Claudio Raimondi

Targeting PDK1 in cancer

Abnormal activation of phosphoinositide 3-kinase (PI3K) signalling is very common in cancer, leading to deregulation of several intracellular processes normally controlled by this enzyme, including cell survival, growth, proliferation and migration. Mutations in the gene encoding the tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which leads to uncontrolled activation of the PI3K pathway, are reported in different cancers. Among the downstream effectors of PI3Ks, 3- phosphoinositide-dependent protein kinase 1 (PDK1) and protein kinase B (PKB)/Akt have a key role in several cancer types. More recent data indicate that alteration of PDK1 is a critical component of oncogenic PI3K signalling in breast cancer, suggesting that inhibition of PDK1 can inhibit breast cancer progression. PDK1 has an essential role in regulating cell migration especially in the context of PTEN deficiency. Downregulation of PDK1 levels inhibits migration and experimental metastasis of human breast cancer cells. PDK1 activates a large number of proteins, including Akt, some PKC isoforms, S6K and SGK. Data also reveal that PDK1 is oncogenic and this is dependent on PI3K pathway. Therefore, accumulating evidence demonstrates that PDK1 is a valid therapeutic target and suggests that PDK1 inhibitors may be useful to prevent cancer progression and abnormal tissue dissemination. This review will focus on published data on the role of PDK1 in cancer and approaches used to inhibit PDK1.

Class II phosphoinositide 3-kinase regulates exocytosis of insulin granules in pancreatic beta cells

Phosphoinositide 3-kinases (PI3Ks) are critical regulators of pancreatic β cell mass and survival, whereas their involvement in insulin secretion is more controversial. Furthermore, of the different PI3Ks, the class II isoforms were detected in β cells, although their role is still not well understood. Here we show that down-regulation of the class II PI3K isoform PI3K-C2α specifically impairs insulin granule exocytosis in rat insulinoma cells without affecting insulin content, the number of insulin granules at the plasma membrane, or the expression levels of key proteins involved in insulin secretion. Proteolysis of synaptosomal-associated protein of 25 kDa, a process involved in insulin granule exocytosis, is impaired in cells lacking PI3K-C2α. Finally, our data suggest that the mRNA for PI3K-C2α may be down-regulated in islets of Langerhans from type 2 diabetic compared with non-diabetic individuals. Our results reveal a critical role for PI3K-C2α in β cells and suggest that down-regulation of PI3K-C2α may be a feature of type 2 diabetes.

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR‐574‐3p and miR‐720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63‐mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.

Imatinib inhibits VEGF-independent angiogenesis by targeting neuropilin 1–dependent ABL1 activation in endothelial cells

Neuropilin 1 regulates angiogenesis in a VEGF-independent manner via association with ABL1, suggesting that Imatinib represents a novel opportunity for anti-angiogenic therapy.

Neuropilin signalling in vessels, neurons and tumours.

The neuropilins NRP1 and NRP2 are transmembrane proteins that regulate many different aspects of vascular and neural development. Even though they were originally identified as adhesion molecules, they are most commonly studied as co-receptors for secreted signalling molecules of the class 3 semaphorin (SEMA) and vascular endothelial growth factor (VEGF) families. During nervous system development, both classes of ligands control soma migration, axon patterning and synaptogenesis in the central nervous system, and they additionally help to guide the neural crest cell precursors of neurons and glia in the peripheral nervous system. Both classes of neuropilin ligands also control endothelial cell behaviour, with NRP1 acting as a VEGF-A isoform receptor in blood vascular endothelium and as a semaphorin receptor in lymphatic valve endothelium, and NRP2 promoting lymphatic vessel growth induced by VEGF-C. Here we provide an overview of neuropilin function in neurons and neural crest cells, discuss current knowledge of neuropilin signalling in the vasculature and conclude with a summary of neuropilin roles in cancer.

A Phosphoinositide 3-Kinase/Phospholipase Cgamma1 Pathway Regulates Fibroblast Growth Factor-Induced Capillary Tube Formation

Background The fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)γ1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions. Methodology/Principal Findings We show that FGF-2 activates PLCγ1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-P3 using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLCγ1 activation and the PI3K/PLCγ1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLCγ1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)α activation since dowregulation of PKCα expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKCα blocks FGF-2-dependent cell migration. Conclusion/Significance These data elucidate the role of PLCγ1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLCγ1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes.

