW620 and HCT116. MTT assay was used to measure the inhibitory effect of p110β knockdown on the proliferation of colon cancer cell lines. SubG1 assay and Annexin-V FITC/PI double-labeling cytometry were applied to detect cell apoptosis. And cell cycle was evaluated by using PI staining and flow cytometry. The expression of caspase 3, cleaved PARP, p-Akt, T-Akt and p110β was determined by western blotting. The results suggested that down-regulation of p110β expression by siRNA obviously reduced cell number via accumulation in G(0)-G(1) phase of the cell cycle in the absence of notablely increased apoptosis in colon cancer cell lines HT29 and SW620 (S phase arrest in HCT116). Moreover, inhibition of p110β expression increased oxaliplatin-induced cell apoptosis and cell cycle arrest in HT29, HCT116 and SW620 cell lines. In addition, increases of cleaved caspase-3 and cleaved PARP induced by oxaliplatin treatment were determined by immunoblotting in p110β knockdown group compared with normal control group and wild-type group. It is concluded that down-regulated expression of p110β could inhibit colon cancer cells proliferation and result in increased chemosensitivity of colorectal cancer cells to oxaliplatin through augmentation of oxaliplatin-induced cell apoptosis and cell cycle arrest.

MiR-125b Inhibits Tumor Growth and Promotes Apoptosis of Cervical Cancer Cells by Targeting Phosphoinositide 3-Kinase Catalytic Subunit Delta

Backgroud: microRNAs (miRNAs) are involved in cancer-related processes. The miRNA-125b (miR-125b) has been identified as miRNA over-expressed in a wide variety of cancers. However, the role of miR-125b in the context of cervical carcinoma remains unknown. Methods: In this study, the effect of miR-125b on the proliferation and apoptosis of human cervical cells was analyzed by MTT assay and Flow cytometry analysis. we identified phosphoinositide 3-kinase catalytic subunit delta (PIK3CD) as a novel miR-125b target. Results: overexpression of miR- 125b in HeLa cervical cancer cells decreased cell proliferation, induced apoptosis and down-regulated expression of PIK3CD. To identify the mechanisms responsible, we investigated the PI3K/Akt pathway and found that PI3K, phospho-Akt, and phospho-mTOR were all down-regulated, while Bid was up-regulated in miR-125b-overexpressing subclones. In vivo, over expression of miR-125b in HeLa cells markedly reduced their ability to form tumors. Conclusion: these results suggest that miR-125b suppresses tumor growth activity by targeting the PI3K/ Akt/mTOR signaling pathway, and may provide a target for effective therapies.

The phosphoinositide 3-kinase signaling pathway in normal and malignant B cells: activation mechanisms, regulation and impact on cellular functions

The phosphoinositide 3-kinase (PI3K) pathway is a central signal transduction axis controlling normal B cell homeostasis and activation in humoral immunity. The p110δ PI3K catalytic subunit has emerged as a critical mediator of multiple B cell functions. The activity of this pathway is regulated at multiple levels, with inositol phosphatases PTEN and SHIP both playing critical roles. When deregulated, the PI3K pathway can contribute to B cell malignancies and autoantibody production. This review summarizes current knowledge on key mechanisms that activate and regulate the PI3K pathway and influence normal B cell functional responses including the development of B cell subsets, antigen presentation, immunoglobulin isotype switch, germinal center responses, and maintenance of B cell anergy. We also discuss PI3K pathway alterations reported in select B cell malignancies and highlight studies indicating the functional significance of this pathway in malignant B cell survival and growth within tissue microenvironments. Finally, we comment on early clinical trial results, which support PI3K inhibition as a promising treatment of chronic lymphocytic leukemia.

