P-REX2 PH Domain Inhibition of PTEN Regulates Transformation, Insulin Signaling, and Glucose Homeostasis

PTEN, a tumor suppressor lost in multiple cancers, antagonizes PI3-kinase signaling by dephosphorylating the second messenger phosphatidylinositol (3,4,5) trisphophate. PTEN expression and enzymatic activity is regulated through various mechanisms, including oxidation, phosphorylation, and protein-protein interactions. Our lab has recently identified a PTEN interacting protein, the Rac GEF P-REX2, which inhibits PTEN phosphatase activity in a non-competitive manner. This thesis focuses on understanding the physiological relevance of this interaction in the regulation of PI3K signaling, as well as determining the mechanism of P-REX2 mediated PTEN inhibition.The first chapter focuses on the role of P-REX2 over expression in PI3K signaling, proliferation, and transformation. We first find that P-REX2 Rac GEF activity is dispensable for PTEN inhibition by utilizing a P-REX2 GEF dead mutant N212A. Next, we determined the effect of P-REX2 overexpression on PI3K signaling in normal mammary epithelial cells. Expression of P-REX2 or the DHPH inhibitory domain increased AKT phosphorylation, promoted cellular proliferation, and disrupted acini morphogenesis. Furthermore, P-REX2 cooperated with other oncogenes, including the PI3K E545K oncogenic mutant, c-MYC, and HER2 to promote proliferation, colony formation in soft agar, and tumor formation in mice. We also analyzed the effects of expression of P-REX2 cancer mutants, and discovered two transforming mutants, V432M and R498I that cooperated with PI3K E545K to increase anchorage independent growth and cellular proliferation.The next chapter examines the role of P-rex2 in PI3K signaling regulation in vivo. We generated Prex2 knockout mice using a gene trap method, and found that baseline signaling and proliferation in fibroblasts was not affected by P-rex2 deletion. However, insulin and IGF-1, but not PDGF or EGF stimulated PI3K signaling was reduced in Prex2-/- fibroblasts. The activity of PTEN from Prex2+/+ fibroblasts was reduced following insulin stimulation, but remained elevated in Prex2-/- cells, suggesting that insulin stimulated PTEN inhibition is dependent on P-rex2. Furthermore, P-REX2 interacted with phosphorylated insulin receptor and recruited PTEN to the membrane following insulin stimulation. Prex2-/- mice are intolerant to insulin and glucose, and have reduced PI3K signaling in the fat and liver following insulin stimulation. Furthermore, the activity of PTEN from Prex2-/- liver samples is elevated, and correlated with a decrease in cellular PIP3 levels. After uncovering an essential role for P-REX2 in PI3K signal transduction, we next examined the mechanism and regulation of P-REX2 mediated PTEN inhibition. We found that P-REX2 interacts with two different sites on PTEN. The PH domain of P-REX2 bound to the phosphatase and C2 domains of PTEN, while the inositol polyphosphate-4 phosphatase domain interacted with the PDZ-binding domain on the PTEN C-terminal tail. We discovered that the PH domain was the minimal domain that constitutively inhibited PTEN. However, the DHPH domain and full length P-REX2 required phosphorylation of the PTEN C-terminal tail for inhibition, suggesting the DH domain of P-REX2 restricts PH domain inhibition of PTEN when the C-terminal tail of PTEN is unphosphorylated. Furthermore, the PH domain of P-REX1 was not able to inhibit PTEN, and full length P-REX1 did not interact with PTEN, suggesting that there is a level of specificity involved in P-REX2 PH domain mediated phosphatase inhibition and binding. Overall, this thesis identifies P-REX2 as a dynamic inhibitor of PTEN phosphatase activity that regulates PI3K mediated cellular transformation, insulin signaling, and glucose metabolism

Regulation of PTEN Inhibition by the Pleckstrin Homology Domain of P-REX2 During Insulin Signaling and Glucose Homeostasis

Insulin activation of phosphoinositide 3-kinase (PI3K) signaling regulates glucose homeostasis through the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3). The dual-specificity phosphatase and tensin homolog deleted on chromosome 10 (PTEN) blocks PI3K signaling by dephosphorylating PIP3, and is inhibited through its interaction with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 (P-REX2). The mechanism of inhibition and its physiological significance are not known. Here, we report that P-REX2 interacts with PTEN via two interfaces. The pleckstrin homology (PH) domain of P-REX2 inhibits PTEN by interacting with the catalytic region of PTEN, and the inositol polyphosphate 4-phosphatase domain of P-REX2 provides high-affinity binding to the postsynaptic density-95/Discs large/zona occludens-1-binding domain of PTEN. P-REX2 inhibition of PTEN requires C-terminal phosphorylation of PTEN to release the P-REX2 PH domain from its neighboring diffuse B-cell lymphoma homology domain. Consistent with its function as a PTEN inhibitor, deletion of Prex2 in fibroblasts and mice results in increased Pten activity and decreased insulin signaling in liver and adipose tissue. Prex2 deletion also leads to reduced glucose uptake and insulin resistance. In human adipose tissue, P-REX2 protein expression is decreased and PTEN activity is increased in insulin-resistant human subjects. Taken together, these results indicate a functional role for P-REX2 PH-domain-mediated inhibition of PTEN in regulating insulin sensitivity and glucose homeostasis and suggest that loss of P-REX2 expression may cause insulin resistance

Rac-Mediated Macropinocytosis of Extracellular Protein Promotes Glucose Independence in Non-Small Cell Lung Cancer

