PI3K regulation of RAC1 is required for KRAS-induced pancreatic tumorigenesis in mice.

BACKGROUND & AIMS: New drug targets are urgently needed for the treatment of patients with pancreatic ductal adenocarcinoma (PDA). Nearly all PDAs contain oncogenic mutations in the KRAS gene. Pharmacological inhibition of KRAS has been unsuccessful, leading to a focus on downstream effectors that are more easily targeted with small molecule inhibitors. We investigated the contributions of phosphoinositide 3-kinase (PI3K) to KRAS-initiated tumorigenesis. METHODS: Tumorigenesis was measured in the Kras(G12D/+);Ptf1a(Cre/+) mouse model of PDA; these mice were crossed with mice with pancreas-specific disruption of genes encoding PI3K p110α (Pik3ca), p110ß (Pik3cb), or RAC1 (Rac1). Pancreatitis was induced with 5 daily intraperitoneal injections of cerulein. Pancreata and primary acinar cells were isolated; acinar cells were incubated with an inhibitor of p110α (PIK75) followed by a broad-spectrum PI3K inhibitor (GDC0941). PDA cell lines (NB490 and MiaPaCa2) were incubated with PIK75 followed by GDC0941. Tissues and cells were analyzed by histology, immunohistochemistry, quantitative reverse-transcription polymerase chain reaction, and immunofluorescence analyses for factors involved in the PI3K signaling pathway. We also examined human pancreas tissue microarrays for levels of p110α and other PI3K pathway components. RESULTS: Pancreas-specific disruption of Pik3ca or Rac1, but not Pik3cb, prevented the development of pancreatic tumors in Kras(G12D/+);Ptf1a(Cre/+) mice. Loss of transformation was independent of AKT regulation. Preneoplastic ductal metaplasia developed in mice lacking pancreatic p110α but regressed. Levels of activated and total RAC1 were higher in pancreatic tissues from Kras(G12D/+);Ptf1a(Cre/+) mice compared with controls. Loss of p110α reduced RAC1 activity and expression in these tissues. p110α was required for the up-regulation and activity of RAC guanine exchange factors during tumorigenesis. Levels of p110α and RAC1 were increased in human pancreatic intraepithelial neoplasias and PDAs compared with healthy pancreata. CONCLUSIONS: KRAS signaling, via p110α to activate RAC1, is required for transformation in Kras(G12D/+);Ptf1a(Cre/+) mice.

Calpain-dependent cleavage of junctophilin-2 and T-tubule remodeling in a mouse model of reversible heart failure.

BACKGROUND: A highly organized transverse tubule (T-tubule) network is necessary for efficient Ca(2+)-induced Ca(2+) release and synchronized contraction of ventricular myocytes. Increasing evidence suggests that T-tubule remodeling due to junctophilin-2 (JP-2) downregulation plays a critical role in the progression of heart failure. However, the mechanisms underlying JP-2 dysregulation remain incompletely understood. METHODS AND RESULTS: A mouse model of reversible heart failure that is driven by conditional activation of the heterotrimeric G protein Gαq in cardiac myocytes was used in this study. Mice with activated Gαq exhibited disruption of the T-tubule network and defects in Ca(2+) handling that culminated in heart failure compared with wild-type mice. Activation of Gαq/phospholipase Cß signaling increased the activity of the Ca(2+)-dependent protease calpain, leading to the proteolytic cleavage of JP-2. A novel calpain cleavage fragment of JP-2 is detected only in hearts with constitutive Gαq signaling to phospholipase Cß. Termination of the Gαq signal was followed by normalization of the JP-2 protein level, repair of the T-tubule network, improvements in Ca(2+) handling, and reversal of heart failure. Treatment of mice with a calpain inhibitor prevented Gαq-dependent JP-2 cleavage, T-tubule disruption, and the development of heart failure. CONCLUSIONS: Disruption of the T-tubule network in heart failure is a reversible process. Gαq-dependent activation of calpain and subsequent proteolysis of JP-2 appear to be the molecular mechanism that leads to T-tubule remodeling, Ca(2+) handling dysfunction, and progression to heart failure in this mouse model.

Increased persistent sodium current due to decreased PI3K signaling contributes to QT prolongation in the diabetic heart.

Diabetes is an independent risk factor for sudden cardiac death and ventricular arrhythmia complications of acute coronary syndrome. Prolongation of the QT interval on the electrocardiogram is also a risk factor for arrhythmias and sudden death, and the increased prevalence of QT prolongation is an independent risk factor for cardiovascular death in diabetic patients. The pathophysiological mechanisms responsible for this lethal complication are poorly understood. Diabetes is associated with a reduction in phosphoinositide 3-kinase (PI3K) signaling, which regulates the action potential duration (APD) of individual myocytes and thus the QT interval by altering multiple ion currents, including the persistent sodium current INaP. Here, we report a mechanism for diabetes-induced QT prolongation that involves an increase in INaP caused by defective PI3K signaling. Cardiac myocytes of mice with type 1 or type 2 diabetes exhibited an increase in APD that was reversed by expression of constitutively active PI3K or intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate (PIP3), the second messenger produced by PI3K. The diabetic myocytes also showed an increase in INaP that was reversed by activated PI3K or PIP3. The increases in APD and INaP in myocytes translated into QT interval prolongation for both types of diabetic mice. The long QT interval of type 1 diabetic hearts was shortened by insulin treatment ex vivo, and this effect was blocked by a PI3K inhibitor. Treatment of both types of diabetic mouse hearts with an INaP blocker also shortened the QT interval. These results indicate that downregulation of cardiac PI3K signaling in diabetes prolongs the QT interval at least in part by causing an increase in INaP. This mechanism may explain why the diabetic population has an increased risk of life-threatening arrhythmias.

Suppression of phosphoinositide 3-kinase signaling and alteration of multiple ion currents in drug-induced long QT syndrome.

Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K(+)) current I(Kr). Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K(+) currents I(Kr) and I(Ks), the L-type calcium ion (Ca(2+)) current I(Ca,L), and the peak sodium ion (Na(+)) current I(Na) and increased the persistent Na(+) current I(NaP). Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110α catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in I(NaP). These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias.

PI3Ks maintain the structural integrity of T-tubules in cardiac myocytes.

BACKGROUND: Phosphoinositide 3-kinases (PI3Ks) regulate numerous physiological processes including some aspects of cardiac function. Although regulation of cardiac contraction by individual PI3K isoforms has been studied, little is known about the cardiac consequences of downregulating multiple PI3Ks concurrently. METHODS AND RESULTS: Genetic ablation of both p110α and p110ß in cardiac myocytes throughout development or in adult mice caused heart failure and death. Ventricular myocytes from double knockout animals showed transverse tubule (T-tubule) loss and disorganization, misalignment of L-type Ca(2+) channels in the T-tubules with ryanodine receptors in the sarcoplasmic reticulum, and reduced Ca(2+) transients and contractility. Junctophilin-2, which is thought to tether T-tubules to the sarcoplasmic reticulum, was mislocalized in the double PI3K-null myocytes without a change in expression level. CONCLUSIONS: PI3K p110α and p110ß are required to maintain the organized network of T-tubules that is vital for efficient Ca(2+)-induced Ca(2+) release and ventricular contraction. PI3Ks maintain T-tubule organization by regulating junctophilin-2 localization. These results could have important medical implications because several PI3K inhibitors that target both isoforms are being used to treat cancer patients in clinical trials.