SIRT1 Deacetylase in SF1 Neurons Protects against Metabolic Imbalance

Chronic feeding on high-calorie diets causes obesity and type 2 diabetes mellitus (T2DM), illnesses that affect hundreds of millions. Thus, understanding the pathways protecting against diet-induced metabolic imbalance is of paramount medical importance. Here, we show that mice lacking SIRT1 in steroidogenic factor 1 (SF1) neurons are hypersensitive to dietary obesity owing to maladaptive energy expenditure. Also, mutant mice have increased susceptibility to developing dietary T2DM due to insulin resistance in skeletal muscle. Mechanistically, these aberrations arise, in part, from impaired metabolic actions of the neuropeptide orexin-A and the hormone leptin. Conversely, mice overexpressing SIRT1 in SF1 neurons are more resistant to diet-induced obesity and insulin resistance due to increased energy expenditure and enhanced skeletal muscle insulin sensitivity. Our results unveil important protective roles of SIRT1 in SF1 neurons against dietary metabolic imbalance.

Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation

Invadopodia are extracellular matrix-degrading protrusions formed by invasive cancer cells that are thought to function in cancer invasion. Although many invadopodia components have been identified, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. We investigate the role of phosphoinositide 3-kinase (PI3K) signaling during invadopodia formation. We find that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix were blocked by treatment with PI3K inhibitors or sequestration of D-3 phosphoinositides. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α, a frequently mutated gene product in human cancers, was selectively involved in invadopodia formation. The expression of p110α with cancerous mutations promoted invadopodia-mediated invasive activity. Furthermore, knockdown or inhibition of PDK1 and Akt, downstream effectors of PI3K signaling, suppressed invadopodia formation induced by p110α mutants. These data suggest that PI3K signaling via p110α regulates invadopodia-mediated invasion of breast cancer cells.

Arouser Reveals a Role for Synapse Number in the Regulation of Ethanol Sensitivity

A reduced sensitivity to the sedating effects of alcohol is a characteristic associated with alcohol use disorders (AUDs). A genetic screen for ethanol sedation mutants in Drosophila identified arouser (aru), which functions in developing neurons to reduce ethanol sensitivity. Genetic evidence suggests that aru regulates ethanol sensitivity through its activation by Egfr/Erk signaling and its inhibition by PI3K/Akt signaling. The aru mutant also has an increased number of synaptic terminals in the larva and adult fly. Both the increased ethanol sensitivity and synapse number of the aru mutant are restored upon adult social isolation, suggesting a causal relationship between synapse number and ethanol sensitivity. We thus show that a developmental abnormality affecting synapse number and ethanol sensitivity is not permanent and can be reversed by manipulating the environment of the adult fly.

VEGF-mediated PI3K class IA and PKC signaling in cardiomyogenesis and vasculogenesis of mouse embryonic stem cells

VEGF-, phosphoinositide 3-kinase (PI3K)- and protein kinase C (PKC)-regulated signaling in cardiac and vascular differentiation was investigated in mouse ES cells and in ES cell-derived Flk-1⁺ cardiovascular progenitor cells. Inhibition of PI3K by wortmannin and LY294002, disruption of PI3K catalytic subunits p110α and p110δ using short hairpin RNA (shRNA), or inhibition of p110α with compound 15e and of p110δ with IC-87114 impaired cardiac and vascular differentiation. By contrast, TGX-221, an inhibitor of p110β, and shRNA knockdown of p110β were without significant effects. Antagonists of the PKC family, i.e. bisindolylmaleimide-1 (BIM-1), GÖ 6976 (targeting PKCα/βII) and rottlerin (targeting PKCδ) abolished vasculogenesis, but not cardiomyogenesis. Inhibition of Akt blunted cardiac as well as vascular differentiation. VEGF induced phosphorylation of PKCα/βII and PKCδ but not PKCζ. This was abolished by PI3K inhibitors and the VEGFR-2 antagonist SU5614. Furthermore, phosphorylation of Akt and phosphoinositide-dependent kinase-1 (PDK1) was blunted upon inhibition of PI3K, but not upon inhibition of PKC by BIM-1, suggesting that activation of Akt and PDK1 by VEGF required PI3K but not PKC. In summary, we demonstrate that PI3K catalytic subunits p110α and p110δ are central to cardiovasculogenesis of ES cells. Akt downstream of PI3K is involved in both cardiomyogenesis and vasculogenesis, whereas PKC is involved only in vasculogenesis.