NRP1 Regulates CDC42 Activation to Promote Filopodia Formation in Endothelial Tip Cells

Summary Sprouting blood vessels are led by filopodia-studded endothelial tip cells that respond to angiogenic signals. Mosaic lineage tracing previously revealed that NRP1 is essential for tip cell function, although its mechanistic role in tip cells remains poorly defined. Here, we show that NRP1 is dispensable for genetic tip cell identity. Instead, we find that NRP1 is essential to form the filopodial bursts that distinguish tip cells morphologically from neighboring stalk cells, because it enables the extracellular matrix (ECM)-induced activation of CDC42, a key regulator of filopodia formation. Accordingly, NRP1 knockdown and pharmacological CDC42 inhibition similarly impaired filopodia formation in vitro and in developing zebrafish in vivo. During mouse retinal angiogenesis, CDC42 inhibition impaired tip cell and vascular network formation, causing defects that resembled those due to loss of ECM-induced, but not VEGF-induced, NRP1 signaling. We conclude that NRP1 enables ECM-induced filopodia formation for tip cell function during sprouting angiogenesis.

A novel regulatory mechanism links PLCγ1 to PDK1

Summary 3-Phosphoinositide-dependent protein kinase-1 (PDK1) and phospholipase C (PLC)&ggr;1 are two key enzymes in signal transduction that control several intracellular processes. Despite the fact that PLC&ggr;1 has been investigated for several years, the mechanisms of activation of this enzyme are still not completely clear. Similarly, although PDK1 has been mostly investigated for its role in activation of Akt, a crucial enzyme in regulation of several cellular processes, it has become evident recently that the role of PDK1 in physiological and pathological conditions is not limited to Akt activation. Here we demonstrate that PDK1 regulates PLC&ggr;1 activation in a mechanism involving association of the two enzymes and modulation of PLC&ggr;1 tyrosine phosphorylation. We further show that this novel PDK1–PLC&ggr;1 pathway is important for cancer cell invasion. The identification of a PDK1–PLC&ggr;1 pathway reveals the existence of a previously undetected link between two of the most important enzymes in signal transduction. This is likely to have profound consequences for our understanding of several cellular functions that are dependent on phosphoinositides and controlled by PDK1 and PLC&ggr;1.

iASPP is a novel autophagy inhibitor in keratinocytes.

ABSTRACT The protein iASPP (encoded by PPP1R13L) is an evolutionarily conserved p53 inhibitor, the expression of which is often upregulated in human cancers. We have recently shown that iASPP is a crucial regulator of epidermal homeostasis. Here, we report that iASPP also acts as autophagy inhibitor in keratinocytes. Our data show that depletion of iASPP protects keratinocytes from apoptosis by modulating the expression of Noxa (also known as PMAIP1). In our model, iASPP expression can affect the fission-fusion cycle, mass and shape of mitochondria. iASPP-silenced keratinocytes display disorganization of cytosolic compartments and increased metabolic stress caused by deregulation of mTORC1 signaling. Moreover, increased levels of lipidated LC3 protein confirmed the activation of autophagy in iASPP-depleted cells. We have identified a novel mechanism modulating autophagy in keratinocytes that relies upon iASPP expression specifically reducing the interaction of Atg5–Atg12 with Atg16L1, an interaction that is essential for autophagosome formation or maturation. Using organotypic culture, we further explored the link between autophagy and differentiation, and we showed that impairing autophagy affects epidermal terminal differentiation. Our data provide an alternative mechanism to explain how epithelial integrity is maintained against environmental stressors and might also improve the understanding of the etiology of skin diseases that are characterized by defects in differentiation and DNA damage responses.

NRP1 function and targeting in neurovascular development and eye disease

Neuropilin 1 (NRP1) is expressed by neurons, blood vessels, immune cells and many other cell types in the mammalian body and binds a range of structurally and functionally diverse extracellular ligands to modulate organ development and function. In recent years, several types of mouse knockout models have been developed that have provided useful tools for experimental investigation of NRP1 function, and a multitude of therapeutics targeting NRP1 have been designed, mostly with the view to explore them for cancer treatment. This review provides a general overview of current knowledge of the signalling pathways that are modulated by NRP1, with particular focus on neuronal and vascular roles in the brain and retina. This review will also discuss the potential of NRP1 inhibitors for the treatment for neovascular eye diseases.