Federico Gulluni

PI3K/AKT signaling pathway and cancer: an updated review.

Abstract Despite development of novel agents targeting oncogenic pathways, matching targeted therapies to the genetic status of individual tumors is proving to be a daunting task for clinicians. To improve the clinical efficacy and to reduce the toxic side effects of treatments, a deep characterization of genetic alterations in different tumors is required. The mutational profile often evidences a gain of function or hyperactivity of phosphoinositide 3-kinases (PI3Ks) in tumors. These enzymes are activated downstream tyrosine kinase receptors (RTKs) and/or G proteins coupled receptors (GPCRs) and, via AKT, are able to induce mammalian target of rapamycin (mTOR) stimulation. Here, we elucidate the impact of class I (p110α, β, γ, and δ) catalytic subunit mutations on AKT-mediated cellular processes that control crucial mechanisms in tumor development. Moreover, the interrelation of PI3K signaling with mTOR, ERK, and RAS pathways will be discussed, exploiting the potential benefits of PI3K signaling inhibitors in clinical use.

Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate

Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic. Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits. No phosphatidylinositol other than PI(4,5)P2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P). How phosphatidylinositol conversion from PI(4,5)P2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) by class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P2 or PI(3)K C2α impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P2 by PI(3)K C2α is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P2 in endocytosis and unravel a novel discrete function of PI(3,4)P2 in a central cell physiological process.

PI3K Class II α Controls Spatially Restricted Endosomal PtdIns3P and Rab11 Activation to Promote Primary Cilium Function

Summary Multiple phosphatidylinositol (PtdIns) 3-kinases (PI3Ks) can produce PtdIns3P to control endocytic trafficking, but whether enzyme specialization occurs in defined subcellular locations is unclear. Here, we report that PI3K-C2α is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium, where it regulates production of a specific pool of PtdIns3P. Loss of PI3K-C2α-derived PtdIns3P leads to mislocalization of PRE markers such as TfR and Rab11, reduces Rab11 activation, and blocks accumulation of Rab8 at the primary cilium. These changes in turn cause defects in primary cilium elongation, Smo ciliary translocation, and Sonic Hedgehog (Shh) signaling and ultimately impair embryonic development. Selective reconstitution of PtdIns3P levels in cells lacking PI3K-C2α rescues Rab11 activation, primary cilium length, and Shh pathway induction. Thus, PI3K-C2α regulates the formation of a PtdIns3P pool at the PRE required for Rab11 and Shh pathway activation.

Targeting PI3K in Cancer: Any Good News?

The phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates several cellular processes and it’s one of the most frequently deregulated pathway in human tumors. Given its prominent role in cancer, there is great interest in the development of inhibitors able to target several members of PI3K signaling pathway in clinical trials. These drug candidates include PI3K inhibitors, both pan- and isoform-specific inhibitors, AKT, mTOR, and dual PI3K/mTOR inhibitors. As novel compounds progress into clinical trials, it’s becoming urgent to identify and select patient population that most likely benefit from PI3K inhibition. In this review we will discuss individual PIK3CA mutations as predictors of sensitivity and resistance to targeted therapies, leading to use of novel PI3K/mTOR/AKT inhibitors to a more “personalized” treatment.

In Vivo Role of INPP4B in Tumor and Metastasis Suppression through Regulation of PI3K–AKT Signaling at Endosomes

UNLABELLED The phosphatases PTEN and INPP4B have been proposed to act as tumor suppressors by antagonizing PI3K-AKT signaling and are frequently dysregulated in human cancer. Although PTEN has been extensively studied, little is known about the underlying mechanisms by which INPP4B exerts its tumor-suppressive function and its role in tumorigenesis in vivo. Here, we show that a partial or complete loss of Inpp4b morphs benign thyroid adenoma lesions in Pten heterozygous mice into lethal and metastatic follicular-like thyroid cancer (FTC). Importantly, analyses of human thyroid cancer cell lines and specimens reveal INPP4B downregulation in FTC. Mechanistically, we find that INPP4B, but not PTEN, is enriched in the early endosomes of thyroid cancer cells, where it selectively inhibits AKT2 activation and in turn tumor proliferation and anchorage-independent growth. We therefore identify INPP4B as a novel tumor suppressor in FTC oncogenesis and metastasis through localized regulation of the PI3K-AKT pathway at the endosomes. SIGNIFICANCE Although both PTEN and INPP4B can inhibit PI3K-AKT signaling through their lipid phosphatase activities, here we demonstrate lack of an epistatic relationship between the two tumor suppressors. Instead, the qualitative regulation of PI3K-AKT2 signaling by INPP4B provides a mechanism for their cooperation in suppressing thyroid tumorigenesis and metastasis.

Phosphoinositide 3-Kinase-C2α Regulates Polycystin-2 Ciliary Entry and Protects against Kidney Cyst Formation

Signaling from the primary cilium regulates kidney tubule development and cyst formation. However, the mechanism controlling targeting of ciliary components necessary for cilium morphogenesis and signaling is largely unknown. Here, we studied the function of class II phosphoinositide 3-kinase-C2α (PI3K-C2α) in renal tubule-derived inner medullary collecting duct 3 cells and show that PI3K-C2α resides at the recycling endosome compartment in proximity to the primary cilium base. In this subcellular location, PI3K-C2α controlled the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial reduction of PI3K-C2α was sufficient to impair elongation of the cilium and the ciliary transport of polycystin-2, as well as to alter proliferation signals linked to polycystin activity. In agreement, heterozygous deletion of PI3K-C2α in mice induced cilium elongation defects in kidney tubules and predisposed animals to cyst development, either in genetic models of polycystin-1/2 reduction or in response to ischemia/reperfusion-induced renal damage. These results indicate that PI3K-C2α is required for the transport of ciliary components such as polycystin-2, and partial loss of this enzyme is sufficient to exacerbate the pathogenesis of cystic kidney disease.

Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function

Proper organization of the mitotic spindle is key to genetic stability, but molecular components of inter-microtubule bridges that crosslink kinetochore fibers (K-fibers) are still largely unknown. Here we identify a kinase-independent function of class II phosphoinositide 3-OH kinase α (PI3K-C2α) acting as limiting scaffold protein organizing clathrin and TACC3 complex crosslinking K-fibers. Downregulation of PI3K-C2α causes spindle alterations, delayed anaphase onset, and aneuploidy, indicating that PI3K-C2α expression is required for genomic stability. Reduced abundance of PI3K-C2α in breast cancer models initially impairs tumor growth but later leads to the convergent evolution of fast-growing clones with mitotic checkpoint defects. As a consequence of altered spindle, loss of PI3K-C2α increases sensitivity to taxane-based therapy in pre-clinical models and in neoadjuvant settings.

Methods to Measure the Enzymatic Activity of PI3Ks

Phosphoinositide-3-kinase (PI3K) signaling has been implicated in a panoply of cellular responses including survival, proliferation, protein synthesis, migration, and vesicular trafficking. In addition, alterations in the enzymatic activity of PI3Ks have been involved in the pathogenesis of multiple diseases, ranging from cancer to chronic inflammation. The emerging interest in PI3K as a pharmacological target has prompted the development of several molecules with inhibitory activity. In this context, the quantification of the second messenger generated by PI3Ks, phosphoinositide-3-phosphate, offers an opportunity to directly test variations in the lipid kinase activity of PI3K in physiological as well as pathological conditions. Here, we will describe common methods to measure the lipid kinase activity of PI3K in vitro and new techniques to follow the production of phosphoinositide-3-phosphate in vivo. These methods are relevant to study the alterations of the PI3K systems at the interface between signaling and oncometabolism.

Cytokinetic Abscission: Phosphoinositides and ESCRTs Direct the Final Cut

Cytokinetic abscission involves the fine and regulated recruitment of membrane remodeling proteins that participate in the abscission of the intracellular bridge that connects the two dividing cells. This essential process is mediated by the concomitant activity of the endosomal sorting complex required for transport (ESCRT) and the vesicular trafficking directed to the midbody. Phosphoinositides (PtdIns), produced at plasma membrane, and endosomes, act as molecular intermediates by recruiting effector proteins involved in multiple cellular processes, such as intracellular signaling, endo‐ and exo‐cytosis, and membrane remodeling events. Emerging evidences suggest that PtdIns have an active role in recruiting key elements that control the stability and the remodeling of the cytoskeleton from the furrow ingression to the abscission, at the end of cytokinesis. Accordingly, a possible concomitant and coordinated activity between PtdIns production and ESCRT machinery assembly could also exist and recent findings are pointing the attention on poorly understood ESCRT subunits potentially able to associate with PtdIns rich membranes. Although further studies are required to link PtdIns to ESCRT machinery during abscission, this might represent a promising field of study. J. Cell. Biochem. 118: 3561–3568, 2017.

PO-466 PI3K-C2A regulates mitotic spindle assembly and chemotherapy response in breast cancer

Introduction Proper organisation of the mitotic spindle is key to genetic stability but the molecular components of inter-microtubule (MT) bridges that crosslink kinetochore fibres (K-fibres) are still largely unknown. Here, we identify class II phosphoinositide 3-OH kinase a (PI3K-C2α) as a limiting scaffold protein organising the clathrin and TACC3 complex crosslinking K-fibres. Material and methods Pik3c2a+/- mice were intercrossed with a transgenic strain expressing the activated HER-2/Neu oncogene in the mammary gland. Mice were weekly followed for survival, tumour appearance and growth. Primary Murine Mammary Epithelial Tumour (MMET) cells were derived from early and late stage tumours. Truncating PI3KC2α mutants were generated and interaction with TACC3 was tested. Levels of PI3K-C2α expression were assessed by IHC in breast cancer tissue microarrays (TMA) and correlated with response to chemotherapy. Results and discussions Loss of PI3K-C2α expression is a frequent occurrence in breast cancer patients (48%) and correlates with local recurrence and metastatic disease. The heterozygous loss of PI3K-C2α initially delays tumour onset but, on the long run, leads to the convergent evolution of aggressive clones with mitotic checkpoint defects. In line with this, downregulation of PI3K-C2α promotes spindle alterations and aneuploidy, indicating that PI3K-C2α expression is a key determinant of genomic stability. As a consequence of the altered spindle, reduction of PI3K-C2α expression increases the sensitivity to anti-MT drugs, such as paclitaxel, in pre-clinical models and in breast cancer patients. Conclusion Loss of PI3K-C2α expression is a frequent occurrence in breast cancer patients (48%) and correlates with local recurrence and metastatic disease. The heterozygous loss of PI3K-C2α initially delays tumour onset but, on the long run, leads to the convergent evolution of aggressive clones with mitotic checkpoint defects. In line with this, downregulation of PI3K-C2α promotes spindle alterations and aneuploidy, indicating that PI3K-C2α expression is a key determinant of genomic stability. As a consequence of the altered spindle, reduction of PI3K-C2α expression increases the sensitivity to anti-MT drugs, such as paclitaxel, in pre-clinical models and in breast cancer patients.