Ablation of PI3K p110-α Prevents High-Fat Diet–Induced Liver Steatosis

OBJECTIVETo determine whether the phosphoinositide 3-kinase (PI3K) catalytic subunits p110-α and p110-β play a role in liver steatosis induced by a high-fat diet (HFD).RESEARCH DESIGN AND METHODSLiver-specific p110-α and p110-β knockout mice and control animals for each group were fed an HFD or normal chow for 8 weeks. Biochemical assays and quantitative real-time PCR were used to measure triglyceride, expression of lipogenic and gluconeogenic genes, and activity of protein kinases downstream of PI3K in liver lysates. Fatty acid uptake and incorporation into triglycerides were assessed in isolated hepatocytes.RESULTSHepatic triglyceride levels in HFD-fed p110-α−/− mice were 84 ± 3% lower than in p110-α+/+ mice, whereas the loss of p110-β did not significantly alter liver lipid accumulation. p110-α−/− livers also showed a reduction in atypical protein kinase C activity and decreased mRNA and protein expression of several lipogenic genes. Hepatocytes isolated from p110-α−/− mice exhibited decreased palmitate uptake and reduced fatty acid incorporation into triglycerides as compared with p110-α+/+ cells, and hepatic expression of liver fatty acid binding protein was lower in p110-α−/− mice fed the HFD as compared with controls. Ablation of neither p110-α nor p110-β ameliorated glucose intolerance induced by the HFD, and genes involved in gluconeogenesis were upregulated in the liver of both knockout animals.CONCLUSIONSPI3K p110-α, and not p110-β, promotes liver steatosis in mice fed an HFD. p110-α might exert this effect in part through activation of atypical protein kinase C, upregulation of lipogenesis, and increased uptake of fatty acids.

Abstract 3179: ACB-PCR quantification of PIK3CA codon 1047 CAT to CGT mutant fraction in human tumor and non-tumor tissues

Abstract Phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) is a proto-oncogene encoding the p110 catalytic subunit of phosphoinositide 3-kinase (PI3K). A variety of tumors carry PIK3CA mutations. These mutations are thought to increase PI3K activity, increase cell proliferation, and may contribute to anti-cancer drug resistance. Three specific hotspot mutations within the PIK3CA gene are localized within the helical and kinase domains and account for >90% of all reported mutations in PIK3CA. One PIK3CA mutation in the kinase domain (codon 1047 CAT to CGT) accounts for more than 30% of all reported mutations. Because PIK3CA mutation may have clinical significance in terms of patient prognosis and treatment selection, we are using a sensitive allele-specific amplification approach called allele-specific competitive blocker-PCR (ACB-PCR) to analyze the PIK3CA codon 1047 CAT to CGT mutant fractions (MFs) in various human tissues. Specific goals of this study are to: 1) define normal and pathological levels of PIK3CA mutation in various human tissues, and 2) obtain data on the co-occurrence of PIK3CA mutation with other mutations (e.g., the KRAS codon 12 GAT and GTT mutations). To date, PIK3CA codon 1047 CAT to CGT MF has been measured in 50 human colon tissue samples, including 17 normal-appearing colonic mucosa samples, 13 normal tumor-adjacent samples, 10 adenomas, and 10 adenocarcinomas. The PIK3CA codon 1047 CAT to CGT mutation was present at measurable levels in all normal appearing colonic mucosa samples. The PIK3CA geometric mean MF in normal colonic mucosa ranged from 3.24 × 10-4 to 6.48 × 10-4, with an average (geometric mean) MF of 5.12 × 10-4. The PIK3CA geometric mean MFs measured in normal tumor-adjacent samples, adenomas, and adenocarcinomas were 6.45 × 10-4, 7.32 × 10-4, and 3.45 × 10-4, respectively. All colon tumors were found to carry PIK3CA codon 1047 CAT to CGT mutations as mutant subpopulations. The levels of PIK3CA mutation in normal-appearing colonic mucosa samples are similar to the levels of KRAS GAT mutation (∼10-4) measured previously in the same samples. Analyses of PIK3CA mutation in breast, pancreas, and thyroid are ongoing. In conjunction with analyses of other mutational targets, these types of data should aid in the identification of the best mutational biomarkers for particular tissues types (those mutations with consistently higher levels in tumors than in normal tissues). Also, by describing the prevalence of different molecular lesions within functional pathways related to carcinogenesis, the most promising molecular targets for therapeutic intervention will be identified. The contents of the abstract do not necessarily reflect the views or policies of the U.S. FDA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3179. doi:10.1158/1538-7445.AM2011-3179