Cancer cells can adapt to nutrient poor conditions by rewiring their metabolism and using alternate fuel sources. Identifying these adaptive metabolic pathways may provide novel targets for cancer therapy. Here, we identify a subset of non-small cell lung cancer (NSCLC) cell lines that survive in the absence of glucose by internalizing and metabolizing extracellular protein via macropinocytosis. Macropinocytosis is increased in these glucose independent cells, and is regulated by phosphoinositide 3-kinase (PI3K) activation of Rac-Pak signaling. Furthermore, inhibition of Rac-dependent macropinocytosis blocks glucose-independent proliferation. We find that degradation of internalized protein produces amino acids, including alanine, which generates TCA cycle and glycolytic intermediates in the absence of glucose. In this process, the conversion of alanine to pyruvate by alanine transaminase 2 (ALT2) is critical for survival during glucose starvation. Collectively, Rac driven macropinocytosis of extracellular protein is an adaptive metabolic pathway used by a subset of lung cancers to survive states of glucose deprivation, and may serve as a potential drug target for cancer therapy

Activation of the PI3K pathway in cancer through inhibition of PTEN by exchange factor P-REX2a

PTEN (Phosphatase and tensin homolog on chromosome ten) is a tumor suppressor whose cellular regulation remains incompletely understood. We identified Phosphatidylinositol-3,4,5-trisphosphate RAC Exchanger 2a (P-REX2a) as a PTEN-interacting protein. P-REX2a mRNA was more abundant in cancer, and significantly increased in tumors with wild type PTEN that expressed an activated mutant of PIK3CA encoding the p110 subunit of phosphoinositide 3-kinase-α (PI3Kα). P-REX2a inhibited PTEN lipid phosphatase activity and stimulated the PI3K pathway only in the presence of PTEN. P-REX2a stimulated cell growth and cooperated with a PIK3CA mutant to promote growth factor-independent proliferation and transformation. Depletion of P-REX2a reduced amounts of phosphorylated AKT and growth in cell lines with intact PTEN. Thus P-REX2a is a component of the PI3K pathway that can antagonize PTEN in cancer cells

PIP4Ks Suppress Insulin Signaling through a Catalytic-Independent Mechanism

Summary: Insulin stimulates the conversion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), which mediates downstream cellular responses. PI(4,5)P2 is produced by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Here, we show that the loss of PIP4Ks (PIP4K2A, PIP4K2B, and PIP4K2C) in vitro results in a paradoxical increase in PI(4,5)P2 and a concomitant increase in insulin-stimulated production of PI(3,4,5)P3. The reintroduction of either wild-type or kinase-dead mutants of the PIP4Ks restored cellular PI(4,5)P2 levels and insulin stimulation of the PI3K pathway, suggesting a catalytic-independent role of PIP4Ks in regulating PI(4,5)P2 levels. These effects are explained by an increase in PIP5K activity upon the deletion of PIP4Ks, which normally suppresses PIP5K activity through a direct binding interaction mediated by the N-terminal motif VMLΦPDD of PIP4K. Our work uncovers an allosteric function of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P2 synthesis and insulin-dependent conversion to PI(3,4,5)P3 and suggests that the pharmacological depletion of PIP4K enzymes could represent a strategy for enhancing insulin signaling. : PI(4,5)P2 is produced by both phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Wang et al. report an allosteric function of a conserved N-terminal motif of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P2 synthesis and insulin-dependent conversion to PI(3,4,5)P3. This non-catalytic role has implications for the development of PIP4K targeted therapies. Keywords: PIP4K, PI5P4K, PIP5K, PI3K, Akt, insulin, signaling, PI(4,5)P2, PI(3,4,5)P3, RT

Suppression of insulin feedback enhances the efficacy of PI3K inhibitors.

Mutations in PIK3CA, which encodes the p110α subunit of the insulin-activated phosphatidylinositol-3 kinase (PI3K), and loss of function mutations in PTEN, which encodes a phosphatase that degrades the phosphoinositide lipids generated by PI3K, are among the most frequent events in human cancers. However, pharmacological inhibition of PI3K has resulted in variable clinical responses, raising the possibility of an inherent mechanism of resistance to treatment. As p110α mediates virtually all cellular responses to insulin, targeted inhibition of this enzyme disrupts glucose metabolism in multiple tissues. For example, blocking insulin signalling promotes glycogen breakdown in the liver and prevents glucose uptake in the skeletal muscle and adipose tissue, resulting in transient hyperglycaemia within a few hours of PI3K inhibition. The effect is usually transient because compensatory insulin release from the pancreas (insulin feedback) restores normal glucose homeostasis. However, the hyperglycaemia may be exacerbated or prolonged in patients with any degree of insulin resistance and, in these cases, necessitates discontinuation of therapy. We hypothesized that insulin feedback induced by PI3K inhibitors may reactivate the PI3K-mTOR signalling axis in tumours, thereby compromising treatment effectiveness. Here we show, in several model tumours in mice, that systemic glucose-insulin feedback caused by targeted inhibition of this pathway is sufficient to activate PI3K signalling, even in the presence of PI3K inhibitors. This insulin feedback can be prevented using dietary or pharmaceutical approaches, which greatly enhance the efficacy/toxicity ratios of PI3K inhibitors. These findings have direct clinical implications for the multiple p110α inhibitors that are in clinical trials and provide a way to increase treatment efficacy for patients with many types of tumour