Abstract 4748: Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation

Abstract Degradation of extracellular matrix (ECM) that is present in the basement membrane and tumor stroma is essential for local invasion and formation of metastatic sites by malignant cancer cells. Invadopodia are ECM-degrading protrusions formed by a variety of invasive cancer cell types and are thought to function in cancer invasion and metastasis. Many components of invadopodia, such as proteins involved in actin polymerization, cell signaling, and ECM degradation, have been reported to date. However, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. The phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that produce D-3 phosphoinositides. PI3Ks mediate the signal transduction of extracellular stimuli and regulate diverse cellular events. Uncontrolled activation of the PI3K signaling pathway leads to several pathological phenomena, including tumorigenesis and tumor malignancy. We investigated the role of PI3K signaling during invadopodia formation. We found that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix was blocked by treatment with PI3K inhibitors or sequestration of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. Mammals have 8 PI3K enzymes that are subdivided into three general classes (I-III) on the basis of their domain structures and substrate specificities. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α is selectively involved in invadopodia formation. p110α is encoded by the PIK3CA gene, which is one of the most frequently amplified and mutated genes identified in human cancers. Clinical studies revealed that activating mutations of PIK3CA are associated with the development of invasive and metastatic phenotypes and poor patient prognosis. The expression of p110α with the activating mutations promoted invadopodia-mediated invasive activity. Furthermore, knocking down PDK1 and Akt, the downstream effectors of PI3K signaling, suppressed invadopodia formation induced by the p110α mutants. These data suggest that the PI3K signaling pathway via p110α regulates invadopodia-mediated invasion of breast cancer cells. Targeting p110α may be a potential therapeutic approach for controlling invasion of malignant cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4748. doi:10.1158/1538-7445.AM2011-4748

Insulin/FOXO Signaling Regulates Ovarian Prostaglandins Critical for Reproduction

Abnormalities in insulin/IGF-1 signaling are associated with infertility, but the molecular mechanisms are not well understood. Here we use liquid chromatography with electrospray ionization tandem mass spectrometry to show that the C. elegans insulin/FOXO pathway regulates the metabolism of locally acting lipid hormones called prostaglandins. C. elegans prostaglandins are synthesized without prostaglandin G/H synthase homologs, the targets of nonsteroidal anti-inflammatory drugs. Our results support the model that insulin signaling promotes the conversion of oocyte polyunsaturated fatty acids (PUFAs) into F-series prostaglandins that guide sperm to the fertilization site. Reduction in insulin signaling activates DAF-16/FOXO, which represses the transcription of germline and intestinal genes required to deliver PUFAs to oocytes in lipoprotein complexes. Nutritional and neuroendocrine cues target this mechanism to control prostaglandin metabolism and reproductive output. Prostaglandins may be conserved sperm guidance factors and widespread downstream effectors of insulin actions that influence both reproductive and nonreproductive processes.

A biochemical mechanism for the oncogenic potential of the p110beta catalytic subunit of phosphoinositide 3-kinase.

Class I PI3-kinases signal downstream of receptor tyrosine kinases and G protein-coupled receptors and have been implicated in tumorigenesis. Although the oncogenic potential of the PI3-kinase subunit p110α requires its mutational activation, other p110 isoforms can induce transformation when overexpressed in the wild-type state. In wild-type p110α, N345 in the C2 domain forms hydrogen bonds with D560 and N564 in the inter-SH2 (iSH2) domain of p85, and mutations of p110α or p85 that disrupt this interface lead to increased basal activity and transformation. Sequence analysis reveals that N345 in p110α aligns with K342 in p110β. This difference makes wild-type p110β analogous to a previously described oncogenic mutant, p110α-N345K. We now show that p110β is inhibited by p85 to a lesser extent than p110α and is not differentially inhibited by wild-type p85 versus p85 mutants that disrupt the C2-iSH2 domain interface. Similar results were seen in soft agar and focus-formation assays, where p110β was similar to p110α-N345K in transforming potential. Inhibition of p110β by p85 was enhanced by a K342N mutation in p110β, which led to decreased activity in vitro, decreased basal Akt and ribosomal protein S6 kinase (S6K1) activation, and decreased transformation in NIH 3T3 cells. Moreover, unlike wild-type p110β, p110β-K342N was differentially regulated by wild-type and mutant p85, suggesting that the inhibitory C2-iSH2 interface is functional in this mutant. This study shows that the enhanced transforming potential of p110β is the result of its decreased inhibition by p85, due to the disruption of an inhibitory C2-iSH2 domain interface.

Excess Phosphoinositide 3-Kinase Subunit Synthesis and Activity as a Novel Therapeutic Target in Fragile X Syndrome

Fragile X syndrome (FXS) is an inherited neurologic disease caused by loss of fragile X mental retardation protein (FMRP), which is hypothesized to mediate negative regulation of mRNA translation at synapses. A prominent feature of FXS animal models is exaggerated signaling through group 1 metabotropic glutamate receptors (gp1 mGluRs), and therapeutic strategies to treat FXS are targeted mainly at gp1 mGluRs. Recent studies, however, indicate that a variety of receptor-mediated signal transduction pathways are dysregulated in FXS, suggesting that FMRP acts on a common downstream signaling molecule. Here, we show that deficiency of FMRP results in excess activity of phosphoinositide 3-kinase (PI3K), a downstream signaling molecule of many cell surface receptors. In Fmr1 knock-out neurons, excess synaptic PI3K activity can be reduced by perturbation of gp1 mGluR-mediated signaling. Remarkably, increased PI3K activity was also observed in FMRP-deficient non-neuronal cells in the absence of gp1 mGluRs. Here, we show that FMRP regulates the synthesis and synaptic localization of p110beta, the catalytic subunit of PI3K. In wild type, gp1 mGluR activation induces p110beta translation, p110beta protein expression, and PI3K activity. In contrast, both p110beta protein synthesis and PI3K activity are elevated and insensitive to gp1 mGluR stimulation in Fmr1 knock-out. This suggests that dysregulated PI3K signaling may underlie the synaptic impairments in FXS. In support of this hypothesis, we show that PI3K antagonists rescue three FXS-associated phenotypes: dysregulated synaptic protein synthesis, excess AMPA receptor internalization, and increased spine density. Targeting excessive PI3K activity might thus be a potent therapeutic strategy for FXS.

Regulation of mammary epithelial architecture by PTEN

The PTEN tumour suppressor is a core component of the phosphoinositide 3-kinase (PI3K) signalling pathway. PTEN is mutated, deleted, or otherwise silenced in over one-third of breast cancers, with another one-third carrying other activating mutations in the core PI3K signalling pathway, most of these being activating mutations in the p110α catalytic subunit of PI3K itself. The best characterised tumour suppressor roles for PTEN are the suppression of cell growth, survival, proliferation and metabolic deregulation. However, we have found that in three-dimensional cultured mammary epithelial cells, knockdown of PTEN leads to the loss of cell polarity and tissue architecture. PTEN knockdown also causes a dramatic disruption of normal tight junction formation in adherent mammary epithelial cell cultures. Since the deregulation of cell polarity is becoming recognised as a potential driving force behind the formation of some tumours, we have been further studying the mechanisms by which PTEN controls mammary epithelial cell polarity.